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VEGFD Protects Retinal Ganglion Cells and, consequently, Capillaries against Excitotoxic Injury
Journal Pre-proof VEGFD protects retinal ganglion cells and, consequently, capillaries against excitotoxic injury Annabelle Schlüter, Bahar Aksan, Ricarda Diem, Richard Fairless, Daniela Mauceri PII:
S2329-0501(19)30158-5
DOI:
https://doi.org/10.1016/j.omtm.2019.12.009
Reference:
OMTM 367
To appear in:
Molecular Therapy: Methods & Clinical Development
Received Date: 19 December 2019 Accepted Date: 19 December 2019
Please cite this article as: Schlüter A, Aksan B, Diem R, Fairless R, Mauceri D, VEGFD protects retinal ganglion cells and, consequently, capillaries against excitotoxic injury, Molecular Therapy: Methods & Clinical Development (2020), doi: https://doi.org/10.1016/j.omtm.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s).
1
VEGFD protects retinal ganglion cells and, consequently, capillaries against excitotoxic
2
injury
3 4
Annabelle Schlüter1, Bahar Aksan1, Ricarda Diem2,3, Richard Fairless2,3, Daniela Mauceri1*
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1
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University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany
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2
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Heidelberg, Germany
Department of Neurobiology, Interdisciplinary Center for Neurosciences, Heidelberg
Department of Neurology, University Clinic Heidelberg, Im Neuenheimer Feld 368, 69120
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3
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Centre (DKFZ), 69120 Heidelberg, Germany
CCU Neurooncology, German Cancer Consortium (DKTK), German Cancer Research
12 13 14
* Correspondence:
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Dr. Daniela Mauceri
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Email: [email protected],
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Phone: +49 6221 5416508
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Short title:
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VEGFD spares neurons and capillaries from damage
22 23 24
Keywords:
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VEGFD, retinal ganglion cells, endothelial cells, excitotoxicity, neuroprotection.
1
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Abstract
27
In the central nervous system, neurons and the vasculature influence each other. While it is
28
well described that a functional vascular system is trophic to neurons and that vascular
29
damage contributes to neurodegeneration; the opposite scenario in which neural damage
30
might impact the microvasculature is less defined. Here, using an in vivo excitotoxic
31
approach in adult mice as a tool to cause specific damage to retinal ganglion cells, we
32
detected a subsequent damage to endothelial cells in retinal capillaries. Further, we detected
33
decreased expression of Vascular Endothelial Growth Factor D (VEGFD) in retinal ganglion
34
cells. In vivo VEGFD supplementation via neuronal-specific viral-mediated expression or
35
acute intravitreal delivery of the mature protein preserved the structural and functional
36
integrity of retinal ganglion cells against excitotoxicity and, additionally spared endothelial
37
cells from degeneration. Viral-mediated suppression of expression of the VEGFD-binding
38
receptor VEGFR3 in retinal ganglion cells revealed that VEGFD exerts its protective
39
capacity directly on retinal ganglion cells while protection of endothelial cells is the result of
40
upheld neuronal integrity. These findings suggest that VEGFD supplementation might be a
41
novel, clinically applicable approach for neuronal and vascular protection.
2
42
Introduction
43
In the central nervous system (CNS), the neuronal and vascular components have a strong
44
structural and functional interdependence during both development and adulthood 1. In the
45
retina, in particular, a tight coupling between retinal ganglion cells (RGCs) and capillaries
46
has been described 2 but the molecular and cellular players underlying this link have not been
47
completely elucidated. Members of the Vascular Endothelial Growth Factor (VEGF) family
48
have been reported to be possible modulators of the communication between the vascular and
49
neuronal compartments 3-6.
50
The retinal neurovascular interface plays a role not only in physiology but also in several
51
pathologies. Dysfunctions of both the neural and vascular components have been implicated
52
in pathologies of the visual system such as glaucoma, diabetic retinopathy and macular
53
degeneration 2. It is well known that vascular occlusion and damage can contribute to the
54
progression of neural retina injury
55
integrity of the capillaries is unclear. In experimental models of neural retinal injury, delayed
56
damage to the retinal microvasculature has been observed 8-10, suggesting that neural integrity
57
may indeed be critical for vascular health.
58
Capillaries have a small diameter and consist of a single endothelial cell (EC) layer forming
59
the inner lining, which are anchored to the basement membrane (BM). A well-reported
60
phenomenon, which has yet no clear explanation for its occurrence, is the detected higher
61
number of empty sleeves throughout the CNS – retina, hippocampus, cortex – in relation to
62
both physiological aging and to several neurodegenerative diseases
63
named string vessels or acellular capillaries, consist of remnants of BM, which have lost the
64
inner ECs and, in several cases, have collapsed
65
plasma as they are not perfused
66
tissue and enhance degeneration. It is not known what drives the formation of empty sleeves;
7
11
while the putative impact of neuronal damage onto the
11
11-13
. Empty sleeves, also
. Empty sleeves cannot transport blood or
and thus may have noxious effects on the surrounding
3
67
however, their increased presence following neuronal degeneration indicates that neurons
68
might play a role.
69
Here, we used an in vivo approach to cause specific damage to RGCs, which we found to be
70
accompanied by a reduction in the expression of VEGFD, a member of the VEGF family, in
71
RGCs and formation of empty sleeves. We additionally reveal the protective capacity of
72
VEGFD against degeneration of RGCs and, as a result of the maintained neuronal integrity,
73
also against capillary damage. Our data indicate that VEGFD delivery might provide a
74
promising therapeutic strategy for the treatment of degenerative diseases of the neuronal and
75
vascular system.
76 77
Results
78
Excitotoxicity is a pathological phenomenon leading to neuronal damage or death that has
79
been associated with several disorders of the nervous system
80
(NMDA) receptors (NMDARs), calcium-permeable channels, are the principal initiators of
81
excitotoxic cell death induced by excessive receptor activation either by high glutamate
82
levels or NMDA exposure
83
retinal ischemia, and diabetic retinopathy
84
NMDA delivered via intravitreal injections can be utilized as a tool to cause RGCs death in
85
vivo
86
analyzed NMDA-induced RGC death at two different time points (day 1 (d1) and day 7 (d7))
87
(Fig. 1). Brn3a served as a marker to identify and quantify RGCs 27 in central and peripheral
88
regions of retinal whole mounts (Fig. 1A,B). One day after intravitreal NMDA injections, the
89
number of RGCs was significantly decreased compared to the PBS-injected control eyes (Fig.
90
1A-D). RGC death did not worsen over time as observed by comparable RGC survival seven
91
days after the NMDAR-mediated excitotoxic insult (Fig. 1C,D). We further detected a
21-26.
14, 15
14
. N-methyl-D-aspartate
. In the eye, excitotoxicity has been linked to glaucoma, 16-20
. Indeed, high concentrations of glutamate or
We performed a single intravitreal injection of 10 nmol NMDA in adult mice and
4
92
significant reduction in the mRNA level of the RGCs markers thy1 and nefl (Fig. 1E) in
93
retinal homogenates. To investigate whether intravitreal injection of NMDA damages other
94
retinal neurons than RGCs, we measured the thickness of the inner nuclear layer (INL) as
95
indication of possible cell death in the INL 28. We did not detect any significant changes after
96
intravitreal NMDA injections (Fig. 1F). TUNEL assay carried out on retinal sections
97
confirmed that the extent of apoptosis in retinal tissue following intravitreal injection of 10
98
nmol NMDA was negligible in the INL (percentage of TUNEL+ cells: d1, PBS (0.02949081
99
%) vs NMDA (0.6696871 %), p= 0.2019; d7, PBS (0.09155836 %) vs NMDA (0.08281018
100
%), p= 0.7552; unpaired student’s t-test, n=3) and outer nuclear layer (ONL; percentage of
101
TUNEL+ cells: d1, PBS (0.07544173 %) vs NMDA (0.05547914 %), p= 0.7181; d7, PBS
102
(0.05516085 %) vs NMDA (0.0662406 %), p= 0.6445; unpaired student’s t-test, n=3). Taken
103
together, these data demonstrate that intravitreal injection of NMDA can be used to trigger
104
degeneration of RGCs in the retina in vivo.
105 106
In the retina, RGCs are structurally and functionally coupled with capillaries and
107
dysfunctions of both the neural and vascular components are present in pathologies of the
108
visual system 2. We proceeded to analyze the possible effects of the in vivo NMDA insult on
109
the retinal capillaries. We found that intravitreal injection of NMDA promoted the formation
110
of collapsed empty sleeves at both d1 and d7 assessed by immunostaining of the BM with
111
anti-Collagen-IV and labeling of the ECs with Isolectin-B4 (Fig. 2A-C). Using electron
112
microscopy, it was possible to observe, in some cases, occluded lumen of the capillaries due
113
to swelling of the damaged ECs prior to their death and removal 29, 30, whereas tight junctions
114
remained intact (Fig. 2D). The increased presence of collapsed empty sleeves appeared to be
115
a specific phenomenon, as other vascular parameters, such as vessel surface area, vessel
116
diameter, and vessel length were not affected by the NMDA insult at any of the analyzed
5
117
time points (Fig. 2E-G). To assess potential NMDA-mediated effects on the blood-retinal
118
barrier (BRB), we first analyzed in retinal homogenates the mRNA levels of ocld, cld5, or
119
tjp1, encoding for Occludin, Claudin-5, and ZO-1 respectively, molecules important for the
120
functionality of endothelial tight junctions. None of the analyzed genes was affected by
121
intravitreal NMDA injection (Fig. 2H). To clarify whether intravitreal NMDA injection could
122
prompt extravasation of cells, we assessed the percentage of erythrocytes within the retina
123
capillaries 31 in relation to the total amount of erythrocytes within and outside vessels (Figure
124
2I,J). We found no difference between PBS-injected controls and NMDA-injected retinas
125
(Figure 2I,J). Further, in whole-retina extracts, we examined the extravasation of
126
intravenously injected 3 kDa-dextran-fluorescein as well as of endogenous albumin and IgG
127
following intravitreal NMDA injections 30, which we found similar to PBS-injected controls
128
(Fig. 2K-N). These data indicate that NMDA-triggered excitotoxicity, in addition to
129
promoting death of RGCs, causes increased formation of empty sleeves without affecting
130
BRB functionality or the overall structure of the vascular network.
131
ECs of the CNS express NMDA receptors
132
NMDAR-mediated toxicity. To understand whether the observed degeneration of ECs
133
following NMDA intravitreal injection is the result of a direct or indirect effect of NMDA,
134
we used a different approach and directly exposed only ECs to NMDA in vitro. We
135
confirmed the expression of the GluN1 channel-forming subunit of the NMDA receptor as
136
well as of the regulatory subunits GluN2A and GluN2D (Fig 2O) in a mouse brain EC line
137
(bEnd.3) 34, 37. GluN2B and GluN2C subunits were not detected. We decided to compare the
138
sensitivity of cultured neurons and ECs to NMDA using exposure to 20 µM NMDA, which
139
typically causes fast neuronal death
140
minutes or for 24 hrs, the vitality of the cultured mouse brain ECs remained unaltered (Fig.
141
2P) while neurons considerably die
38
38
32-36
and might therefore be a direct target of
. When treated with 20 µM NMDA acutely for 10
. Even using higher NMDA concentration (1 mM),
6
142
close to the one reached in vivo after intravitreal injection of 10nmol NMDA in the relatively
143
small volume of the mouse eye; while neuronal death rate reached 100%, the vitality of
144
cultured endothelial cells was still unaffected (Untreated vs NMDA 10 min, n=3, p=0.9868.
145
Untreated vs NMDA 24 hrs, n=3, p=0.6556. Unpaired Student’s t-test.) The expression levels
146
of ocld, cld5, and tjp1 of NMDA-treated cultured ECs were also similar to control-treated
147
cells (Fig. 2Q). In the mammalian neurovascular unit, astrocytes represent a considerable
148
percentage of the present cells and can exchange signals with ECs
149
studies revealed the functional expression of NMDARs on various types of glia cells in the
150
nervous system 40. In a similar manner to that observed for cultured brain ECs, we found that
151
application of NMDA for 24 hrs did not affect the survival of primary cultured astrocytes
152
(Fig. 2R). Collectively, these data indicate that the in vivo detected increased number of
153
empty sleeves, due to loss of ECs, after NMDA-based insult (Fig. 2A-D) is likely mediated
154
by an indirect mechanism.
39
. Moreover, numerous
155 156
By reducing the time of analysis following intravitreal injection of NMDA to 6 hrs, we found
157
that loss of RGCs (40.2 ± 6.3%; NMDA vs PBS, n=3, p=0.0005) temporally precedes the
158
formation of empty sleeves (105.9 ± 5.8% number of empty sleeves after NMDA treatment;
159
NMDA vs PBS, n=3, p=0.5249). In experimental models of neural retinal damage,
160
degeneration of the capillaries in correlation to RGC death has been observed 7-10, but it is not
161
yet understood if and how RGCs might impact the retinal microvasculature. We hypothesized
162
that degeneration of RGCs could negatively impact capillaries via an alteration in the
163
molecular crosstalk between RGCs and ECs. The vascular endothelial growth factor (VEGF)
164
family members and their receptors (VEGFRs) play roles both in the vascular and nervous
165
system where they regulate, for example, vascular development, angiogenesis as well as
166
axonal path finding, dendritic morphology, synaptic plasticity, neuroprotection and cognition 7
167
41-43
168
cross-talk between neurons and endothelial cells (ECs) in the retina has been described 6, and
169
VEGFR3 has been detected outside the vasculature in neural elements of the retina 44, 45. We
170
therefore evaluated the potential effects of NMDA intravitreal injection on the expression
171
levels of VEGF family members and receptors. While no change was found for the mRNA
172
levels of vegf, vegfc, vegfr2 (kdr) or vegfr3 (flt4) in retinal homogenates, we detected a
173
significant reduction in vegfd levels (Fig. 3A). VEGFD, beyond its previously documented
174
functions in angiogenesis and lymphangiogenesis 42, is expressed also in neurons
175
fundamental for the maintenance of neuronal dendritic architecture and cognition 43, 49-51. We
176
successfully detected VEGFD mRNA (Fig. 3C) and VEGFD protein in RGCs in post-mortem
177
human retinas (Fig. 3B) as well as in adult mouse RGCs (Fig. 3E). After intravitreal injection
178
of NMDA, we found that VEGFD protein levels were specifically reduced in RGCs (Fig.
179
3D,E). Recently, in agreement with our present findings, it was shown that bath application
180
of NMDA to primary cultured neurons caused a rapid and progressive decline of vegfd
181
mRNA levels 52. Using a similar experimental strategy, namely NMDA treatment of cultured
182
cells, we found that vegfd expression levels remained constant in both mouse cultured ECs
183
(Fig. 3F) and primary astrocytic cultures (Fig. 3G) following 24 hrs of exposure to NMDA.
184
Thus, an NMDA-mediated insult caused reduction of VEGFD expression in RGCs but not in
185
other cell types.
. Recently, a novel function of neuronal VEGFR2 expression in the regulation of the
46-48
and is
186 187
We hypothesized that preventing VEGFD down-regulation in RGCs might be beneficial for
188
RGC survival after NMDA-triggered injury. We intravitreally injected adult wildtype mice
189
with recombinant adeno-associated viruses (rAAVs) containing neuron-specific expression
190
cassettes for VEGFD (rAAV-VEGFD)
191
additional HA- (for rAAV-VEGFD) or Flag- (for rAAV-LacZ) tags to facilitate their
43, 50
8
or as control, LacZ (rAAV-LacZ), with
192
detection. Intravitreal injection of rAAVs is a robust method used successfully to achieve
193
long-lasting, stable gene delivery
194
VEGFD has been previously used effectively to boost neuronal expression of VEGFD
195
and does not infect cells of the microvasculature (Supplementary Fig. 1A). To investigate the
196
protective capacities of neuronal overexpressed VEGFD against excitotoxic retinal damage,
197
we subjected rAAV-LacZ- and rAAV-VEGFD-injected mice to NMDA-triggered
198
excitotoxicity and quantified RGC degeneration at d7 after the excitotoxic insult. While
199
NMDA injections caused robust RGC loss in rAAV-LacZ-injected mice, RGC survival after
200
NMDA treatment in rAAV-VEGFD-injected mice was similar to PBS-injected control eyes
201
(Fig. 4A,C,D). Further, VEGFD overexpression in RGCs significantly protected RGC axons
202
in the optic nerve from the NMDA-mediated excitotoxic damage assessed by measuring the
203
increased presence of dephosphorylated neurofilaments (detected by SMI-32), as indication
204
for axonal stress. After intravitreal NMDA injections, in rAAV-LacZ-injected mice, SMI-32-
205
labelled clusters greatly increased in comparison to PBS-injected controls, while in rAAV-
206
VEGFD-injected animals, their NMDA-triggered formation was blunted (Fig. 4B,E,F).
207
We proceeded to test whether providing VEGFD in an acute treatment, at the same time of
208
the insult, would still spare RGCs from degeneration. We intravitreally co-injected
209
recombinant mature VEGFD protein (rVEGFD) with either PBS or NMDA and determined
210
RGC survival at d1 and d7 after injection (Fig. 5). At both time points, rVEGFD prevented
211
excitotoxic degeneration of RGCs (Fig. 5A,C-E). Further, we found that acute delivery of
212
rVEGFD was capable to reduce the formation of NMDA-triggered SMI-32-positive clusters,
213
indicative of axonal damage (Fig. 5B,F,G). As control, we performed similar experiments by
214
co-injecting a different recombinant protein, recombinant GFP (rGFP), instead of rVEGFD,
215
with NMDA or PBS (Fig. 5H-L). rGFP treatment did not have any effects on RGC survival
216
(Fig. 5H-J; Supplementary Fig. 2A) or axonal injury (Fig. 5K,L; Supplementary Fig. 2B). In
53
and is under consideration in clinical trials
9
54
. rAAV43, 50
217
conclusion, these findings show the specific protective capacity of VEGFD on RGCs and
218
axonal integrity in an NMDA-triggered excitotoxicity model.
219
We investigated whether VEGFD supplementation could also restore the functional
220
properties of RGCs in addition to sparing them from structural degeneration and death (Fig.
221
4, 5). For this purpose, we measured the function of RGCs within the retina by performing
222
pattern electroretinography in mice
223
without co-injection of rVEGFD (Fig. 6). NMDA-triggered excitotoxicity led to significantly
224
decreased amplitudes at complex spatial frequencies (0.10 cyc/deg), which represent reduced
225
neuronal activity in the retina. The supplementation of rVEGFD with NMDA was capable to
226
restore amplitudes to values comparable to those in PBS- or rVEGFD-injected control mice
227
(Fig. 6B, C). After sacrificing the mice, we confirmed, in agreement to what was assessed
228
previously (Fig. 5), that intravitreal NMDA injections resulted in significant RGC loss (48.52
229
± 11.52%), which was reduced after rVEGFD supplementation (19.78 ± 4.55%). In
230
summary, our data show the potential therapeutic capacity of intravitreal VEGFD
231
supplementation in the maintenance of functional and intact RGCs.
232
Next, we tested whether supplementation of VEGFD could also spare the retinal
233
microvasculature from NMDA-triggered excitotoxic damage (Fig. 2). When VEGFD was
234
specifically overexpressed in RGCs via rAAV-VEGFD (Supplementary Fig. 1), the
235
formation of collapsed empty sleeves at d7 after the NMDA-triggered excitotoxic insult was
236
reduced in comparison to control mice injected with rAAV-LacZ (Fig. 7A,C,D). Acute
237
VEGFD treatment via co-injection of rVEGFD led to similar results displaying a significant
238
decrease in the number of empty sleeves at d1 and d7 after NMDA treatment (Fig. 7B,E-G).
239
Delivery of rGFP did not affect the NMDA-triggered formation of empty sleeves (Fig. 7 H-J;
240
Supplementary Fig. 2C) indicating a specific protective effect of VEGFD against NMDA-
55
subjected to NMDA-triggered excitotoxicity with or
10
241
triggered vascular damage.
242
Our data show that an NMDA-mediated excitotoxic insult decreases the levels of VEGFD
243
specifically in RGCs and triggers RGC degeneration prior to promoting formation of empty
244
sleeves, likely via an indirect mechanism. Increasing VEGFD levels blunts the excitotoxicity-
245
induced degeneration of both RGCs and ECs. In humans, VEGFD binds and activates both
246
VEGFR2 and VEGFR3, whereas murine VEGFD exclusively activates VEGFR3 56. To better
247
elucidate whether capillaries are spared from excitotoxic damage as a consequence of a
248
VEGFD-mediated effects on neuronal survival or via a possible direct trophic simultaneous
249
action of VEGFD on ECs, we used an approach to specifically reduce the expression of
250
VEGFR3 in RGCs 45 while leaving the expression unaltered in ECs. There are no satisfactory
251
inducible cre-driver lines, which would guarantee efficient and extensive manipulation of the
252
diverse RGC subtypes without affecting other retinal neurons and cell types
253
decided to employ intravitreal injection of rAAV-shVEGFR3
254
the mouse Flt4 (VEGFR3) mRNA inserted downstream of the U6 promoter of the rAAV
255
vector or, as control, a scrambled version rAAV-shSCR. The used rAAVs have been
256
previously extensively characterized 43 and carry an mCherry expression cassette allowing for
257
detection of infected neurons. Three weeks after intravitreal rAAV delivery, the infection rate
258
of Brn3a-positive RGCs was above 60%. Other cell types within the retina, including cells of
259
the vascular system, were not transduced by the used viruses (Supplementary Fig. 1C). Three
260
weeks after infection, we performed intravitreal NMDA injections with or without co-
261
injection of rVEGFD. Using this strategy, VEGFD cannot act on RGCs any longer - as they
262
do not express VEGFR3 - while they are still vulnerable to NMDA. In control mice that were
263
intravitreally injected with rAAV-shSCR, NMDA caused significant RGC loss, which was
264
prevented by intravitreal supplementation of rVEGFD (Fig. 8A-C), as expected. When
265
VEGFR3 expression in RGCs was specifically reduced via rAAV-shVEGFR3, the VEGFD11
43
57-59
. Thus, we
encoding shRNAs targeting
266
mediated protection of RGCs from excitotoxicity was completely lost (Fig. 8A,B,D). In
267
rAAV-shSCR-injected mice, rVEGFD still interfered with formation of empty sleeves (Fig.
268
8E,F,G,). In rAAV-shVEGFR3 injected mice, however, ECs were no longer protected from
269
the NMDA-triggered degeneration by the administration of rVEGFD, although rVEGFD
270
could activate endothelial VEGFR3 (Fig. 8E-H). Thus, our data indicate that VEGFD-
271
mediated safeguard of RGCs and capillaries from damage takes place by VEGFD directly
272
exerting its protective functions on RGCs and, as a consequence of this phenomenon, sparing
273
ECs from degeneration (Fig. 8I)
274
Discussion
275
In this study, we found that an injury to RGCs triggers downregulation of neuronal VEGFD
276
and is followed by capillary damage. Moreover, we demonstrate that VEGFD
277
supplementation promotes RGC survival, which is crucial to prevent the formation of
278
collapsed, acellular capillaries. Taken together, VEGFD might have clinical relevance to
279
robustly promote neuronal and vascular integrity.
280
Degeneration of neurons and regression of capillaries have been reported for several CNS
281
disorders
282
cause for many disorders of the CNS
283
excitotoxic death of neurons along with damage to the microvasculature has been described 8,
284
10
285
available. Here, we detected RGC damage after a single intravitreal NMDA injection within
286
6hrs from delivering the insult, which did not significantly progress over time. Degeneration
287
of ECs followed neuronal death temporally. Unlike the widely acknowledged contribution of
288
vascular abnormalities to neurodegeneration
289
following neural degeneration has been less discussed although it has been observed, for
290
instance, in diabetes and glaucoma
4, 60
. Noxious NMDAR-mediated excitotoxicity has been put forward as a possible 61-63
. Also for pathologies of the visual system,
; however, no detailed description of the possible influence of neurons on EC integrity is
7, 65
64
, delayed damage to the microvasculature
. Our data suggest that signals deriving from 12
291
degenerating RGCs might be the cause of capillary damage. Such a hypothesis is further
292
supported by our observations that a direct NMDA insult to cultured brain ECs did not affect
293
their survival, although they expressed different GluN subunits ensuring a functional
294
NMDAR
295
that RGCs are degenerating to the ECs. This process might be ascribed to an imbalance in the
296
expression of particular factors and could involve multiple cell types. In this view, it is
297
appealing to speculate that the reduction of VEGFD levels in RGCs, which we detected,
298
might be playing a role; however, such a hypothesis calls for future in-depth investigations.
299
Of the several parameters measured, including expression of markers and functionality of the
300
BRB as well as detailed analysis of the microvascular architecture, we only observed
301
alterations in the increased presence of string vessels as a consequence of injured RGCs. Our
302
data therefore suggest that EC damage is not necessarily correlated to BRB disruption. BRB
303
breakdown might be staggered in time or uncoupled; a phenomenon which has been
304
previously observed
305
capillaries would collapse and stop being perfused before leakage and disruption could take
306
place 68.
307
Neurons can influence the vascular system in different ways. While synaptic activity drives
308
vascular patterning during CNS development, neural cells provide feedback to vessels
309
regarding their metabolic needs in the mature brain allowing regional adjustments of blood
310
flow in response to changing neuronal activity
311
damaged neurons could have a noxious impact on the vasculature. In addition, we provide
312
information on the possible causes driving the formation of empty sleeves, a phenomenon
313
largely uncharacterized and unexplained. Empty sleeves can be present in low number in
314
physiological conditions, but their number dramatically increases throughout the CNS during
315
aging and in neurodegenerative diseases such as diabetic retinopathy, Alzheimer’s disease,
39, 66
. An open question remains which nature of signals might carry the message
30, 67
. Mechanistically, a possible explanation may be that the affected
13
1, 69
. Our study showed for the first time that
316
stroke, glaucoma and Parkinson’s disease 70-73. Our data point towards the idea that structural
317
and functional integrity of neurons is critical for the maintenance of healthy capillaries and
318
that, when neurons are compromised or damaged, this negatively affects ECs. The resulting
319
creation of collapsed capillaries might in turn exacerbate neurodegeneration and create a
320
vicious circle of vascular and neural dysfunctions. Future work needs to extend our findings
321
obtained with an acute tool to promote RGC degeneration to different models of CNS
322
disorders and to different brain regions.
323
Interestingly, among the members of the VEGF family and their receptors, known key
324
modulators of neurovascular communication 3-6, our study identified VEGFD as the only one
325
whose expression in the retina was affected by the excitotoxic insult to RGCs. Such
326
observations are in agreement with our recent findings showing that VEGFD expression, but
327
not that of other VEGFs or VEGFRs, is shut-off following toxic activation of NMDARs in
328
pyramidal neurons in vitro or in vivo following stroke
329
activity, nuclear calcium signaling and epigenetic regulators can modulate VEGFD
330
expression in neurons while leaving expression of VEGFC, VEGF and VEGFRs unaltered 43,
331
51
332
regulation. Here, we showed for the first time that VEGFD is expressed in RGCs in the
333
human retina and we found that the NMDA-triggered down regulation of VEGFD is taking
334
place specifically in RGCs and not in other cell types of the neurovascular unit, in spite of the
335
fact that ECs and astrocytes carry functional NMDARs 32, 40, 66, 74. Excitotoxicity in the retina
336
alters the subcellular distribution, and thus activity, of the epigenetic regulators histone
337
deacetylases (HDACs) in RGCs
338
regulates the expression of VEGFD
339
regulators are involved in the decrease of VEGFD levels described in the present study.
52
. Several factors, such as synaptic
; thus suggesting that VEGFD neuronal expression is under a peculiar and dynamic
26
. The activity of one particular HDAC, namely HDAC4, 51
. It remains an open question, if such epigenetic
14
75
340
RGCs lose dendrites before they die due to NMDA excitotoxicity
341
models of excitotoxicity-associated disorders such as glaucoma and optic nerve crush 76-80. In
342
a mouse model of stroke, a pathology also characterized by excitotoxic mechanisms, VEGFD
343
supplementation, but not VEGFC or VEGFA, spared cortical neurons from stroke-induced
344
damage
345
connections of cortical neurons, and, as result, preventing neurodegeneration
346
different approaches, we uncovered the protective functions of VEGFD on both the
347
degenerating neural and vascular components of the retina. It is highly likely that the detected
348
beneficial effects of VEGFD on RGCs, as it happens for pyramidal neurons, may be due to
349
the specific properties of VEGFD to preserve and maintain dendritic connections
350
recent years, rAAVs received increasing attention as tools for gene delivery for the treatment
351
of several diseases
352
in clinical trials and rapidly approaching medical praxis 82. We first used rAAVs to boost the
353
production of VEGFD in RGCs without affecting VEGFD expression in other cell types.
354
This approach significantly blunted the excitotoxic damage to RGCs, optic nerve axons, and
355
the microvasculature. Even if we did not detect any effects as a result of the sole
356
overexpression of VEGFD in absence of the toxic insult, we cannot completely rule out that
357
some additional mechanisms owing to the long-term expression of VEGFD might be
358
participating in the protective effects. Notably, however, acute VEGFD treatment via
359
intravitreal injections of the mature form of VEGFD (rVEGFD) recapitulated the same
360
protective properties as achieved via rAAV-mediated neuronal VEGFD overexpression.
361
Moreover, rVEGFD administration also restored the impaired functionality of RGCs
362
following excitotoxicity. As RGCs are responsible for transmitting visual information to the
363
brain and their death is accompanied by devastating effects on vision 83, therapies designed to
364
promote survival of RGCs are of utmost relevance. Our work highlights the possible
52
and in several animal
. This is mechanistically implemented by VEGFD preserving the dendritic
81, 82
52
. Here, using
43, 49-51
. In
. For eye diseases, in particular, they are already under investigation
15
365
therapeutic capacity of VEGFD supplementation to treat vision deficiency associated to RGC
366
degeneration.
367
Our results revealed that, in addition to protecting the neural components of the retina,
368
VEGFD prevented the formation of empty sleeves. These findings might be interpreted as a
369
possible direct mechanism of VEGFD on ECs, sparing them from degeneration. However,
370
multiple evidences argue against this line of thought. First, our data indicate that in our model
371
empty sleeves formation follows RGC damage and that VEGFD levels in ECs are not
372
affected by the insult. Although rAAV-mediated neuronal overexpression of VEGFD would
373
increase the levels of available VEGFD free to act on both RGCs and ECs, as the
374
overexpressed protein undergoes proteolytic maturation and is secreted
375
primarily via an autocrine mechanism in neurons 43 as well as in other cell types 84-87. Thus, it
376
is probable that VEGFD produced by RGCs would act primarily on autocrine receptors of
377
RGCs, protects them from NMDA-mediated damage and, consequently, prevents the
378
formation of empty sleeves. The most conclusive piece of evidence, however, derives from
379
our observations that decreasing VEGFR3 expression specifically in RGCs prevents the
380
VEGFD-mediated protection of RGCs, and as a result, leads also to loss of protection of ECs.
381
In conclusion, an insult targeting specifically RGCs is accompanied by a reduction of
382
VEGFD levels in RGCs and, eventually, by damage to the capillaries. Boosting VEGFD
383
levels protects the structure and function of RGCs from the injury and, as a result, spares the
384
capillaries from degeneration. Our findings about the VEGFD-mediated prevention of RGC
385
loss and capillary damage is particularly interesting in light of potential clinical applications
386
targeting CNS diseases in which dysfunctions of the neurovascular system have been
387
implicated. Strategies aiming at enhancing neuronal and capillary survival through VEGFD
388
supplementation may therefore represent approaches for the development of therapies for
389
age- and disease-related dysfunctions of the neurovascular system.
16
43
, VEGFD acts
390 391
Materials and Methods
392
Animals
393
All experiments were performed in accordance with ethical guidelines imposed by the local
394
governing body (Regierungspräsidium Karlsruhe, Germany). Adult 8-week-old male mice of
395
the strain C57BL/6J (Charles River, Sulzfeld, Germany) were used in this study. Mice were
396
housed in groups of maximal 4 animals in the animal facility at the Heidelberg University in
397
standard cages (15 cm×21 cm×13.5 cm) on a normal 12:12 hours light: dark cycle with ad
398
libitum access to water and food. Each cage contained nesting material. Animals were
399
randomly allocated to treatment groups. After intravitreal injections, mice were kept under
400
frequent observation to ensure animal welfare.
401 402
Human retina samples
403
All eyes used in this study were taken under the legal responsibility of the Department of
404
Ophthalmology, Lions Eye Bank (Federal authority Paul-Ehrlich-Institut: PEI.G.11601.01.1)
405
within the facilities of the University Hospital Heidelberg for the purpose of cornea
406
transplantation. Human retina samples were obtained from two cornea donations within 48
407
hours of death after cornea trephination. Informed consent of relatives to use the tissues for
408
scientific purposes was present for all eyes used in this study.
409 410
Intravitreal injections of chemicals and proteins
411
For intravitreal injections, mice were anesthetized by intraperitoneal injection of 0.3 mg/kg
412
BW Medetomidine (Alvetra und Werfft GmbH, Vienna, Austria), 3 mg/kg BW Midazolam
413
(Hameln Pharmaceuticals GmbH, Hameln, Germany) and 0.03 mg/kg BW Fentanyl (Janssen
414
Pharmaceutica, Beerse, Belgium) to induce deep anesthesia. To induce excitotoxicity in the
17
415
retina, 10 nmol NMDA (1 µl of 10 mM, dissolved in sterile 1x PBS; Biotrend,
416
Wangen/Zurich, Switzerland) was injected in one eye with a 32-gauge needle (Nanofil
417
syringe, 10 µl; World Precision Instruments, Sarasota, Florida, USA) under a
418
stereomicroscope. The other eye received the same volume of 1x PBS (vehicle) as a control.
419
After the injection, eyes were treated with eye ointment (Bepanthen; Roche, Basel,
420
Switzerland). Afterwards, anesthesia was antagonized by 450 µg/kg BW Atipamezole
421
(Prodivet pharmaceuticals S.A.; Raeren, Belgium), 0.3 mg/kg BW Flumazenil (Fresenius
422
Kabi, Bad Homburg, Germany) and 0.7 mg/kg BW Narcanti/Naloxone (Inresa Arzneimittel
423
GmbH, Freiburg, Germany). For rVEGFD treatment, 100 ng recombinant mouse VEGFD (1
424
µl of 100 ng/µl, dissolved in sterile 1x PBS; 469-VD-025/CF; R&D Systems, Minneapolis,
425
Minnesota, USA) was co-injected with PBS or NMDA. Control experiments were performed
426
using 100 ng recombinant green fluorescent protein (1 µl of 100 ng/µl, dissolved in sterile 1x
427
PBS; recombinant A. victoria GFP protein, ab116434, Abcam, Cambridge, UK) was co-
428
injected with PBS or NMDA.
429 430
Intravitreal injections of rAAVs
431
2 µl of rAAVs were intravitreally injected prior to the excitotoxic insult. To determine
432
efficient duration of protein expression after transduction, protein products were analyzed via
433
Western blot of retinal lysates. For rAAV-LacZ and rAAV-VEGFD, incubation of 14 days
434
was sufficient to detect expression whereas for rAAV-shSCR and rAAV-shVEGFR3 an
435
incubation of 21 days prior to the insult was necessary. Typical infection rate of the
436
employed rAAV range was >60% of Brn3a-positive RGCs.
437 438
Tissue preparation
18
439
Retinas from mice were dissected 6 hrs, one day (d1) or seven days (d7) after intravitreal
440
injections. For qRT-PCR analysis and Western blot, animals were euthanized with CO2;
441
animals were decapitated and heads were cooled in iced water. For histology, animals were
442
euthanized by intraperitoneal injection with 400 mg/kg BW pentobarbital (Narcoren; Merial
443
GmbH, Hallbergmoos, Germany), followed by a transcardial perfusion with ice-cold 1x PBS
444
and 10% formalin for 5min. Eyes were enucleated and post-fixed for 15 min. In order to
445
dissect the retina, the cornea, iris, lens, and vitreous body were removed under a
446
stereomicroscope. For the preparation of sagittal retina sections, the retina was kept in the
447
surrounding sclera and pigment epithelium. Samples were embedded in Tissue-Tek® (Leica,
448
Wetzlar, Germany) frozen and stored at -80°C until cryo-sectioning. For retina whole mount
449
immunostainings, the retina was isolated from the sclera and pigment epithelium and
450
transferred into PBS. Human retina samples were dissected and shock-frozen in liquid
451
nitrogen for qRT-PCR analysis or post-fixed for 15 min in 10% formalin for whole mount
452
immunostainings.
453 454
Cryo-sectioning
455
Sagittal retina sections of 16 µm and optic nerve sections of 10 µm thickness were obtained
456
with a Leica CM1950 cryostat (Leica) and mounted on superfrost plus slides (Thermo
457
Scientific; Waltham, Massachusetts, USA). Slides were stored at -20°C until analysis.
458 459
TUNEL assay
460
TUNEL staining was done on retinal sections on microscope slides. Sections were
461
permeabilized in 0.1% sodium citrate and 0.5% Triton-X100 for 30 min at room temperature
462
and incubated with TUNEL solution for 1 h at 37°C using the In Situ Cell Death Detection
19
463
Kit, TMR red (Roche, Basel, Switzerland). Nuclei were labeled with Hoechst (1:6000 in 1x
464
PBS, Serva Electrophoresis GmbH, Heidelberg, Germany) for 10 min.
465 466
Immunohistochemistry
467
Retinal sections were immunostained directly on microscope slides. Slides were incubated in
468
0.5% Triton-X100 and 10% FCS in 1x PBS for 1.5 hrs at room temperature. Primary
469
antibodies were diluted in blocking solution and incubated overnight at 4°C in a humidified
470
chamber. For retinal whole mounts, free-floating immunostaining was performed. Retinas
471
were blocked in 1% Triton-X100 and 10% FCS in 1x PBS overnight at 4°C. Primary
472
antibodies were diluted in blocking solution and incubated overnight at room temperature.
473
The following antibodies were used: mouse-anti-Brn3a (1:500; sc-8429, Santa Cruz
474
Biotechnology, Dallas, Texas, USA), rabbit-anti-Collagen-IV (1:500; 2150-1470, Bio-Rad
475
Laboratories, Inc., Hercules, California, USA), mouse-anti-VEGFD (1:100; sc-373866, Santa
476
Cruz Biotechnology), rabbit-anti-SMI-32 (1:1000; 801802, BioLegend, San Diego,
477
California, USA), mouse-anti-HA-probe (Y-11) (1:500; sc-805, Santa Cruz Biotechnology),
478
rabbit-anti-RBPMS (1:250; ab194213, Abcam), rat-anti-TER119 (1:200; MAB1125, R&D
479
Systems, Minneapolis, Minnesota, USA). For VEGFD immunostaining, heat-induced epitope
480
retrieval was performed prior to blocking by incubating the slides in pre-heated sodium
481
citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95°C for 10 min. ECs
482
were labeled with Alexa Fluor 488- or 594-conjugated Isolectin-B4 (1:100; I21411, I21413,
483
Life Technologies, Carlsbad, California, USA). The following secondary antibodies were
484
used: Goat-anti-rabbit IgG (H+L) Alexa Fluor 488 (1:1000; A11008, Life Technologies),
485
goat-anti-mouse IgG (H+L) Alexa Fluor 594 (1:1000; A11005, Life Technologies) and
486
donkey-anti-rat IgG (H+L) Alexa Fluor 488 (1:700; A21208, Life Technologies). Nuclei
20
487
were labeled with Hoechst (1:6000 in 1x PBS, Serva Electrophoresis GmbH, Heidelberg,
488
Germany) for 10 min.
489 490
Transmission Electron Microscopy (TEM)
491
Mice were transcardially perfused with 2.5% glutaraldehyde /2% polyvidone 25 in 0.1 M
492
sodium phosphate buffer, pH 7.4. The eyes were enucleated and the retinas were dissected as
493
described in the section “tissue preparation”. The tissue was processed for TEM as previously
494
published
495
with uranyl acetate, dehydrated with a graded dilution series of ethanol, and embedded into
496
glycid ether 100-based resin. Ultrathin retinal cross sections of 85 nm were cut with a
497
Reichert Ultracut S ultramicrotome (Leica Microsystems) and contrasted with uranyl acetate
498
and lead citrate. Sections were examined in an electron microscope (Zeiss EM 10 CR) at an
499
acceleration voltage of 60 kV. The representative electron microscopy pictures derive from
500
the same mouse but two mice were prepared for analysis.
88, 89
. Retinas were post-fixed with 1% OsO4, 1.5% K4Fe(CN)6, contrasted en bloc
501 502
bEND.3 cells
503
bEND.3 cells were cultured in high-glucose-containing (4.5 g/l) Dulbecco’s Modified Eagle
504
Medium (DMEM; Life Technologies, Carlsbad, California, USA) supplemented with 10%
505
FBS, 100 units/ml penicillin and 100 µg/ml streptomycin (Sigma, Kawasaki, Kanagawa,
506
Japan) on 0.1% gelatin-coated flasks. For passaging, cells were washed with PBS and
507
incubated with Trypsin (#25300-054; Gibco, Life Technologies, Carlsbad, California, USA)
508
for 3 min. Cells were resuspended in medium and centrifuged at 800xg for 3 min. The cell
509
pellet was resuspended in cell culture medium and cells were seeded at an appropriate
510
dilution according to their growth rate. For bEND.3 mortality assay, cells were treated with
511
20 µM NMDA or 1 mM NMDA for 10 min and 24 hrs. Untreated cells served as controls.
21
512
After 24 hrs, cells were fixed for 10 min with 4% PFA and labeled with Hoechst for cell
513
survival analysis. Hoechst-labeled cells were automatically counted in 16 images out of 4
514
coverslips using a self-written macro in ImageJ/Fiji. Healthy nuclei were differentiated from
515
fragmented or condensed nuclei by their morphology.
516 517
Primary astrocyte cultures
518
Neurons and glial cells from new born C57BL/6N mice were isolated as described
519
Medium was changed after 2.5 hrs from plating with DMEM supplemented with 10% FCS
520
and Pen-Strep. Every 3 days, cultures were washed with ice-cold PBS to promote death and
521
detachment of neuronal cells, and growth of astrocytes. When confluence was reached,
522
experiments were performed. Cultures were treated for 24 hrs with 20 µM NMDA and then
523
either harvested for qRT-PCR analysis or fixed for immunocitochemistry. We confirmed the
524
exclusive presence of glia cells using the marker GFAP (1:500; 3670, Cell Signaling
525
Technology, Danvers, Massachusetts, USA). Hoechst labeling was used to count and assess
526
cellular vitality via nuclear morphology.
49
.
527 528
Recombinant adeno-associated viruses
529
The method used to construct, package, and purify recombinant adeno-associated viruses
530
(rAAVs) has been previously described 90, 91. Recombinant adeno-associated viruses (rAAVs)
531
serotype 1/2 were produced by co-transfection of HEK293 cells by standard calcium
532
phosphate precipitation. Before transfection, culture medium was replaced with fresh
533
modified Dulbecco’s medium (Iscove’s Modified Dulbecco's Medium; Life Technologies,
534
Carlsbad, California, USA) containing 5% FBS without antibiotics. Packaging of rAAVs was
535
carried out with helper plasmids pF∆6, pRV1, and pH21 together with either pAAV-VEGFD,
536
pAAV-LacZ, pAAV-shVEGFR3 or pAAV-shSCR. After transfection, the medium was
22
537
replaced with fresh DMEM containing 10% FBS and antibiotics. Cells were collected at low
538
speed centrifugation, resuspended in 150 mM NaCl-10 mM Tris-HCl (pH 8.5) and lysed by
539
incubation with 0.5% sodium deoxycholate followed by freeze-thaw cycles. rAAVs were
540
purified using heparin affinity columns (HiTrap Heparin HP; GE Healthcare, Chicago,
541
Illinois, USA). rAAVs stocks were concentrated using Amicon Ultra-4 centrifugal filter
542
devices (Merck Millipore, Burlington, Massachusetts, USA).
543
The rAAVs used in this work have been previously extensively characterized in vitro and in
544
vivo for their specificity and efficacy
545
or FLAG-tag, respectively, allowing for detection of infected cells. rAAV-shVEGFR3 and
546
rAAV-shSCR carry an additional cassette for mCherry expression for rapid and specific
547
detection of the infected cells. The transduction efficiency was calculated as percentage of
548
Brn3a+ cells also positive for the transgene expression. The virus batches used for the
549
experiments had a titre of: rAAV-LacZ 6*1011 molecules/ml; rAAV-VEGFD 3*1011
550
molecules/ml, which had, however, identical infection efficiency in both cultured neurons
551
and in vivo in RGCs. For the shRNA constructs, the virus batches used for the experiments
552
had a titre of: rAAV-shSCR 6*1012 molecules/ml; rAAV-shVEGFR3 2*1012 molecules/ml,
553
which had, however, identical infection efficiency in both cultured neurons and in vivo in
554
RGCs.
555
The number of mice and eyes injected with rAAV-LacZ was 9 mice / 18 eyes (9 eyes were
556
injected with PBS, 9 eyes were injected with NMDA and of each experimental group 6 eyes
557
were co-stained for RGCs and empty sleeves quantification while 3 only for RGCs
558
quantification). The number of mice and eyes injected with rAAV-VEGFD was 9 mice / 18
559
eyes (9 eyes were injected with PBS, 9 eyes were injected with NMDA and of each
560
experimental group 6 eyes were co-stained for RGCs and empty sleeves quantification while
561
3 only for RGCs quantification). The number of mice and eyes treated with rAAV-shSCR
43, 50, 51
. rAAV-VEGFD and rAAV-LacZ carry an HA-
23
562
was 16 mice / 32 eyes for quantification of RGC survival (8 eyes were injected with PBS, 8
563
eyes were injected with NMDA, 8 eyes were injected with rVEGFD, 8 eyes were injected
564
with NMDA+rVEGFD) and 12 mice / 24 eyes for empty sleeves analysis (6 eyes were
565
injected with PBS, 6 eyes were injected with NMDA, 6 eyes were injected with rVEGFD, 6
566
eyes were injected with NMDA+rVEGFD). The number of mice and eyes treated with
567
rAAV-shVEGFR3 was 16 mice / 32 eyes for quantification of RGC survival (8 eyes were
568
injected with PBS, 8 eyes were injected with NMDA, 8 eyes were injected with rVEGFD, 8
569
eyes were injected with NMDA+rVEGFD) and 12 mice / 24 eyes for empty sleeves analysis
570
(6 eyes were injected with PBS, 6 eyes were injected with NMDA, 6 eyes were injected with
571
rVEGFD, 6 eyes were injected with NMDA+rVEGFD).
572 573
qRT-PCR
574
Total RNA was extracted from bEnd.3 cells, primary astrocytes, mouse or human retinas
575
using the RNeasy Mini Kit (Qiagen, Hilden, Germany) including an optional DNase I
576
treatment at room temperature for 15 min according to manufacturer’s instructions (Qiagen).
577
Extracted RNA was reverse transcribed into first-strand cDNA using High Capacity cDNA
578
Reverse Transcription kit (Applied Biosystems, Foster City, California, USA). Quantitative
579
reverse transcriptase PCR (qRT-PCR) was performed on a StepOne plus Real Time PCR
580
system using TaqMan Gene Expression Assays for the indicated genes (Applied Biosystems).
581
The following TaqMan Gene Expression Assays were used in this study: Thy1
582
(Mm01174153_m1),
nefl
(Mm01315666_m1),
583
(Mm00727012_m1),
tjp1
Mm00493699_s1),
584
(Mm01202432_m1),
vegf
585
(Mm00438980_m1), kdr (Mm00440099_m1). Expression of target genes was normalized
586
against the expression of Gapdh (Mm99999915_g1) or, in the case of bEnd.3 cells, Gusb
(Mm01281449_m1),
24
ocld vegfd flt4
(Mm01282968_m1), (Mm00438963_m1), (Mm01292618_m1),
cld5 vegfc flt1
587
(Mm00446953_m1), which were used as endogenous control genes. For detection of VEGFD
588
in human retina, the following probes were used: vegfd (Hs01128659_m1), vegf
589
(Hs00900055_m1), actb (Hs99999903_m1). For each treatment, 6 animals, 3 technical
590
replicates or 4 human retinas from 2 donors were analyzed.
591 592
Western blot analysis
593
bEnd.3 cells were homogenized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.57%
594
sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8, 1x CompleteTM cocktail of protease
595
inhibitors; Roche, Basel, Switzerland). Homogenates centrifuged with 13000rpm for 10min.
596
Supernatants were mixed with 4x Laemmli sample buffer (200 mM Tris/HCl pH 6.8, 8%
597
SDS, 40% Glycerol, 50 mM EDTA, 0.08% bromophenol blue) and 1% dithiothreitol. Lysates
598
were heated at 95°C for 10 min before performing standard SDS-PAGE and western blotting.
599
The following primary antibodies were used: rabbit-anti-GAPDH (1:20000 in 5% BSA;
600
2118, Cell signaling Technology), rabbit anti-GluN1 (1:2000 in 5% milk; 5704, Cell
601
signaling Technology), rabbit-GluN2A (1:500 in 5% milk; AB1555, Merck Millipore), rabbit
602
anti-GluN2B (1:1000 in 5% milk; NB300-106, Novus Biologicals, Cenennial, Colorado,
603
USA), rabbit anti-GluN2C (1:1000 in 5% milk; PAB9705, Abnova, Taiwan, China), rabbit
604
anti-GluN2D (1:1000 in 5% milk; AP21181PU-N, Acris OriGene, Herford, Germany). The
605
following secondary antibody was used: Peroxidase AffiniPure goat anti-rabbit IgG (H+L)
606
(1:5000 in 5% milk; 111-035-144, Jackson Immuno Research Laboratories Inc., West Grove,
607
Pennsylvania, USA). Membranes were developed with Enhanced Chemiluminescence (ECL)
608
solution (GE Healthcare, Chicago, Illinois, USA) and the ChemiDoc Imaging System (Bio-
609
Rad Laboratories).
610 611
Analysis of BRB functionality
25
612
For extravasation experiments, animals received intravenous injection of fluorescein-
613
conjugated dextran (3 kDa, 50 µg/µl in 1x PBS, 50 µg/g BW; D3306; Thermo Scientific).
614
After 15 min, mice were euthanized by intraperitoneal injection with 400 mg/kg BW
615
pentobarbital (Narcoren; Merial GmbH, Hallbergmoos, Germany), followed by a transcardial
616
perfusion with 10 ml ice-cold 1x PBS to clear the remaining intravascular dextran. A blood
617
sample, serving as positive control, was collected immediately before perfusion. The blood
618
sample was centrifuged at 15000xg for 5 min at 4°C, and the supernatant was frozen at -20°C
619
until measurements. Blood sera were diluted 1:100 and 1:1000. Immediately after perfusion,
620
retinas were dissected, weighed, and homogenized in 100 µl ice-cold 1x PBS. The extracts
621
were centrifuged at 10000xg for 15 min at 4°C. The supernatant was kept undiluted or was
622
diluted 1:100. The fluorescence in each 100 µl sample was measured (excitation, 492 nm;
623
emission, 521 nm) using a spectrofluorometer (SpectraMax M5; Molecular Devices, LLC,
624
San José, California, USA) with 1x PBS as a blank. Retinal or serum autofluorescence was
625
corrected based on not-injected controls. For normalization, the corrected retinal fluorescence
626
was divided by the retinal weight and by the corrected serum fluorescence, with the results
627
being expressed in relative fluorescence unit (RFU).
628
For extravasation analysis of albumin or IgG via western blotting 30, animals were euthanized
629
as described above, followed by a transcardial perfusion with 10 ml ice-cold 1x PBS to
630
reduce blood contaminants. Eyes were enucleated, retinas were immediately dissected under
631
a stereomicroscope and shock-frozen in liquid nitrogen. Retinas were processed for standard
632
Western blot analysis. Western blotting was performed as described above. As controls,
633
recombinant albumin (45µg, 8076, Roth, Karlsruhe, Germany), mouse IgG (9 µg; sc-2025,
634
Santa Cruz Biotechnology), and retinal lysates from non-perfused animals were loaded. The
635
following primary and secondary antibodies were used to detect endogenous, extravasated
636
albumin and IgG: goat anti-mouse serum albumin (1:1000; ab19194, Abcam), Peroxidase
26
637
AffiniPure goat anti-mouse IgG (H+L) (1:5000 in 5% milk; 705-035-147, Jackson Immuno
638
Research Laboratories Inc.). For protein expression analysis, relative protein band densities
639
were calculated by normalizing to the loading control (GAPDH) using ImageJ/Fiji. For each
640
time point d1 and d7, relative protein levels of the NMDA-treated eyes were normalized to
641
relative protein levels of the PBS control eye of the same animal. For all image analyses,
642
backgrounds were subtracted. For each treatment, 3 animals were analyzed.
643 644
Image acquisition
645
For each analysis, images were acquired with constant microscopy settings (exposure time,
646
pixel size, bit depth, binning) across experiments. For fluorescence intensity analysis, all
647
images were acquired on the same day to avoid possible variations in fluorescence lamp
648
excitation. Images of sagittal retina sections and primary astrocytes were acquired with Leica
649
DM IRBE inverted fluorescent microscope with a 40x oil immersion objective (Leica) and a
650
Spot Insight FireWire 2 camera with VisiView software (Visitron Systems GmbH,
651
Diagnostic Instruments, Puchheim, Germany). Images of mouse and human retina whole
652
mounts were acquired with a Nikon A1R confocal laser scanning microscope (at the Nikon
653
Imaging Center of Heidelberg University, Germany) with a 63x oil immersion objective
654
(Nikon, Minato, Tokio, Japan) or with a Leica SP8 confocal laser scanning microscope with a
655
63x oil immersion objective (Leica).
656 657
Quantification of RGCs
658
For RGC number quantification in retinal whole mounts, Brn3a-positive cells were manually
659
counted in 8 images per retina equally acquired in the central and peripheral part of the retina
660
using ImageJ/Fiji (see schema Fig. 1B). For each eye, RGC number was summed. Relative
661
RGC numbers were calculated by normalizing RGC number in the NMDA-treated eye to the
27
662
number of RGCs in the PBS-treated eye of the same animal. For each treatment, 6 animals
663
were analyzed.
664 665
INL Analysis
666
For INL analysis, the thickness of the INL was measured at 5 different positions across the
667
INL in 3 sagittal retinal sections of 10 µm thickness using ImageJ/Fiji. For each section,
668
values were averaged and converted into µm taking the microscope resolution into account.
669
For each treatment, 3 animals were analyzed.
670 671
Quantification of axonal damage
672
For axonal damage analysis, the area of SMI-32-positive clusters was quantified in 3-4
673
sagittal optic nerve sections of 10 µm thickness using ImageJ/Fiji. For each optic nerve,
674
cluster area was summed and normalized to the analyzed area. Relative cluster areas were
675
calculated by normalizing cluster area in the NMDA-treated optic nerve to the cluster area in
676
the PBS-treated optic nerve of the same animal. For each treatment, 6 animals were analyzed.
677 678
Quantification of vascular injury
679
For vascular injury analysis in retinal whole mounts, the number of regressed vessels – so
680
called-empty sleeves, which represent collapsed Collagen-IV-positive / Isolectin-B4-negative
681
vessels – was quantified using ImageJ/Fiji. For each eye, empty sleeves number was
682
summed. Relative empty sleeves numbers were calculated by normalizing number of empty
683
sleeves in the NMDA-treated eye to number of empty sleeves in the PBS-treated eye of the
684
same animal. For each treatment, 6 animals were analyzed.
685 686
Quantification of vessel architecture
28
687
For vessel architecture in retinal whole mounts, the isosurface of Collagen-IV
688
immunolabeled vessels was measured with ImageJ/Fiji and the plugin BoneJ. Vessel
689
diameter and length were measured with ImageJ/Fiji and the plugin DiameterJ. Relative
690
values were calculated by normalizing the mean values in the NMDA-treated eye to mean
691
values in the PBS-treated eye of the same animal. For each treatment, 6 animals were
692
analyzed.
693 694
Analysis of VEGFD expression
695
Somatic levels of VEGFD in sagittal retinal sections were analyzed as previously published
696
92
697
drawing regions of interests around the ganglion cell layer in 4-5 images, each corresponding
698
to one individual 16 µm sagittal retina section using ImageJ/Fiji. For each eye/condition,
699
mean integrated densities were calculated out of the single fluorescence values within each
700
sagittal retinal section. The mean integrated density of theNMDA-treated eye was then
701
normalized to the mean integrated densityof the PBS-treated eye of the same animal.
702
Background subtraction was performed prior to all analyses. For negative controls, a
703
blocking peptide (sc-373866, Santa Cruz Biotechnology) was pre-absorbed with the primary
704
antibody in a five-fold excess (by weight) resulting in absence of specific immunolabeling of
705
the primary antibody. For each treatment, n=3-4 animals were analyzed.
. Fluorescent signals were measured in cells located in the ganglion cell layer by manually
706 707
Electroretinography
708
Mice were anaesthetized with ketamine (150 mg/kg; Atarost GmbH and Co., Twistringen,
709
Germany) and xylazine (10 mg/kg; Albrecht, Aulendorf, Germany). ERG recordings were
710
recorded using the UTAS Visual Diagnostic System (LKC Technologies, Gaithersburg, MD,
711
USA) using a contact lens electrode with gold contact (LKC Technologies). The electrical
29
712
contact, as well as prevention of eye desiccation, was facilitated through application of
713
Liquifilm O.K. (Allergen, Westport, Ireland). Reference and ground needle electrode were
714
placed subcutaneously in the neck and tail, respectively. A heating pad controlled by rectal
715
temperature probe was maintained at 37oC. Mice were centered at a distance of 15 cm in
716
front of a 17-inch cathode ray tube monitor. In order to achieve maximum focus of the
717
pattern stimulation, pupils were not dilated. Vertical square wave grating stimulations (66%
718
contrast, temporal frequency of 1 Hz – equivalent to 2 pattern reversals per second) were
719
presented at different spatial frequencies (0.05, 0.10, and 0.20 cyc/deg), and noise levels were
720
measured with the monitor turned off. Due to the small amplitudes of pattern ERGs, 250
721
sweeps were averaged to increase the signal-to-noise ratio. Responses were sampled at
722
1000x, band-pass filtered between 0.3 and 300 Hz, and amplified (64 x gain). The amplitude
723
of the second harmonic (2 Hz) was analyzed with the fast Fourier Transformation function
724
using the EMWin software provided with the UTAS visual diagnostic system.
725 726
Statistical analysis
727
Mean values and standard error of the mean (SEM) were calculated in Excel (Microsoft),
728
plotted and analyzed in GraphPad Prism 7 software (GraphPad Software, Inc.). Values were
729
tested for normal distribution by performing Shapiro-Wilk normality test. Unpaired t-test,
730
one-way or two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test for
731
multiple comparisons were used. Details on the test used and N of each experiment are
732
indicated in the respective figure legends. Bars represent mean values. Error bars indicate
733
SEM. Asterisks indicate significant differences (****p<0.0001, ***p<0.001, **p<0.01,
734
*p<0.05).
735 736
30
737
Author Contributions
738
Conceptualization, D.M.; Methodology, A.S., D.M.; Formal Analysis, A.S., B.A., R.F.,
739
D.M.; Investigation, A.S., B.A., R.F, D.M.; Writing – Original Draft, A.S., D.M.; Writing –
740
Review & Editing, A.S., B.A., R.F., R.D., D.M.; Visualization, A.S., D.M.; Supervision,
741
A.S., D.M.; Project Administration, D.M., Funding Acquisition, D.M.
742 743
Acknowledgments
744
The authors wish to thank Dr. Patrick Merz from the Department of Ophthalmology, Lions
745
Eye Bank, Heidelberg University Hospital, for providing human retina samples. The authors
746
are extremely grateful towards Andrea Hellwig for providing the electron microscopy images
747
shown in Figure 2. The authors additionally thank Eva Bindewald and Viktoria Greeck for
748
technical assistance, Iris Bünzli-Ehret and Dr. Anna M. Hagenston for their help with the
749
preparation of astrocytic primary cultures, Dr. Carmen Ruiz de Almodovar for kindly
750
providing the TER119 antibody.
751
DM is a member of the Excellence Cluster CellNetworks at Heidelberg University. Part of the
752
pictures was acquired at the Nikon Imaging Center at Heidelberg University. We also
753
acknowledge support from the Interdisciplinary Neurobehavioral Core (INBC) at Heidelberg
754
University. This work was supported by the FOR2325 grant (project 2) and SFB1158 (project
755
A08) of the Deutsche Forschungsgemeinschaft (DFG) to DM, the FRONTIER grant of
756
Heidelberg University to DM.
757 758
Conflicts of interest statement
759
DM is one of the founders and shareholders of FundaMental Pharma GmbH.
760 761
31
762
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38
1022
Figure legends
1023
Figure 1: NMDA-triggered excitotoxicity induces RGC death.
1024
A) Representative images of retinal whole mounts at d1 and d7 after intravitreal injections of
1025
NMDA or PBS. RGCs were immunolabeled using an antibody against Brn3a (green), nuclei
1026
were labeled with Hoechst (blue). Scale bar=20 µm. B) Schematic of the fields used for
1027
quantification in retinal whole mounts in central and peripheral parts of the retina. C)
1028
Quantification of RGCs in retinas injected as indicated. Unpaired t-test. n=6. PBS vs NMDA
1029
(d1), p=0.0274; PBS vs NMDA (d7), p=0.0038. D) Quantification of RGCs in retinas
1030
injected as indicated. RGC numbers in the NMDA-treated eye were normalized to the PBS
1031
control eye from the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1), p=0.0318; PBS
1032
vs NMDA (d7), p=0.0051. E) QRT-PCR analysis of thy1 and nefl expression in retinas one
1033
day after intravitreal injection of NMDA. Unpaired t-test. n=6. Thy1, p=0.0002; nefl,
1034
p=0.0002. F) Quantification of INL thickness in sagittal retina sections injected as indicated.
1035
Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.4015; PBS vs NMDA (d7), p=0.8741. Bars
1036
represent mean ± SEM. Dots represent single values. ***p<0.001, **p<0.01, *p<0.05.
1037 1038
Figure 2: Intravitreal injection of NMDA induces formation of empty sleeves via an
1039
indirect mechanism.
1040
A) Representative images of retinal whole mounts at d7 after intravitreal injections of NMDA
1041
or PBS. Vessels were immunolabeled with antibodies against Collagen-IV (green) and
1042
Isolectin-B4 (red). Arrows indicate empty sleeves. Scale bar=20 µm. B) Quantification of
1043
empty sleeves in retinas injected as indicated. Unpaired t-test. n=6. PBS vs NMDA (d1),
1044
p=0.0129; PBS vs NMDA (d7), p<0.0001. C) Quantification of empty sleeves in retinas
1045
injected as indicated. Number of empty sleeves in the NMDA-treated eye were normalized to
1046
the PBS control eye from the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1),
39
1047
p<0.0001; PBS vs NMDA (d7), p=0.0002. D) Representative images of electron microscopy
1048
of retinas injected as indicated. Left: Normal capillary in retinas of PBS-injected eyes with
1049
ECs (e), tight junctions (tj), and pericyte (p). Scale bar=5000 nm. Right: Capillary with
1050
swollen EC (e) in retinas of NMDA-injected eyes. Asterisks indicate occluded lumen. Tight
1051
junctions (tj) and pericytes (p) were still intact. Scale bar=500 nm. E) Quantification of vessel
1052
surface area in retinas injected as indicated. Surface area in the NMDA-treated eye was
1053
normalized to the PBS control eye from the same animal. Unpaired t-test. n=6. PBS vs
1054
NMDA (d1), p=0.0626; PBS vs NMDA (d7), p=0.5847. F) Quantification of vessel diameter
1055
in retinas injected as indicated. Diameter in the NMDA-treated eye was normalized to the
1056
PBS control eye from the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1), p=0.5313;
1057
PBS vs NMDA (d7), p=0.3695. G) Quantification of vessel fiber length in retinas injected as
1058
indicated. Fiber length in the NMDA-treated eye was normalized to the PBS control eye from
1059
the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1), p=0.2375; PBS vs NMDA (d7),
1060
p=0.6235. H) QRT-PCR analysis of ocld, cld5 and tjp1 expression in retinas of mice seven
1061
days after intravitreal injection of PBS and NMDA. Unpaired t-test. n=6. ocld, p=0.2803;
1062
cld5, p=0.8959; tjp1, p=0.1733. I) Representative images of retinal whole mounts at d7 after
1063
intravitreal injections of NMDA or PBS. Vessels were immunolabeled with antibodies
1064
against Collagen-IV (red) and erythrocytes with antibodies against TER119 (green). Scale
1065
bar=50 µm. J) Quantification of vascular erythrocytes in retinas injected as indicated and
1066
expressed as percentage of the total N of erythrocytes present in the analyzed images (within
1067
and outside vessels). Unpaired t-test. n=4. PBS vs NMDA (d7), p=0.088. K) Quantification
1068
of dextran-fluorescein (3 kDa) extravasation in retinal extracts one and seven days after
1069
intravitreal PBS and NMDA injections. Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.3799;
1070
PBS vs NMDA (d7), p=0.6891. L) Representative immunoblots of albumin, IgG and
1071
GAPDH in retinas of unperfused mice (control) or in retinas of perfused mice one and seven
40
1072
days after intravitreal PBS and NMDA injections. M) Quantification of albumin levels in
1073
retinal lysates injected as indicated. GAPDH was used for control of the amount of protein.
1074
Samples from NMDA-treated eye were normalized to the PBS control eye from the same
1075
animal. Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.4845; PBS vs NMDA (d7), p=0.5231.
1076
N) Quantification of IgG levels in retinal lysates injected as indicated. GAPDH was used for
1077
control of the amount of protein. Samples from NMDA-treated eye were normalized to the
1078
PBS control eye from the same animal. Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.7794;
1079
PBS vs NMDA (d7), p=0.5372. O) Representative immunoblots of different NMDAR
1080
(GluN) subunits and GAPDH in brain ECs (bEnd.3, left lane) and primary hippocampal
1081
neurons (right lane). P) Quantification of cultured brain ECs (bEnd.3) survival after 20 µM
1082
NMDA exposure in vitro for 10 min or 24 hrs. The number of living bEnd.3 cells was
1083
normalized to the number of total bEnd.3 cells as well as to the ratio of living/total cells in
1084
the control (untreated). One-way ANOVA, Bonferroni. n=3. Untreated vs NMDA 10 min,
1085
p>0.9999. Untreated vs NMDA 24 hrs, p>0.9999. NMDA 10 min vs NMDA 24 hrs,
1086
p>0.9999. Q) QRT-PCR analysis of ocld, cld5 and tjp1 expression in bEnd.3 with or without
1087
NMDA treatment. Unpaired t-test. n=6. ocld, p=0.7118; cld5, p=0.1998, tjp1, p=0.4434. R)
1088
Quantification of cultured primary astrocyte survival after 20 µM NMDA exposure for 24
1089
hrs. The number of living astrocytes was normalized to the number of total astrocytes as well
1090
as to the ratio of living/total astrocytes in the control (untreated). Unpaired t-test. n=3.
1091
Untreated vs NMDA 24 hrs, p=0.7016. Bars represent mean ± SEM. Dots represent single
1092
values. ****p<0.0001, *p<0.05.
1093 1094
Figure 3: NMDA-triggered excitotoxicity reduces VEGFD expression in RGCs.
1095
A) QRT-PCR analysis of vegfd, vegfc, vegfa, flt4, flt1, and kdr expression in retinal
1096
homogenates after intravitreal injection of NMDA. Unpaired t-test. n=6. vegfd, p=0.0085;
41
1097
vegfc, p=0.2052; vegfa, p=0.8524; flt4, p=0.9414; flt1, p=0.6074; kdr, p=0.2016. B)
1098
Representative images of human retinal whole mounts. Retinas were immunolabeled with
1099
antibodies against Bn3a (red) and VEGFD (green). Scale bars=10 µm. Negative controls of
1100
human retinas were immunolabeled only with secondary antibodies. C) Representative bands
1101
of VEGFD, VEGFA, and ß-actin cDNA products following qRT-PCR analysis of human
1102
retinal extracts obtained from two donors (subject 1, subject 2). D) Quantification of VEGFD
1103
protein levels in cells in the ganglion cell layer of retinas of mice injected as indicated.
1104
Unpaired t-test. n=3. PBS vs NMDA, p=0.0192. E) Representative images of mouse sagittal
1105
retina sections at d7 after intravitreal injections of NMDA or PBS. Retinas were
1106
immunolabeled for VEGFD (green). Nuclei were labeled with Hoechst (blue). GCL:
1107
Ganglion cell layer. Scale bar=20 µm. F) QRT-PCR analysis of vegfd, vegfc, vegfa, and flt4
1108
expression in brain endothelial cells (b.END3) 24 hrs after 20 µM NMDA treatment.
1109
Unpaired t-test. n=3. vegfd, p=0.1744; vegfc, p=0.7598; vegfa, p=0.8998; flt4, p=0.4457. G)
1110
QRT-PCR analysis of vegfd, vegfc, vegfa, and flt4 expression in primary astrocytes 24 hrs
1111
after 20 µM NMDA treatment. Unpaired t-test. n=3. vegfd, p=0.8716; vegfc, p=0.4918; vegfa,
1112
p=0.9902; flt4, p=0.9331. Bars represent mean ± SEM. Dots represent single values.
1113
**p<0.01.
1114 1115
Figure 4: VEGFD overexpression in RGCs protects them from NMDA-mediated
1116
damage. A) Representative images of retinal whole mounts at d7 after intravitreal injections
1117
of NMDA or PBS from mice injected either with rAAV-LacZ or rAAV-VEGFD as indicated.
1118
RGCs were immunolabeled against Brn3a (green). Scale bar=15 µm. B) Representative
1119
images of sagittal optic nerve sections injected as indicated. Stressed axons were
1120
immunolabeled using SMI-32 (green). Nuclei were labeled using Hoechst (blue). Scale
1121
bar=50 µm. C) Quantification of RGCs injected as indicated. Two-way ANOVA, Bonferroni.
42
1122
n=9. rAAV-LacZ+PBS vs rAAV-LacZ+NMDA, p<0.0001; rAAV-VEGFD+PBS vs rAAV-
1123
VEGFD+NMDA, p=0.5035. rAAV-LacZ+PBS vs rAAV-VEGFD+PBS, p>0.9999 rAAV-
1124
LacZ+NMDA vs rAAV-VEGFD+NMDA, p=0.0006. D) Quantification of RGCs injected as
1125
indicated. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye
1126
from the same animal. n=9. E) Quantification of SMI-32+ clusters in optic nerves from eyes
1127
injected as indicated. Two-way ANOVA, Bonferroni. n=6. rAAV-LacZ+PBS vs rAAV-
1128
LacZ+NMDA, p<0.0001; rAAV-VEGFD+PBS vs rAAV-VEGFD+NMDA, p=0.7794;
1129
rAAV-LacZ+PBS vs rAAV-VEGFD+PBS, p>0.9999; rAAV-LacZ+NMDA vs rAAV-
1130
VEGFD+NMDA, p<0.0001. F) Quantification of SMI-32+ clusters in optic nerves from eyes
1131
injected as indicated. SMI-32+ clusters in the NMDA-treated eye were normalized to the PBS
1132
control eye from the same animal. n=6. Bars represent mean ± SEM. Dots represent single
1133
values. ****p<0.0001, ***p<0.001. See also Figure S1.
1134 1135
Figure 5: Recombinant VEGFD protects RGCs from NMDA-mediated damage.
1136
A) Representative images of retinal whole mounts at d7 after intravitreal injections of PBS,
1137
NMDA, rVEGFD or rVEGFD+NMDA as indicated. RGCs were immunolabeled against
1138
Brn3a (green). Nuclei were immunolabeled with Hoechst (blue). Scale bar=40 µm. B)
1139
Representative images of sagittal optic nerve sections injected as indicated in A. Stressed
1140
axons were immunolabeled using SMI-32 (green). Nuclei were labeled using Hoechst (blue).
1141
Scale bar=50 µm. C) Quantification of RGCs at d1 after intravitreal injections, injected as
1142
indicated in A. Two-way ANOVA, Bonferroni. n=6. PBS vs NMDA (sham), p=0.0002; PBS
1143
vs NMDA (rVEGFD), p>0.9999; sham vs rVEGFD (PBS), p>0.9999; sham vs rVEGFD
1144
(NMDA), p=00013. D) Quantification of RGCs at d7 after intravitreal injections, injected as
1145
indicated in A. Two-way ANOVA, Bonferroni. n=6. PBS vs NMDA (sham), p=0.0012; PBS
1146
vs NMDA (rVEGFD), p=0.0973; sham vs rVEGFD (PBS), p>0.9999; sham vs rVEGFD
43
1147
(NMDA), p=0.1102. E) Quantification of RGCs at d7 after intravitreal injections, injected as
1148
indicated in A. RGC numbers in the NMDA-treated eye were normalized to the PBS control
1149
eye from the same animal. n=6. F) Quantification of SMI-32+ clusters in optic nerves at d7
1150
after intravitreal injections, injected as indicated in A. Two-way ANOVA, Bonferroni. n=6.
1151
PBS vs NMDA (sham), p=0.0012; PBS vs NMDA (rVEGFD), p=0.6742; sham vs rVEGFD
1152
(PBS), p>0.9999; sham vs rVEGFD (NMDA), p=0.0047. G) Quantification of SMI-32+
1153
clusters in optic nerves from eyes at d7 after intravitreal injections, injected as indicated in A.
1154
SMI-32+ clusters in the NMDA-treated eye were normalized to the PBS control eye from the
1155
same animal. n=6. H) Quantification of RGCs at d1 after intravitreal injections of PBS,
1156
NMDA, rGFP or rGFP+NMDA. Two-way ANOVA, Bonferroni. n=6. PBS vs NMDA
1157
(sham), p=0.0013; PBS vs NMDA (rVEGFD), p=0.0059; sham vs rVEGFD (PBS),
1158
p>0.9999; sham vs rVEGFD (NMDA), p=0.8657. I) Quantification of RGCs at d7 after
1159
intravitreal injections, injected as indicated in H. Two-way ANOVA, Bonferroni. n=6. PBS
1160
vs NMDA (sham), p<0.0001; PBS vs NMDA (rGFP), p<0.0001; sham vs rGFP (PBS),
1161
p>0.9999; sham vs rGFP (NMDA), p>0.9999. J) Quantification of RGCs at d7 after
1162
intravitreal injections, injected as indicated in H. RGC numbers in the NMDA-treated eye
1163
were normalized to the PBS control eye from the same animal. n=6. K) Quantification of
1164
SMI-32+ clusters in optic nerves at d7 after intravitreal injections, injected as indicated in H.
1165
Two-way ANOVA, Bonferroni. n=5. PBS vs NMDA (sham), p=0.0036; PBS vs NMDA
1166
(rGFP), p=0.0041; sham vs rGFP (PBS), p>0.9999; sham vs rGFP (NMDA), p>0.9999. L)
1167
Quantification of SMI-32+ clusters in optic nerves from eyes at d7 after intravitreal injections,
1168
injected as indicated in H. SMI-32+ clusters in the NMDA-treated eye were normalized to the
1169
PBS control eye from the same animal. n=6. Bars represent mean ± SEM. Dots represent
1170
single values. ****p<0.0001, ***p<0.001, **p<0.01. See also Figure S2.
1171
44
1172
Figure 6: Recombinant VEGFD prevents decrease of pattern electroretinography
1173
amplitudes after NMDA-triggered damage to RGCs.
1174
A) Representative images of retinal whole mounts at d7 after intravitreal injections of PBS,
1175
NMDA, rVEGFD or rVEGFD+NMDA. RGCs were immunolabeled against Brn3a. Scale
1176
bar=20 µm. B) Amplitude of the second harmonic of the Fast Fourier Transformation plotted
1177
against spatial frequency of stimulation. Asterisks indicate statistical significance between
1178
NMDA- and rVEGFD+NMDA-treated animals. Two-way ANOVA, Bonferroni. n=4. PBS vs
1179
NMDA (0.05 cyc/deg), p=0.6877; PBS vs NMDA (0.10 cyc/deg), p=0.0076; PBS vs NMDA
1180
(0.20 cyc/deg), p=0.0964; PBS vs NMDA (noise), p=0.8182; PBS vs rVEGFD (0.05
1181
cyc/deg), p=0.9669; PBS vs rVEGFD (0.10 cyc/deg), p=0.9140; PBS vs rVEGFD (0.20
1182
cyc/deg), p=0.5811; PBS vs rVEGFD (noise), p=0.9509; rVEGFD vs rVEGFD+NMDA (0.05
1183
cyc/deg), p=0.4433; rVEGFD vs rVEGFD+NMDA (0.10 cyc/deg), p=0.9976; rVEGFD vs
1184
rVEGFD+NMDA (0.20 cyc/deg), p=0.8797; rVEGFD vs rVEGFD+NMDA (noise),
1185
p=0.9980;
1186
rVEGFD+NMDA (0.10 cyc/deg), p=0.0296; NMDA vs rVEGFD+NMDA (0.20 cyc/deg),
1187
p=0.2776; NMDA vs rVEGFD+NMDA (noise), p=0.9980. C) Representative pattern
1188
electroretinography waveforms recorded from mice injected as indicated in A. Bars represent
1189
mean ± SEM. Dots represent single values. **p<0.01, *p<0.05.
NMDA
vs
rVEGFD+NMDA
(0.05
cyc/deg),
p=0.0144;
NMDA
vs
1190 1191
Figure 7: VEGFD reduces formation of empty sleeves triggered by intravitreal NMDA
1192
injection
1193
A) Representative images of retinal whole mounts at d7 after intravitreal injections of NMDA
1194
or PBS from mice injected either with rAAV-LacZ or rAAV-VEGFD as indicated. Vessels
1195
were immunolabeled against CollagenIV (Coll-IV, green) and using Isolectin-B4 (IB4, red).
1196
Scale bar=30 µm. B) Representative images of retinal whole mounts at d7 after intravitreal
45
1197
injections of PBS, NMDA, rVEGFD or rVEGFD+NMDA. Vessels were immunolabeled
1198
against CollagenIV (Coll-IV, green) and using Isolectin-B4 (IB4, red). Scale bar=30 µm. C)
1199
Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in A.
1200
Two-way ANOVA, Bonferroni. n=6. rAAV-LacZ+PBS vs rAAV-LacZ+NMDA, p=0.0008;
1201
rAAV-VEGFD+PBS vs rAAV-VEGFD+NMDA, p=0.6565; rAAV-LacZ+PBS vs rAAV-
1202
VEGFD+PBS, p=0.2950; rAAV-LacZ+NMDA vs rAAV-VEGFD+NMDA, p=0.1484. D)
1203
Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in A.
1204
Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye
1205
from the same animal. n=6. E) Quantification of empty sleeves at d1 after intravitreal
1206
injections with PBS, NMDA, rVEGFD, or rVEGFD+NMDA. Two-way ANOVA,
1207
Bonferroni. n=6. PBS vs NMDA (sham), p=0.0003; PBS vs NMDA (rVEGFD), p=0.5067;
1208
sham vs rVEGFD (PBS), p>0.9999; sham vs rVEGFD (NMDA), p=0.0022. F) Quantification
1209
of empty sleeves at d7 after intravitreal injections, injected as indicated in E. Two-way
1210
ANOVA, Bonferroni. n=6. PBS vs NMDA (sham), p=0.0001; PBS vs NMDA (rVEGFD),
1211
p=0.1673; sham vs rVEGFD (PBS), p=0.8438; sham vs rVEGFD (NMDA), p=0.0329. G)
1212
Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in E.
1213
Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye
1214
from the same animal. n=6. H) Quantification of empty sleeves at d1 after intravitreal
1215
injections of PBS, NMDA, rGFP or rGFP+NMDA. Two-way ANOVA, Bonferroni. n=6.
1216
PBS vs NMDA (sham), p=0.0019; PBS vs NMDA (rGFP), p=0.0004; sham vs rGFP (PBS),
1217
p>0.9999; sham vs rGFP (NMDA), p=0.6279. I) Quantification of empty sleeves at d7 after
1218
intravitreal injections, injected as indicated in H. Two-way ANOVA, Bonferroni. n=6. PBS
1219
vs NMDA (sham), p=0.0143; PBS vs NMDA (rGFP), p=0.0050; sham vs rGFP (PBS),
1220
p=0.5439; sham vs rGFP (NMDA), p=0.1376. J) Quantification of empty sleeves at d7 after
1221
intravitreal injections, injected as indicated in H. Number of empty sleeves in the NMDA-
46
1222
treated eye was normalized to the PBS control eye from the same animal. n=6. Graphs
1223
represent mean ± SEM. ***p<0.001, **p<0.01, *p<0.05. See also Figure S1.
1224 1225
Figure 8: VEGFD protection against NMDA-triggered excitotoxic retinal damage is
1226
mediated by neuronal VEGFR3 expression.
1227
A) Representative confocal scans of retinal whole mounts at d7 after intravitreal injections of
1228
PBS, NMDA, rVEGFD or rVEGFD+NMDA from mice intravitreally injected either with
1229
rAAV-shSCR- (control) or rAAV-shVEGFR3. RGCs were immunolabeled using Brn3a.
1230
Scale bar=20 µm. B) Quantification of RGCs injected as indicated in A. Two-way ANOVA,
1231
Bonferroni.
1232
shSCR+PBS vs rAAV-shSCR+rVEGFD, p=0.9583; rAAV-shSCR+PBS vs rAAV-
1233
shSCR+rVEGFD+NMDA, p=0.4943; shVEGFR3+PBS vs rAAV-shVEGFR3+NMDA,
1234
p=0.0046; rAAV-shVEGFR3+PBS vs rAAV-shVEGFR3r+VEGFD, p=0.7359; rAAV-
1235
shVEGFR3+PBS vs rAAV-shVEGFR3+rVEGFD+NMDA, p=0.0017. C) Quantification of
1236
RGCs at d7 after intravitreal injections, injected as indicated in A, in rAAV-shSCR-injected
1237
eyes. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye from
1238
the same animal. n=8. D) Quantification of RGCs at d7 after intravitreal injections injected as
1239
indicated in A, in rAAV-shVEGFR3-injected eyes. RGC numbers in the NMDA-treated eye
1240
were normalized to the PBS control eye from the same animal. n=8. E) Representative
1241
confocal scans of retinal whole mounts at d7 after intravitreal injections of PBS, NMDA,
1242
rVEGFD or rVEGFD+NMDA from mice injected either with rAAV-shSCR or rAAV-
1243
shVEGFR3 as indicated. Vessels were immunolabeled using Collagen-IV (CollIV, green) and
1244
Isolectin-B4 (IB4, red). Scale bar=30 µm. F) Quantification of empty sleeves injected as
1245
indicated in A. Two-way ANOVA, Bonferroni. n=6. rAAV-shSCR+PBS vs rAAV-
1246
shSCR+NMDA, p=0.0018; rAAV-shSCR+PBS vs rAAV-shSCR+rVEGFD, p=0.5622;
n=8.
rAAV-shSCR+PBS
vs
rAAV-shSCR+NMDA,
47
p=0.0067;
rAAV-
1247
rAAV-shSCR+PBS vs rAAV-shSCR+rVEGFD+NMDA, p=0.1073; rAAV-shVEGFR3+PBS
1248
vs
1249
shVEGFR3r+VEGFD,
1250
shVEGFR3+rVEGFD+NMDA, p=0.0001. G) Quantification of empty sleeves at d7 after
1251
intravitreal injections, injected as indicated in A, in rAAV-shSCR-injected eyes. Number of
1252
empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the
1253
same animal. n=8. H) Quantification of empty sleeves at d7 after intravitreal injections,
1254
injected as indicated in A, in rAAV-shVEGFR3-injected eyes. Number of empty sleeves in
1255
the NMDA-treated eye was normalized to the PBS control eye from the same animal. n=8. I)
1256
Schema of neuronal-mediated VEGFD protection against excitotoxic retinal damage. Upper
1257
row, left box: NMDA triggers RGC and EC degeneration in rAAV-shSCR-injected mice by
1258
direct action on RGCs (red arrows). RGCs and ECs express VEGFR3 (VEGFR3+). Lower
1259
row, left box: VEGFD prevents RGCs and ECs from NMDA-triggered degeneration in
1260
rAAV-shSCR-injected mice. RGCs and ECs express VEGFR3 (VEGFR3+). VEGFD can act
1261
on VEGFR3 in both RGCs and ECs (green arrows). Upper row, right box: NMDA triggers
1262
RGC and EC degeneration in rAAV-shVEGFR3-injected mice by direct action on RGCs (red
1263
arrows). RGCs do not express VEGFR3 (VEGFR3-), ECs express VEGFR3 (VEGFR3+).
1264
Lower row, right box: In rAAV-shVEGFR3-injected mice, VEGFD does not prevent RGCs
1265
and ECs from NMDA-triggered degeneration. RGCs do not express VEGFR3 (VEGFR3-),
1266
ECs express VEGFR3 (VEGFR3+). VEGFD cannot act on VEGFR3 in RGCs, but on
1267
VEGFR3 in ECs (green arrows). Graphs represent mean ± SEM. ***p<0.001, **p<0.01,
1268
*p<0.05. See also Figure S1.
rAAV-shVEGFR3+NMDA,
p=0.0009;
p=0.2623;
rAAV-shVEGFR3+PBS
rAAV-shVEGFR3+PBS
48
vs vs
rAAVrAAV-
Figure 1 d1
PBS
NMDA
PBS
d7
NMDA
Hoechst
Brn3a
A
Brn3a Hoechst
Brn3a Hoechst
Brn3a Hoechst
merge
Brn3a Hoechst
800 600 400
1.0
0.6 0.4
0
0.0
d1 d7
0.2
E 1.2
PBS NMDA
0.8
200
d1 d7
**
time after injection time after injection
*** *** PBS
1.0 0.8 0.6 0.4 0.2 0.0
F 60
NMDA
50
PBS NMDA
40 30 20 10 0
d1 d7
1000
*
INL thickness (µm)
1200
D 1.2
PBS NMDA
relative mRNA level
**
th y1 ne fl
*
relative N RGC
C 1400 N RGC
B
time after injection
PBS NMDA
rel. vessel surface area
1.2
PBS NMDA
1.0
d7
d1
0.6 0.4 0.2 0.0
d7
d7
d1
d7
time after injection
time after injection
J
100
PBS NMDA
vascular TER119+ cells (% of total)
Coll-IV TER119
80
K
60
0.5 0.0
GAPDH
36 kDa
1.0
1.0
0.0
0.0
Q 1.5
untreat. NMDA
1.0
0.8 0.6
0.5
0.2
un tre a 2 0 t. µM
0.0
NMDA
0.0
oc cl l d d tjp5 1
0.4
time after injection
R relative mRNA level
1.0
d7
GluN1
120 kDa
GluN2A
170 kDa
GluN2B
180 kDa
GluN2C
140 kDa
d7
d7
rel. ratio living/total Bend.3 cells
1.2
O
0.5
0.5
time after injection 10 min 24 h
time after injection
d1
25 kDa
PBS NMDA
un rel. ratio living/total astrocytes tre 2 0 at. µM
IgG
2.0 1.5
d1
69 kDa
N
rel. IgG level
rel. albumin level
albumin
PBS NMDA
1.5
NMDA
NMDA
PBS
PBS
d7
d1 conrol
d1
d7
oc cl ld d tjp5 1
time after injection 2.0
PBS NMDA
1.0
0
0.0
2.0 1.5
20
M
PBS NMDA
1.2 0.8
40
L
1.4
0.0
NMDA
* p
1.0
0.2
0.5
P
G
0.4
d1
relative mRNA level
Coll-IV TER119
1.0
PBS NMDA
1.2
0.0
PBS
1.4
0.6
0.2
d7
tj
d1
merge
F
0.8
0.6
I
*
*
time after injection
1.0
0.8
0.4
PBS NMDA
e
*
rel. vessel diameter
d7
d1
Isolectin-B4
time after injection
1.5
e
0
time after injection
H
NMDA
p
tj
1
50 0
Coll-IV IB4
PBS
2
100
Coll-IV IB4
PBS NMDA
3
150
E
D
* ****
4
bEND.3
Collagen-IV
200
C
neurons
* ****
250
rel. vessel length
B
NMDA
N empty sleeves
PBS
rel. fluorescence unit
d7
rel. N empty sleeves
Figure 2 A
1.2
24 h
1.0 0.8 0.6 0.4 0.2 0.0
NMDA
GluN2D GAPDH
170 kDa 36 kDa
Figure 3 2.5
relative mRNA level
A
B
PBS NMDA
2.0
Brn3a
VEGFD
1.5
**
neg. control
neg. control
human
1.0
merge
Brn3a VEGFD
0.5
0.0
E
PBS
NMDA
relative m RNA level
VEGFD
2.0
0.5
0.8
0.4 0.2 0.0
merge
0.6
VEGFD Hoechst
VEGFD Hoechst
GCL
GCL
2.0
untreated NMDA
1.5 1.0 0.5
0.0
0.0
ve v e g fd ve g fc gf a f lt 4
PBS NMDA
2.5
ve v e g fd ve g fc gf a f lt 4
rel. VEGFD protein level
1.0
**
1.0
Hoechst
Beta-actin
untreated NMDA
1.5
VEGF
1.2
G 2.5
VEGFD
D
F
relative m RNA level
subject 2
subject 1
C
d c a 4 1 r g f e gfegf f lt f lt kd e v v v
Figure 4
rAAV-LacZ 2000
N RGC
1500
**** ***
1000 500
rAAV-LacZ +PBS rAAV-LacZ +NMDA rAAV-VEGFD +PBS rAAV-VEGFD +NMDA
D 1.2
SMI-32 Hoechst
SMI-32 Hoechst
SMI-32 Hoechst
rAAV-VEGFD rAAV-LacZ rAAV-VEGFD
1.0 0.8 0.6 0.4 0.2
NM DA
time after injection
0.0
PB S
d7
0
E
Area SMI-32+ cluster (µm2)
C
Brn3a
NMDA
SMI-32 Hoechst
10000
**** ****
5000 1500 1000 500 400
rAAV-LacZ +PBS rAAV-LacZ +NMDA rAAV-VEGFD +PBS rAAV-VEGFD +NMDA
200 0
d7
Brn3a
relative N RGC
rAAV-VEGFD
PBS
time after injection
F
rAAV-LacZ rAAV-VEGFD
120 100 80 60 40 20 0
NM DA
NMDA
Brn3a
d7
PB S
PBS
B rAAV-LacZ
Brn3a
d7
rel. area SMI32+ cluster
A
Figure 5 A
d7
PBS
B
NMDA
Brn3a Hoechst
Brn3a Hoechst
Brn3a Hoechst
SMI-32 Hoechst
rVEGFD 500
E 1.2
PBS/NMDA rVEGFD/NMDA
1.0 0.8 0.6 0.4 0.2
0
1000
I
**** 2000 **** **** 1500 1000 500
0
0
d1
500
time after injection
PBS NMDA rGFP NMDA+ rGFP
J 1.2
treatm.
PBS/NMDA rGFP/NMDA
1.0 0.8 0.6 0.4 0.2 0.0
d7
N RGC
1500
PBS NMDA rGFP NMDA+ rGFP
N RGC
2000 ** **
time after injection
ctrl.
relative N RGC
time after injection
H
0.0
d7
d1
0
time after injection
ctrl.
treatm.
F
12000 ** 10000 8000 6000 4000 2000
**
G
PBS NMDA rVEGFD NMDA+ rVEGFD
200 100 0
rel. area SMI-32+ cluster
1000
PBS NMDA rVEGFD NMDA+ rVEGFD
PBS/NMDA rVEGFD/NMDA
110 100 90 40 20 0
time after injection
K
12000 ** ** 10000 8000 6000 4000 2000
L
PBS NMDA rGFP NMDA+ rGFP
rel. area SMI-32+ cluster
**
d7
1500
40
ctrl.
treatm.
PBS/NMDA rGFP/NMDA
30 20 10
200 100 0
d7
500
D
Area SMI-32+ cluster (µm2)
N RGC
1000
PBS NMDA rVEGFD NMDA+ rVEGFD
Area SMI-32+ cluster (µm2)
**
relative N RGC
**
N RGC
1500
NMDA
SMI-32 Hoechst
PBS SMI-32 Hoechst
rVEGFD
C
PBS
SMI-32 Hoechst
PBS
Brn3a Hoechst
d7
time after injection
0
ctrl.
treatm.
Figure 6 A
PBS
NMDA
B
Brn3a
PBS
Brn3a
d7
60
* **
40
Brn3a
30
rVEGFD
Brn3a
Amplitude (µV)
50
PBS NMDA rVEGFD rVEGFD+NMDA
20 10 0
0.05
0.10
0.20
noise
Spatial frequency (cyc/deg)
C
PBS cyc/deg 0.05
0.10
0.20
noise
25 µV 500 ms
NMDA
rVEGFD
rVEGFD+NMDA
Figure 7 A
PBS
d7
B
NMDA
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
NMDA
PBS
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
rVEGFD
rAAV-VEGFD
100
1.0
time after injection 3.0
PBS/NMDA rVEGFD/NMDA
H 400 300 **
2.5 2.0
***
200
1.5 1.0
NMDA
PBS I NMDA rGFP NMDA+ rGFP
100
0.5 0.0
PBS
ctrl.
treatm.
0
50 0
150
PBS NMDA rVEGFD NMDA+ rVEGFD
50 0
time after injection 400 300
*
**
200
time after injection
PBS J NMDA rGFP NMDA+ rGFP
3.0
PBS/NMDA rGFP/NMDA
2.5 2.0 1.5 1.0 0.5 0.0
0
time after injection
*
100
100
d1
G
N empty sleeves
d7
0.0
200
***
d7
1.5
250
N empty sleeves
2.0
0.5
0
rel. N empty sleeves
150
F PBS NMDA rVEGFD NMDA+ rVEGFD
rel. N empty sleeves
50
200 **
**
d1
100
2.5
250
d7
150
3.0
rAAV-LacZ rAAV-VEGFD
E
N empty sleeves
200
rAAV-LacZ D +PBS rAAV-LacZ +NMDA rAAV-VEGFD +PBS rAAV-VEGFD +NMDA
N empty sleeves
**
rel. N empty sleeves
C 250 N empty sleeves
PBS
Coll-IV IB4
rAAV-LacZ
Coll-IV IB4
d7
time after injection
ctrl.
treatm.
NMDA
rVEGFD
Brn3a
Brn3a
Brn3a
Brn3a
Brn3a
** **
N RGC
1000
PBS NMDA rVEGFD NMDA+ rVEGFD
500
C
rAAV-shSCR: 1.2
PBS/NMDA rVEGFD/NMDA
D
0.6
0.6
0.4 0.2
0.2
PBS
NMDA
0.0
treatm.
rVEGFD
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
400
**
300 200
*** ****
PBS NMDA rVEGFD NMDA+ rVEGFD
100 0
I
G rAAV-shSCR: rel. N empty sleeves
F
ctrl.
Coll-IV IB4
rAAV-shVEGFR3 rAAV-shSCR
E
shSCR shVEGFR3
2.0
PBS/NMDA rVEGFD/NMDA
1.5
VEGFR3+ NMDA
rVEGFD+NMDA Coll-IV IB4
Coll-IV IB4
H
rAAV-shVEGFR3: 2.0
PBS/NMDA rVEGFD/NMDA
0.5
ctrl.
0.0
treatm.
rAAV-shSCR
ctrl.
treatm.
rAAV-shVEGFR3
VEGFR3NMDA
VEGFR3+
VEGFR3+
VEGFR3+
VEGFR3-
NMDA
NMDA
VEGFD
VEGFD VEGFR3+
treatm.
1.0
1.0
0.0
ctrl.
1.5
0.5
shSCR shVEGFR3
PBS/NMDA rVEGFD/NMDA
0.8
0.8
0.0
1.2 1.0
1.0
0.4
0
rAAV-shVEGFR3:
relative N RGC
1500 **
Brn3a
Brn3a
relative N RGC
B
rVEGFD+NMDA
r el. N empty sleeves
PBS
Brn3a
rAAV-shVEGFR3 rAAV-shSCR
A
N empty sleeves
Figure 8
VEGFR3+
S2329-0501(19)30158-5
DOI:
https://doi.org/10.1016/j.omtm.2019.12.009
Reference:
OMTM 367
To appear in:
Molecular Therapy: Methods & Clinical Development
Received Date: 19 December 2019 Accepted Date: 19 December 2019
Please cite this article as: Schlüter A, Aksan B, Diem R, Fairless R, Mauceri D, VEGFD protects retinal ganglion cells and, consequently, capillaries against excitotoxic injury, Molecular Therapy: Methods & Clinical Development (2020), doi: https://doi.org/10.1016/j.omtm.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s).
1
VEGFD protects retinal ganglion cells and, consequently, capillaries against excitotoxic
2
injury
3 4
Annabelle Schlüter1, Bahar Aksan1, Ricarda Diem2,3, Richard Fairless2,3, Daniela Mauceri1*
5 6
1
7
University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany
8
2
9
Heidelberg, Germany
Department of Neurobiology, Interdisciplinary Center for Neurosciences, Heidelberg
Department of Neurology, University Clinic Heidelberg, Im Neuenheimer Feld 368, 69120
10
3
11
Centre (DKFZ), 69120 Heidelberg, Germany
CCU Neurooncology, German Cancer Consortium (DKTK), German Cancer Research
12 13 14
* Correspondence:
15
Dr. Daniela Mauceri
16
Email: [email protected],
17
Phone: +49 6221 5416508
18 19 20
Short title:
21
VEGFD spares neurons and capillaries from damage
22 23 24
Keywords:
25
VEGFD, retinal ganglion cells, endothelial cells, excitotoxicity, neuroprotection.
1
26
Abstract
27
In the central nervous system, neurons and the vasculature influence each other. While it is
28
well described that a functional vascular system is trophic to neurons and that vascular
29
damage contributes to neurodegeneration; the opposite scenario in which neural damage
30
might impact the microvasculature is less defined. Here, using an in vivo excitotoxic
31
approach in adult mice as a tool to cause specific damage to retinal ganglion cells, we
32
detected a subsequent damage to endothelial cells in retinal capillaries. Further, we detected
33
decreased expression of Vascular Endothelial Growth Factor D (VEGFD) in retinal ganglion
34
cells. In vivo VEGFD supplementation via neuronal-specific viral-mediated expression or
35
acute intravitreal delivery of the mature protein preserved the structural and functional
36
integrity of retinal ganglion cells against excitotoxicity and, additionally spared endothelial
37
cells from degeneration. Viral-mediated suppression of expression of the VEGFD-binding
38
receptor VEGFR3 in retinal ganglion cells revealed that VEGFD exerts its protective
39
capacity directly on retinal ganglion cells while protection of endothelial cells is the result of
40
upheld neuronal integrity. These findings suggest that VEGFD supplementation might be a
41
novel, clinically applicable approach for neuronal and vascular protection.
2
42
Introduction
43
In the central nervous system (CNS), the neuronal and vascular components have a strong
44
structural and functional interdependence during both development and adulthood 1. In the
45
retina, in particular, a tight coupling between retinal ganglion cells (RGCs) and capillaries
46
has been described 2 but the molecular and cellular players underlying this link have not been
47
completely elucidated. Members of the Vascular Endothelial Growth Factor (VEGF) family
48
have been reported to be possible modulators of the communication between the vascular and
49
neuronal compartments 3-6.
50
The retinal neurovascular interface plays a role not only in physiology but also in several
51
pathologies. Dysfunctions of both the neural and vascular components have been implicated
52
in pathologies of the visual system such as glaucoma, diabetic retinopathy and macular
53
degeneration 2. It is well known that vascular occlusion and damage can contribute to the
54
progression of neural retina injury
55
integrity of the capillaries is unclear. In experimental models of neural retinal injury, delayed
56
damage to the retinal microvasculature has been observed 8-10, suggesting that neural integrity
57
may indeed be critical for vascular health.
58
Capillaries have a small diameter and consist of a single endothelial cell (EC) layer forming
59
the inner lining, which are anchored to the basement membrane (BM). A well-reported
60
phenomenon, which has yet no clear explanation for its occurrence, is the detected higher
61
number of empty sleeves throughout the CNS – retina, hippocampus, cortex – in relation to
62
both physiological aging and to several neurodegenerative diseases
63
named string vessels or acellular capillaries, consist of remnants of BM, which have lost the
64
inner ECs and, in several cases, have collapsed
65
plasma as they are not perfused
66
tissue and enhance degeneration. It is not known what drives the formation of empty sleeves;
7
11
while the putative impact of neuronal damage onto the
11
11-13
. Empty sleeves, also
. Empty sleeves cannot transport blood or
and thus may have noxious effects on the surrounding
3
67
however, their increased presence following neuronal degeneration indicates that neurons
68
might play a role.
69
Here, we used an in vivo approach to cause specific damage to RGCs, which we found to be
70
accompanied by a reduction in the expression of VEGFD, a member of the VEGF family, in
71
RGCs and formation of empty sleeves. We additionally reveal the protective capacity of
72
VEGFD against degeneration of RGCs and, as a result of the maintained neuronal integrity,
73
also against capillary damage. Our data indicate that VEGFD delivery might provide a
74
promising therapeutic strategy for the treatment of degenerative diseases of the neuronal and
75
vascular system.
76 77
Results
78
Excitotoxicity is a pathological phenomenon leading to neuronal damage or death that has
79
been associated with several disorders of the nervous system
80
(NMDA) receptors (NMDARs), calcium-permeable channels, are the principal initiators of
81
excitotoxic cell death induced by excessive receptor activation either by high glutamate
82
levels or NMDA exposure
83
retinal ischemia, and diabetic retinopathy
84
NMDA delivered via intravitreal injections can be utilized as a tool to cause RGCs death in
85
vivo
86
analyzed NMDA-induced RGC death at two different time points (day 1 (d1) and day 7 (d7))
87
(Fig. 1). Brn3a served as a marker to identify and quantify RGCs 27 in central and peripheral
88
regions of retinal whole mounts (Fig. 1A,B). One day after intravitreal NMDA injections, the
89
number of RGCs was significantly decreased compared to the PBS-injected control eyes (Fig.
90
1A-D). RGC death did not worsen over time as observed by comparable RGC survival seven
91
days after the NMDAR-mediated excitotoxic insult (Fig. 1C,D). We further detected a
21-26.
14, 15
14
. N-methyl-D-aspartate
. In the eye, excitotoxicity has been linked to glaucoma, 16-20
. Indeed, high concentrations of glutamate or
We performed a single intravitreal injection of 10 nmol NMDA in adult mice and
4
92
significant reduction in the mRNA level of the RGCs markers thy1 and nefl (Fig. 1E) in
93
retinal homogenates. To investigate whether intravitreal injection of NMDA damages other
94
retinal neurons than RGCs, we measured the thickness of the inner nuclear layer (INL) as
95
indication of possible cell death in the INL 28. We did not detect any significant changes after
96
intravitreal NMDA injections (Fig. 1F). TUNEL assay carried out on retinal sections
97
confirmed that the extent of apoptosis in retinal tissue following intravitreal injection of 10
98
nmol NMDA was negligible in the INL (percentage of TUNEL+ cells: d1, PBS (0.02949081
99
%) vs NMDA (0.6696871 %), p= 0.2019; d7, PBS (0.09155836 %) vs NMDA (0.08281018
100
%), p= 0.7552; unpaired student’s t-test, n=3) and outer nuclear layer (ONL; percentage of
101
TUNEL+ cells: d1, PBS (0.07544173 %) vs NMDA (0.05547914 %), p= 0.7181; d7, PBS
102
(0.05516085 %) vs NMDA (0.0662406 %), p= 0.6445; unpaired student’s t-test, n=3). Taken
103
together, these data demonstrate that intravitreal injection of NMDA can be used to trigger
104
degeneration of RGCs in the retina in vivo.
105 106
In the retina, RGCs are structurally and functionally coupled with capillaries and
107
dysfunctions of both the neural and vascular components are present in pathologies of the
108
visual system 2. We proceeded to analyze the possible effects of the in vivo NMDA insult on
109
the retinal capillaries. We found that intravitreal injection of NMDA promoted the formation
110
of collapsed empty sleeves at both d1 and d7 assessed by immunostaining of the BM with
111
anti-Collagen-IV and labeling of the ECs with Isolectin-B4 (Fig. 2A-C). Using electron
112
microscopy, it was possible to observe, in some cases, occluded lumen of the capillaries due
113
to swelling of the damaged ECs prior to their death and removal 29, 30, whereas tight junctions
114
remained intact (Fig. 2D). The increased presence of collapsed empty sleeves appeared to be
115
a specific phenomenon, as other vascular parameters, such as vessel surface area, vessel
116
diameter, and vessel length were not affected by the NMDA insult at any of the analyzed
5
117
time points (Fig. 2E-G). To assess potential NMDA-mediated effects on the blood-retinal
118
barrier (BRB), we first analyzed in retinal homogenates the mRNA levels of ocld, cld5, or
119
tjp1, encoding for Occludin, Claudin-5, and ZO-1 respectively, molecules important for the
120
functionality of endothelial tight junctions. None of the analyzed genes was affected by
121
intravitreal NMDA injection (Fig. 2H). To clarify whether intravitreal NMDA injection could
122
prompt extravasation of cells, we assessed the percentage of erythrocytes within the retina
123
capillaries 31 in relation to the total amount of erythrocytes within and outside vessels (Figure
124
2I,J). We found no difference between PBS-injected controls and NMDA-injected retinas
125
(Figure 2I,J). Further, in whole-retina extracts, we examined the extravasation of
126
intravenously injected 3 kDa-dextran-fluorescein as well as of endogenous albumin and IgG
127
following intravitreal NMDA injections 30, which we found similar to PBS-injected controls
128
(Fig. 2K-N). These data indicate that NMDA-triggered excitotoxicity, in addition to
129
promoting death of RGCs, causes increased formation of empty sleeves without affecting
130
BRB functionality or the overall structure of the vascular network.
131
ECs of the CNS express NMDA receptors
132
NMDAR-mediated toxicity. To understand whether the observed degeneration of ECs
133
following NMDA intravitreal injection is the result of a direct or indirect effect of NMDA,
134
we used a different approach and directly exposed only ECs to NMDA in vitro. We
135
confirmed the expression of the GluN1 channel-forming subunit of the NMDA receptor as
136
well as of the regulatory subunits GluN2A and GluN2D (Fig 2O) in a mouse brain EC line
137
(bEnd.3) 34, 37. GluN2B and GluN2C subunits were not detected. We decided to compare the
138
sensitivity of cultured neurons and ECs to NMDA using exposure to 20 µM NMDA, which
139
typically causes fast neuronal death
140
minutes or for 24 hrs, the vitality of the cultured mouse brain ECs remained unaltered (Fig.
141
2P) while neurons considerably die
38
38
32-36
and might therefore be a direct target of
. When treated with 20 µM NMDA acutely for 10
. Even using higher NMDA concentration (1 mM),
6
142
close to the one reached in vivo after intravitreal injection of 10nmol NMDA in the relatively
143
small volume of the mouse eye; while neuronal death rate reached 100%, the vitality of
144
cultured endothelial cells was still unaffected (Untreated vs NMDA 10 min, n=3, p=0.9868.
145
Untreated vs NMDA 24 hrs, n=3, p=0.6556. Unpaired Student’s t-test.) The expression levels
146
of ocld, cld5, and tjp1 of NMDA-treated cultured ECs were also similar to control-treated
147
cells (Fig. 2Q). In the mammalian neurovascular unit, astrocytes represent a considerable
148
percentage of the present cells and can exchange signals with ECs
149
studies revealed the functional expression of NMDARs on various types of glia cells in the
150
nervous system 40. In a similar manner to that observed for cultured brain ECs, we found that
151
application of NMDA for 24 hrs did not affect the survival of primary cultured astrocytes
152
(Fig. 2R). Collectively, these data indicate that the in vivo detected increased number of
153
empty sleeves, due to loss of ECs, after NMDA-based insult (Fig. 2A-D) is likely mediated
154
by an indirect mechanism.
39
. Moreover, numerous
155 156
By reducing the time of analysis following intravitreal injection of NMDA to 6 hrs, we found
157
that loss of RGCs (40.2 ± 6.3%; NMDA vs PBS, n=3, p=0.0005) temporally precedes the
158
formation of empty sleeves (105.9 ± 5.8% number of empty sleeves after NMDA treatment;
159
NMDA vs PBS, n=3, p=0.5249). In experimental models of neural retinal damage,
160
degeneration of the capillaries in correlation to RGC death has been observed 7-10, but it is not
161
yet understood if and how RGCs might impact the retinal microvasculature. We hypothesized
162
that degeneration of RGCs could negatively impact capillaries via an alteration in the
163
molecular crosstalk between RGCs and ECs. The vascular endothelial growth factor (VEGF)
164
family members and their receptors (VEGFRs) play roles both in the vascular and nervous
165
system where they regulate, for example, vascular development, angiogenesis as well as
166
axonal path finding, dendritic morphology, synaptic plasticity, neuroprotection and cognition 7
167
41-43
168
cross-talk between neurons and endothelial cells (ECs) in the retina has been described 6, and
169
VEGFR3 has been detected outside the vasculature in neural elements of the retina 44, 45. We
170
therefore evaluated the potential effects of NMDA intravitreal injection on the expression
171
levels of VEGF family members and receptors. While no change was found for the mRNA
172
levels of vegf, vegfc, vegfr2 (kdr) or vegfr3 (flt4) in retinal homogenates, we detected a
173
significant reduction in vegfd levels (Fig. 3A). VEGFD, beyond its previously documented
174
functions in angiogenesis and lymphangiogenesis 42, is expressed also in neurons
175
fundamental for the maintenance of neuronal dendritic architecture and cognition 43, 49-51. We
176
successfully detected VEGFD mRNA (Fig. 3C) and VEGFD protein in RGCs in post-mortem
177
human retinas (Fig. 3B) as well as in adult mouse RGCs (Fig. 3E). After intravitreal injection
178
of NMDA, we found that VEGFD protein levels were specifically reduced in RGCs (Fig.
179
3D,E). Recently, in agreement with our present findings, it was shown that bath application
180
of NMDA to primary cultured neurons caused a rapid and progressive decline of vegfd
181
mRNA levels 52. Using a similar experimental strategy, namely NMDA treatment of cultured
182
cells, we found that vegfd expression levels remained constant in both mouse cultured ECs
183
(Fig. 3F) and primary astrocytic cultures (Fig. 3G) following 24 hrs of exposure to NMDA.
184
Thus, an NMDA-mediated insult caused reduction of VEGFD expression in RGCs but not in
185
other cell types.
. Recently, a novel function of neuronal VEGFR2 expression in the regulation of the
46-48
and is
186 187
We hypothesized that preventing VEGFD down-regulation in RGCs might be beneficial for
188
RGC survival after NMDA-triggered injury. We intravitreally injected adult wildtype mice
189
with recombinant adeno-associated viruses (rAAVs) containing neuron-specific expression
190
cassettes for VEGFD (rAAV-VEGFD)
191
additional HA- (for rAAV-VEGFD) or Flag- (for rAAV-LacZ) tags to facilitate their
43, 50
8
or as control, LacZ (rAAV-LacZ), with
192
detection. Intravitreal injection of rAAVs is a robust method used successfully to achieve
193
long-lasting, stable gene delivery
194
VEGFD has been previously used effectively to boost neuronal expression of VEGFD
195
and does not infect cells of the microvasculature (Supplementary Fig. 1A). To investigate the
196
protective capacities of neuronal overexpressed VEGFD against excitotoxic retinal damage,
197
we subjected rAAV-LacZ- and rAAV-VEGFD-injected mice to NMDA-triggered
198
excitotoxicity and quantified RGC degeneration at d7 after the excitotoxic insult. While
199
NMDA injections caused robust RGC loss in rAAV-LacZ-injected mice, RGC survival after
200
NMDA treatment in rAAV-VEGFD-injected mice was similar to PBS-injected control eyes
201
(Fig. 4A,C,D). Further, VEGFD overexpression in RGCs significantly protected RGC axons
202
in the optic nerve from the NMDA-mediated excitotoxic damage assessed by measuring the
203
increased presence of dephosphorylated neurofilaments (detected by SMI-32), as indication
204
for axonal stress. After intravitreal NMDA injections, in rAAV-LacZ-injected mice, SMI-32-
205
labelled clusters greatly increased in comparison to PBS-injected controls, while in rAAV-
206
VEGFD-injected animals, their NMDA-triggered formation was blunted (Fig. 4B,E,F).
207
We proceeded to test whether providing VEGFD in an acute treatment, at the same time of
208
the insult, would still spare RGCs from degeneration. We intravitreally co-injected
209
recombinant mature VEGFD protein (rVEGFD) with either PBS or NMDA and determined
210
RGC survival at d1 and d7 after injection (Fig. 5). At both time points, rVEGFD prevented
211
excitotoxic degeneration of RGCs (Fig. 5A,C-E). Further, we found that acute delivery of
212
rVEGFD was capable to reduce the formation of NMDA-triggered SMI-32-positive clusters,
213
indicative of axonal damage (Fig. 5B,F,G). As control, we performed similar experiments by
214
co-injecting a different recombinant protein, recombinant GFP (rGFP), instead of rVEGFD,
215
with NMDA or PBS (Fig. 5H-L). rGFP treatment did not have any effects on RGC survival
216
(Fig. 5H-J; Supplementary Fig. 2A) or axonal injury (Fig. 5K,L; Supplementary Fig. 2B). In
53
and is under consideration in clinical trials
9
54
. rAAV43, 50
217
conclusion, these findings show the specific protective capacity of VEGFD on RGCs and
218
axonal integrity in an NMDA-triggered excitotoxicity model.
219
We investigated whether VEGFD supplementation could also restore the functional
220
properties of RGCs in addition to sparing them from structural degeneration and death (Fig.
221
4, 5). For this purpose, we measured the function of RGCs within the retina by performing
222
pattern electroretinography in mice
223
without co-injection of rVEGFD (Fig. 6). NMDA-triggered excitotoxicity led to significantly
224
decreased amplitudes at complex spatial frequencies (0.10 cyc/deg), which represent reduced
225
neuronal activity in the retina. The supplementation of rVEGFD with NMDA was capable to
226
restore amplitudes to values comparable to those in PBS- or rVEGFD-injected control mice
227
(Fig. 6B, C). After sacrificing the mice, we confirmed, in agreement to what was assessed
228
previously (Fig. 5), that intravitreal NMDA injections resulted in significant RGC loss (48.52
229
± 11.52%), which was reduced after rVEGFD supplementation (19.78 ± 4.55%). In
230
summary, our data show the potential therapeutic capacity of intravitreal VEGFD
231
supplementation in the maintenance of functional and intact RGCs.
232
Next, we tested whether supplementation of VEGFD could also spare the retinal
233
microvasculature from NMDA-triggered excitotoxic damage (Fig. 2). When VEGFD was
234
specifically overexpressed in RGCs via rAAV-VEGFD (Supplementary Fig. 1), the
235
formation of collapsed empty sleeves at d7 after the NMDA-triggered excitotoxic insult was
236
reduced in comparison to control mice injected with rAAV-LacZ (Fig. 7A,C,D). Acute
237
VEGFD treatment via co-injection of rVEGFD led to similar results displaying a significant
238
decrease in the number of empty sleeves at d1 and d7 after NMDA treatment (Fig. 7B,E-G).
239
Delivery of rGFP did not affect the NMDA-triggered formation of empty sleeves (Fig. 7 H-J;
240
Supplementary Fig. 2C) indicating a specific protective effect of VEGFD against NMDA-
55
subjected to NMDA-triggered excitotoxicity with or
10
241
triggered vascular damage.
242
Our data show that an NMDA-mediated excitotoxic insult decreases the levels of VEGFD
243
specifically in RGCs and triggers RGC degeneration prior to promoting formation of empty
244
sleeves, likely via an indirect mechanism. Increasing VEGFD levels blunts the excitotoxicity-
245
induced degeneration of both RGCs and ECs. In humans, VEGFD binds and activates both
246
VEGFR2 and VEGFR3, whereas murine VEGFD exclusively activates VEGFR3 56. To better
247
elucidate whether capillaries are spared from excitotoxic damage as a consequence of a
248
VEGFD-mediated effects on neuronal survival or via a possible direct trophic simultaneous
249
action of VEGFD on ECs, we used an approach to specifically reduce the expression of
250
VEGFR3 in RGCs 45 while leaving the expression unaltered in ECs. There are no satisfactory
251
inducible cre-driver lines, which would guarantee efficient and extensive manipulation of the
252
diverse RGC subtypes without affecting other retinal neurons and cell types
253
decided to employ intravitreal injection of rAAV-shVEGFR3
254
the mouse Flt4 (VEGFR3) mRNA inserted downstream of the U6 promoter of the rAAV
255
vector or, as control, a scrambled version rAAV-shSCR. The used rAAVs have been
256
previously extensively characterized 43 and carry an mCherry expression cassette allowing for
257
detection of infected neurons. Three weeks after intravitreal rAAV delivery, the infection rate
258
of Brn3a-positive RGCs was above 60%. Other cell types within the retina, including cells of
259
the vascular system, were not transduced by the used viruses (Supplementary Fig. 1C). Three
260
weeks after infection, we performed intravitreal NMDA injections with or without co-
261
injection of rVEGFD. Using this strategy, VEGFD cannot act on RGCs any longer - as they
262
do not express VEGFR3 - while they are still vulnerable to NMDA. In control mice that were
263
intravitreally injected with rAAV-shSCR, NMDA caused significant RGC loss, which was
264
prevented by intravitreal supplementation of rVEGFD (Fig. 8A-C), as expected. When
265
VEGFR3 expression in RGCs was specifically reduced via rAAV-shVEGFR3, the VEGFD11
43
57-59
. Thus, we
encoding shRNAs targeting
266
mediated protection of RGCs from excitotoxicity was completely lost (Fig. 8A,B,D). In
267
rAAV-shSCR-injected mice, rVEGFD still interfered with formation of empty sleeves (Fig.
268
8E,F,G,). In rAAV-shVEGFR3 injected mice, however, ECs were no longer protected from
269
the NMDA-triggered degeneration by the administration of rVEGFD, although rVEGFD
270
could activate endothelial VEGFR3 (Fig. 8E-H). Thus, our data indicate that VEGFD-
271
mediated safeguard of RGCs and capillaries from damage takes place by VEGFD directly
272
exerting its protective functions on RGCs and, as a consequence of this phenomenon, sparing
273
ECs from degeneration (Fig. 8I)
274
Discussion
275
In this study, we found that an injury to RGCs triggers downregulation of neuronal VEGFD
276
and is followed by capillary damage. Moreover, we demonstrate that VEGFD
277
supplementation promotes RGC survival, which is crucial to prevent the formation of
278
collapsed, acellular capillaries. Taken together, VEGFD might have clinical relevance to
279
robustly promote neuronal and vascular integrity.
280
Degeneration of neurons and regression of capillaries have been reported for several CNS
281
disorders
282
cause for many disorders of the CNS
283
excitotoxic death of neurons along with damage to the microvasculature has been described 8,
284
10
285
available. Here, we detected RGC damage after a single intravitreal NMDA injection within
286
6hrs from delivering the insult, which did not significantly progress over time. Degeneration
287
of ECs followed neuronal death temporally. Unlike the widely acknowledged contribution of
288
vascular abnormalities to neurodegeneration
289
following neural degeneration has been less discussed although it has been observed, for
290
instance, in diabetes and glaucoma
4, 60
. Noxious NMDAR-mediated excitotoxicity has been put forward as a possible 61-63
. Also for pathologies of the visual system,
; however, no detailed description of the possible influence of neurons on EC integrity is
7, 65
64
, delayed damage to the microvasculature
. Our data suggest that signals deriving from 12
291
degenerating RGCs might be the cause of capillary damage. Such a hypothesis is further
292
supported by our observations that a direct NMDA insult to cultured brain ECs did not affect
293
their survival, although they expressed different GluN subunits ensuring a functional
294
NMDAR
295
that RGCs are degenerating to the ECs. This process might be ascribed to an imbalance in the
296
expression of particular factors and could involve multiple cell types. In this view, it is
297
appealing to speculate that the reduction of VEGFD levels in RGCs, which we detected,
298
might be playing a role; however, such a hypothesis calls for future in-depth investigations.
299
Of the several parameters measured, including expression of markers and functionality of the
300
BRB as well as detailed analysis of the microvascular architecture, we only observed
301
alterations in the increased presence of string vessels as a consequence of injured RGCs. Our
302
data therefore suggest that EC damage is not necessarily correlated to BRB disruption. BRB
303
breakdown might be staggered in time or uncoupled; a phenomenon which has been
304
previously observed
305
capillaries would collapse and stop being perfused before leakage and disruption could take
306
place 68.
307
Neurons can influence the vascular system in different ways. While synaptic activity drives
308
vascular patterning during CNS development, neural cells provide feedback to vessels
309
regarding their metabolic needs in the mature brain allowing regional adjustments of blood
310
flow in response to changing neuronal activity
311
damaged neurons could have a noxious impact on the vasculature. In addition, we provide
312
information on the possible causes driving the formation of empty sleeves, a phenomenon
313
largely uncharacterized and unexplained. Empty sleeves can be present in low number in
314
physiological conditions, but their number dramatically increases throughout the CNS during
315
aging and in neurodegenerative diseases such as diabetic retinopathy, Alzheimer’s disease,
39, 66
. An open question remains which nature of signals might carry the message
30, 67
. Mechanistically, a possible explanation may be that the affected
13
1, 69
. Our study showed for the first time that
316
stroke, glaucoma and Parkinson’s disease 70-73. Our data point towards the idea that structural
317
and functional integrity of neurons is critical for the maintenance of healthy capillaries and
318
that, when neurons are compromised or damaged, this negatively affects ECs. The resulting
319
creation of collapsed capillaries might in turn exacerbate neurodegeneration and create a
320
vicious circle of vascular and neural dysfunctions. Future work needs to extend our findings
321
obtained with an acute tool to promote RGC degeneration to different models of CNS
322
disorders and to different brain regions.
323
Interestingly, among the members of the VEGF family and their receptors, known key
324
modulators of neurovascular communication 3-6, our study identified VEGFD as the only one
325
whose expression in the retina was affected by the excitotoxic insult to RGCs. Such
326
observations are in agreement with our recent findings showing that VEGFD expression, but
327
not that of other VEGFs or VEGFRs, is shut-off following toxic activation of NMDARs in
328
pyramidal neurons in vitro or in vivo following stroke
329
activity, nuclear calcium signaling and epigenetic regulators can modulate VEGFD
330
expression in neurons while leaving expression of VEGFC, VEGF and VEGFRs unaltered 43,
331
51
332
regulation. Here, we showed for the first time that VEGFD is expressed in RGCs in the
333
human retina and we found that the NMDA-triggered down regulation of VEGFD is taking
334
place specifically in RGCs and not in other cell types of the neurovascular unit, in spite of the
335
fact that ECs and astrocytes carry functional NMDARs 32, 40, 66, 74. Excitotoxicity in the retina
336
alters the subcellular distribution, and thus activity, of the epigenetic regulators histone
337
deacetylases (HDACs) in RGCs
338
regulates the expression of VEGFD
339
regulators are involved in the decrease of VEGFD levels described in the present study.
52
. Several factors, such as synaptic
; thus suggesting that VEGFD neuronal expression is under a peculiar and dynamic
26
. The activity of one particular HDAC, namely HDAC4, 51
. It remains an open question, if such epigenetic
14
75
340
RGCs lose dendrites before they die due to NMDA excitotoxicity
341
models of excitotoxicity-associated disorders such as glaucoma and optic nerve crush 76-80. In
342
a mouse model of stroke, a pathology also characterized by excitotoxic mechanisms, VEGFD
343
supplementation, but not VEGFC or VEGFA, spared cortical neurons from stroke-induced
344
damage
345
connections of cortical neurons, and, as result, preventing neurodegeneration
346
different approaches, we uncovered the protective functions of VEGFD on both the
347
degenerating neural and vascular components of the retina. It is highly likely that the detected
348
beneficial effects of VEGFD on RGCs, as it happens for pyramidal neurons, may be due to
349
the specific properties of VEGFD to preserve and maintain dendritic connections
350
recent years, rAAVs received increasing attention as tools for gene delivery for the treatment
351
of several diseases
352
in clinical trials and rapidly approaching medical praxis 82. We first used rAAVs to boost the
353
production of VEGFD in RGCs without affecting VEGFD expression in other cell types.
354
This approach significantly blunted the excitotoxic damage to RGCs, optic nerve axons, and
355
the microvasculature. Even if we did not detect any effects as a result of the sole
356
overexpression of VEGFD in absence of the toxic insult, we cannot completely rule out that
357
some additional mechanisms owing to the long-term expression of VEGFD might be
358
participating in the protective effects. Notably, however, acute VEGFD treatment via
359
intravitreal injections of the mature form of VEGFD (rVEGFD) recapitulated the same
360
protective properties as achieved via rAAV-mediated neuronal VEGFD overexpression.
361
Moreover, rVEGFD administration also restored the impaired functionality of RGCs
362
following excitotoxicity. As RGCs are responsible for transmitting visual information to the
363
brain and their death is accompanied by devastating effects on vision 83, therapies designed to
364
promote survival of RGCs are of utmost relevance. Our work highlights the possible
52
and in several animal
. This is mechanistically implemented by VEGFD preserving the dendritic
81, 82
52
. Here, using
43, 49-51
. In
. For eye diseases, in particular, they are already under investigation
15
365
therapeutic capacity of VEGFD supplementation to treat vision deficiency associated to RGC
366
degeneration.
367
Our results revealed that, in addition to protecting the neural components of the retina,
368
VEGFD prevented the formation of empty sleeves. These findings might be interpreted as a
369
possible direct mechanism of VEGFD on ECs, sparing them from degeneration. However,
370
multiple evidences argue against this line of thought. First, our data indicate that in our model
371
empty sleeves formation follows RGC damage and that VEGFD levels in ECs are not
372
affected by the insult. Although rAAV-mediated neuronal overexpression of VEGFD would
373
increase the levels of available VEGFD free to act on both RGCs and ECs, as the
374
overexpressed protein undergoes proteolytic maturation and is secreted
375
primarily via an autocrine mechanism in neurons 43 as well as in other cell types 84-87. Thus, it
376
is probable that VEGFD produced by RGCs would act primarily on autocrine receptors of
377
RGCs, protects them from NMDA-mediated damage and, consequently, prevents the
378
formation of empty sleeves. The most conclusive piece of evidence, however, derives from
379
our observations that decreasing VEGFR3 expression specifically in RGCs prevents the
380
VEGFD-mediated protection of RGCs, and as a result, leads also to loss of protection of ECs.
381
In conclusion, an insult targeting specifically RGCs is accompanied by a reduction of
382
VEGFD levels in RGCs and, eventually, by damage to the capillaries. Boosting VEGFD
383
levels protects the structure and function of RGCs from the injury and, as a result, spares the
384
capillaries from degeneration. Our findings about the VEGFD-mediated prevention of RGC
385
loss and capillary damage is particularly interesting in light of potential clinical applications
386
targeting CNS diseases in which dysfunctions of the neurovascular system have been
387
implicated. Strategies aiming at enhancing neuronal and capillary survival through VEGFD
388
supplementation may therefore represent approaches for the development of therapies for
389
age- and disease-related dysfunctions of the neurovascular system.
16
43
, VEGFD acts
390 391
Materials and Methods
392
Animals
393
All experiments were performed in accordance with ethical guidelines imposed by the local
394
governing body (Regierungspräsidium Karlsruhe, Germany). Adult 8-week-old male mice of
395
the strain C57BL/6J (Charles River, Sulzfeld, Germany) were used in this study. Mice were
396
housed in groups of maximal 4 animals in the animal facility at the Heidelberg University in
397
standard cages (15 cm×21 cm×13.5 cm) on a normal 12:12 hours light: dark cycle with ad
398
libitum access to water and food. Each cage contained nesting material. Animals were
399
randomly allocated to treatment groups. After intravitreal injections, mice were kept under
400
frequent observation to ensure animal welfare.
401 402
Human retina samples
403
All eyes used in this study were taken under the legal responsibility of the Department of
404
Ophthalmology, Lions Eye Bank (Federal authority Paul-Ehrlich-Institut: PEI.G.11601.01.1)
405
within the facilities of the University Hospital Heidelberg for the purpose of cornea
406
transplantation. Human retina samples were obtained from two cornea donations within 48
407
hours of death after cornea trephination. Informed consent of relatives to use the tissues for
408
scientific purposes was present for all eyes used in this study.
409 410
Intravitreal injections of chemicals and proteins
411
For intravitreal injections, mice were anesthetized by intraperitoneal injection of 0.3 mg/kg
412
BW Medetomidine (Alvetra und Werfft GmbH, Vienna, Austria), 3 mg/kg BW Midazolam
413
(Hameln Pharmaceuticals GmbH, Hameln, Germany) and 0.03 mg/kg BW Fentanyl (Janssen
414
Pharmaceutica, Beerse, Belgium) to induce deep anesthesia. To induce excitotoxicity in the
17
415
retina, 10 nmol NMDA (1 µl of 10 mM, dissolved in sterile 1x PBS; Biotrend,
416
Wangen/Zurich, Switzerland) was injected in one eye with a 32-gauge needle (Nanofil
417
syringe, 10 µl; World Precision Instruments, Sarasota, Florida, USA) under a
418
stereomicroscope. The other eye received the same volume of 1x PBS (vehicle) as a control.
419
After the injection, eyes were treated with eye ointment (Bepanthen; Roche, Basel,
420
Switzerland). Afterwards, anesthesia was antagonized by 450 µg/kg BW Atipamezole
421
(Prodivet pharmaceuticals S.A.; Raeren, Belgium), 0.3 mg/kg BW Flumazenil (Fresenius
422
Kabi, Bad Homburg, Germany) and 0.7 mg/kg BW Narcanti/Naloxone (Inresa Arzneimittel
423
GmbH, Freiburg, Germany). For rVEGFD treatment, 100 ng recombinant mouse VEGFD (1
424
µl of 100 ng/µl, dissolved in sterile 1x PBS; 469-VD-025/CF; R&D Systems, Minneapolis,
425
Minnesota, USA) was co-injected with PBS or NMDA. Control experiments were performed
426
using 100 ng recombinant green fluorescent protein (1 µl of 100 ng/µl, dissolved in sterile 1x
427
PBS; recombinant A. victoria GFP protein, ab116434, Abcam, Cambridge, UK) was co-
428
injected with PBS or NMDA.
429 430
Intravitreal injections of rAAVs
431
2 µl of rAAVs were intravitreally injected prior to the excitotoxic insult. To determine
432
efficient duration of protein expression after transduction, protein products were analyzed via
433
Western blot of retinal lysates. For rAAV-LacZ and rAAV-VEGFD, incubation of 14 days
434
was sufficient to detect expression whereas for rAAV-shSCR and rAAV-shVEGFR3 an
435
incubation of 21 days prior to the insult was necessary. Typical infection rate of the
436
employed rAAV range was >60% of Brn3a-positive RGCs.
437 438
Tissue preparation
18
439
Retinas from mice were dissected 6 hrs, one day (d1) or seven days (d7) after intravitreal
440
injections. For qRT-PCR analysis and Western blot, animals were euthanized with CO2;
441
animals were decapitated and heads were cooled in iced water. For histology, animals were
442
euthanized by intraperitoneal injection with 400 mg/kg BW pentobarbital (Narcoren; Merial
443
GmbH, Hallbergmoos, Germany), followed by a transcardial perfusion with ice-cold 1x PBS
444
and 10% formalin for 5min. Eyes were enucleated and post-fixed for 15 min. In order to
445
dissect the retina, the cornea, iris, lens, and vitreous body were removed under a
446
stereomicroscope. For the preparation of sagittal retina sections, the retina was kept in the
447
surrounding sclera and pigment epithelium. Samples were embedded in Tissue-Tek® (Leica,
448
Wetzlar, Germany) frozen and stored at -80°C until cryo-sectioning. For retina whole mount
449
immunostainings, the retina was isolated from the sclera and pigment epithelium and
450
transferred into PBS. Human retina samples were dissected and shock-frozen in liquid
451
nitrogen for qRT-PCR analysis or post-fixed for 15 min in 10% formalin for whole mount
452
immunostainings.
453 454
Cryo-sectioning
455
Sagittal retina sections of 16 µm and optic nerve sections of 10 µm thickness were obtained
456
with a Leica CM1950 cryostat (Leica) and mounted on superfrost plus slides (Thermo
457
Scientific; Waltham, Massachusetts, USA). Slides were stored at -20°C until analysis.
458 459
TUNEL assay
460
TUNEL staining was done on retinal sections on microscope slides. Sections were
461
permeabilized in 0.1% sodium citrate and 0.5% Triton-X100 for 30 min at room temperature
462
and incubated with TUNEL solution for 1 h at 37°C using the In Situ Cell Death Detection
19
463
Kit, TMR red (Roche, Basel, Switzerland). Nuclei were labeled with Hoechst (1:6000 in 1x
464
PBS, Serva Electrophoresis GmbH, Heidelberg, Germany) for 10 min.
465 466
Immunohistochemistry
467
Retinal sections were immunostained directly on microscope slides. Slides were incubated in
468
0.5% Triton-X100 and 10% FCS in 1x PBS for 1.5 hrs at room temperature. Primary
469
antibodies were diluted in blocking solution and incubated overnight at 4°C in a humidified
470
chamber. For retinal whole mounts, free-floating immunostaining was performed. Retinas
471
were blocked in 1% Triton-X100 and 10% FCS in 1x PBS overnight at 4°C. Primary
472
antibodies were diluted in blocking solution and incubated overnight at room temperature.
473
The following antibodies were used: mouse-anti-Brn3a (1:500; sc-8429, Santa Cruz
474
Biotechnology, Dallas, Texas, USA), rabbit-anti-Collagen-IV (1:500; 2150-1470, Bio-Rad
475
Laboratories, Inc., Hercules, California, USA), mouse-anti-VEGFD (1:100; sc-373866, Santa
476
Cruz Biotechnology), rabbit-anti-SMI-32 (1:1000; 801802, BioLegend, San Diego,
477
California, USA), mouse-anti-HA-probe (Y-11) (1:500; sc-805, Santa Cruz Biotechnology),
478
rabbit-anti-RBPMS (1:250; ab194213, Abcam), rat-anti-TER119 (1:200; MAB1125, R&D
479
Systems, Minneapolis, Minnesota, USA). For VEGFD immunostaining, heat-induced epitope
480
retrieval was performed prior to blocking by incubating the slides in pre-heated sodium
481
citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95°C for 10 min. ECs
482
were labeled with Alexa Fluor 488- or 594-conjugated Isolectin-B4 (1:100; I21411, I21413,
483
Life Technologies, Carlsbad, California, USA). The following secondary antibodies were
484
used: Goat-anti-rabbit IgG (H+L) Alexa Fluor 488 (1:1000; A11008, Life Technologies),
485
goat-anti-mouse IgG (H+L) Alexa Fluor 594 (1:1000; A11005, Life Technologies) and
486
donkey-anti-rat IgG (H+L) Alexa Fluor 488 (1:700; A21208, Life Technologies). Nuclei
20
487
were labeled with Hoechst (1:6000 in 1x PBS, Serva Electrophoresis GmbH, Heidelberg,
488
Germany) for 10 min.
489 490
Transmission Electron Microscopy (TEM)
491
Mice were transcardially perfused with 2.5% glutaraldehyde /2% polyvidone 25 in 0.1 M
492
sodium phosphate buffer, pH 7.4. The eyes were enucleated and the retinas were dissected as
493
described in the section “tissue preparation”. The tissue was processed for TEM as previously
494
published
495
with uranyl acetate, dehydrated with a graded dilution series of ethanol, and embedded into
496
glycid ether 100-based resin. Ultrathin retinal cross sections of 85 nm were cut with a
497
Reichert Ultracut S ultramicrotome (Leica Microsystems) and contrasted with uranyl acetate
498
and lead citrate. Sections were examined in an electron microscope (Zeiss EM 10 CR) at an
499
acceleration voltage of 60 kV. The representative electron microscopy pictures derive from
500
the same mouse but two mice were prepared for analysis.
88, 89
. Retinas were post-fixed with 1% OsO4, 1.5% K4Fe(CN)6, contrasted en bloc
501 502
bEND.3 cells
503
bEND.3 cells were cultured in high-glucose-containing (4.5 g/l) Dulbecco’s Modified Eagle
504
Medium (DMEM; Life Technologies, Carlsbad, California, USA) supplemented with 10%
505
FBS, 100 units/ml penicillin and 100 µg/ml streptomycin (Sigma, Kawasaki, Kanagawa,
506
Japan) on 0.1% gelatin-coated flasks. For passaging, cells were washed with PBS and
507
incubated with Trypsin (#25300-054; Gibco, Life Technologies, Carlsbad, California, USA)
508
for 3 min. Cells were resuspended in medium and centrifuged at 800xg for 3 min. The cell
509
pellet was resuspended in cell culture medium and cells were seeded at an appropriate
510
dilution according to their growth rate. For bEND.3 mortality assay, cells were treated with
511
20 µM NMDA or 1 mM NMDA for 10 min and 24 hrs. Untreated cells served as controls.
21
512
After 24 hrs, cells were fixed for 10 min with 4% PFA and labeled with Hoechst for cell
513
survival analysis. Hoechst-labeled cells were automatically counted in 16 images out of 4
514
coverslips using a self-written macro in ImageJ/Fiji. Healthy nuclei were differentiated from
515
fragmented or condensed nuclei by their morphology.
516 517
Primary astrocyte cultures
518
Neurons and glial cells from new born C57BL/6N mice were isolated as described
519
Medium was changed after 2.5 hrs from plating with DMEM supplemented with 10% FCS
520
and Pen-Strep. Every 3 days, cultures were washed with ice-cold PBS to promote death and
521
detachment of neuronal cells, and growth of astrocytes. When confluence was reached,
522
experiments were performed. Cultures were treated for 24 hrs with 20 µM NMDA and then
523
either harvested for qRT-PCR analysis or fixed for immunocitochemistry. We confirmed the
524
exclusive presence of glia cells using the marker GFAP (1:500; 3670, Cell Signaling
525
Technology, Danvers, Massachusetts, USA). Hoechst labeling was used to count and assess
526
cellular vitality via nuclear morphology.
49
.
527 528
Recombinant adeno-associated viruses
529
The method used to construct, package, and purify recombinant adeno-associated viruses
530
(rAAVs) has been previously described 90, 91. Recombinant adeno-associated viruses (rAAVs)
531
serotype 1/2 were produced by co-transfection of HEK293 cells by standard calcium
532
phosphate precipitation. Before transfection, culture medium was replaced with fresh
533
modified Dulbecco’s medium (Iscove’s Modified Dulbecco's Medium; Life Technologies,
534
Carlsbad, California, USA) containing 5% FBS without antibiotics. Packaging of rAAVs was
535
carried out with helper plasmids pF∆6, pRV1, and pH21 together with either pAAV-VEGFD,
536
pAAV-LacZ, pAAV-shVEGFR3 or pAAV-shSCR. After transfection, the medium was
22
537
replaced with fresh DMEM containing 10% FBS and antibiotics. Cells were collected at low
538
speed centrifugation, resuspended in 150 mM NaCl-10 mM Tris-HCl (pH 8.5) and lysed by
539
incubation with 0.5% sodium deoxycholate followed by freeze-thaw cycles. rAAVs were
540
purified using heparin affinity columns (HiTrap Heparin HP; GE Healthcare, Chicago,
541
Illinois, USA). rAAVs stocks were concentrated using Amicon Ultra-4 centrifugal filter
542
devices (Merck Millipore, Burlington, Massachusetts, USA).
543
The rAAVs used in this work have been previously extensively characterized in vitro and in
544
vivo for their specificity and efficacy
545
or FLAG-tag, respectively, allowing for detection of infected cells. rAAV-shVEGFR3 and
546
rAAV-shSCR carry an additional cassette for mCherry expression for rapid and specific
547
detection of the infected cells. The transduction efficiency was calculated as percentage of
548
Brn3a+ cells also positive for the transgene expression. The virus batches used for the
549
experiments had a titre of: rAAV-LacZ 6*1011 molecules/ml; rAAV-VEGFD 3*1011
550
molecules/ml, which had, however, identical infection efficiency in both cultured neurons
551
and in vivo in RGCs. For the shRNA constructs, the virus batches used for the experiments
552
had a titre of: rAAV-shSCR 6*1012 molecules/ml; rAAV-shVEGFR3 2*1012 molecules/ml,
553
which had, however, identical infection efficiency in both cultured neurons and in vivo in
554
RGCs.
555
The number of mice and eyes injected with rAAV-LacZ was 9 mice / 18 eyes (9 eyes were
556
injected with PBS, 9 eyes were injected with NMDA and of each experimental group 6 eyes
557
were co-stained for RGCs and empty sleeves quantification while 3 only for RGCs
558
quantification). The number of mice and eyes injected with rAAV-VEGFD was 9 mice / 18
559
eyes (9 eyes were injected with PBS, 9 eyes were injected with NMDA and of each
560
experimental group 6 eyes were co-stained for RGCs and empty sleeves quantification while
561
3 only for RGCs quantification). The number of mice and eyes treated with rAAV-shSCR
43, 50, 51
. rAAV-VEGFD and rAAV-LacZ carry an HA-
23
562
was 16 mice / 32 eyes for quantification of RGC survival (8 eyes were injected with PBS, 8
563
eyes were injected with NMDA, 8 eyes were injected with rVEGFD, 8 eyes were injected
564
with NMDA+rVEGFD) and 12 mice / 24 eyes for empty sleeves analysis (6 eyes were
565
injected with PBS, 6 eyes were injected with NMDA, 6 eyes were injected with rVEGFD, 6
566
eyes were injected with NMDA+rVEGFD). The number of mice and eyes treated with
567
rAAV-shVEGFR3 was 16 mice / 32 eyes for quantification of RGC survival (8 eyes were
568
injected with PBS, 8 eyes were injected with NMDA, 8 eyes were injected with rVEGFD, 8
569
eyes were injected with NMDA+rVEGFD) and 12 mice / 24 eyes for empty sleeves analysis
570
(6 eyes were injected with PBS, 6 eyes were injected with NMDA, 6 eyes were injected with
571
rVEGFD, 6 eyes were injected with NMDA+rVEGFD).
572 573
qRT-PCR
574
Total RNA was extracted from bEnd.3 cells, primary astrocytes, mouse or human retinas
575
using the RNeasy Mini Kit (Qiagen, Hilden, Germany) including an optional DNase I
576
treatment at room temperature for 15 min according to manufacturer’s instructions (Qiagen).
577
Extracted RNA was reverse transcribed into first-strand cDNA using High Capacity cDNA
578
Reverse Transcription kit (Applied Biosystems, Foster City, California, USA). Quantitative
579
reverse transcriptase PCR (qRT-PCR) was performed on a StepOne plus Real Time PCR
580
system using TaqMan Gene Expression Assays for the indicated genes (Applied Biosystems).
581
The following TaqMan Gene Expression Assays were used in this study: Thy1
582
(Mm01174153_m1),
nefl
(Mm01315666_m1),
583
(Mm00727012_m1),
tjp1
Mm00493699_s1),
584
(Mm01202432_m1),
vegf
585
(Mm00438980_m1), kdr (Mm00440099_m1). Expression of target genes was normalized
586
against the expression of Gapdh (Mm99999915_g1) or, in the case of bEnd.3 cells, Gusb
(Mm01281449_m1),
24
ocld vegfd flt4
(Mm01282968_m1), (Mm00438963_m1), (Mm01292618_m1),
cld5 vegfc flt1
587
(Mm00446953_m1), which were used as endogenous control genes. For detection of VEGFD
588
in human retina, the following probes were used: vegfd (Hs01128659_m1), vegf
589
(Hs00900055_m1), actb (Hs99999903_m1). For each treatment, 6 animals, 3 technical
590
replicates or 4 human retinas from 2 donors were analyzed.
591 592
Western blot analysis
593
bEnd.3 cells were homogenized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.57%
594
sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8, 1x CompleteTM cocktail of protease
595
inhibitors; Roche, Basel, Switzerland). Homogenates centrifuged with 13000rpm for 10min.
596
Supernatants were mixed with 4x Laemmli sample buffer (200 mM Tris/HCl pH 6.8, 8%
597
SDS, 40% Glycerol, 50 mM EDTA, 0.08% bromophenol blue) and 1% dithiothreitol. Lysates
598
were heated at 95°C for 10 min before performing standard SDS-PAGE and western blotting.
599
The following primary antibodies were used: rabbit-anti-GAPDH (1:20000 in 5% BSA;
600
2118, Cell signaling Technology), rabbit anti-GluN1 (1:2000 in 5% milk; 5704, Cell
601
signaling Technology), rabbit-GluN2A (1:500 in 5% milk; AB1555, Merck Millipore), rabbit
602
anti-GluN2B (1:1000 in 5% milk; NB300-106, Novus Biologicals, Cenennial, Colorado,
603
USA), rabbit anti-GluN2C (1:1000 in 5% milk; PAB9705, Abnova, Taiwan, China), rabbit
604
anti-GluN2D (1:1000 in 5% milk; AP21181PU-N, Acris OriGene, Herford, Germany). The
605
following secondary antibody was used: Peroxidase AffiniPure goat anti-rabbit IgG (H+L)
606
(1:5000 in 5% milk; 111-035-144, Jackson Immuno Research Laboratories Inc., West Grove,
607
Pennsylvania, USA). Membranes were developed with Enhanced Chemiluminescence (ECL)
608
solution (GE Healthcare, Chicago, Illinois, USA) and the ChemiDoc Imaging System (Bio-
609
Rad Laboratories).
610 611
Analysis of BRB functionality
25
612
For extravasation experiments, animals received intravenous injection of fluorescein-
613
conjugated dextran (3 kDa, 50 µg/µl in 1x PBS, 50 µg/g BW; D3306; Thermo Scientific).
614
After 15 min, mice were euthanized by intraperitoneal injection with 400 mg/kg BW
615
pentobarbital (Narcoren; Merial GmbH, Hallbergmoos, Germany), followed by a transcardial
616
perfusion with 10 ml ice-cold 1x PBS to clear the remaining intravascular dextran. A blood
617
sample, serving as positive control, was collected immediately before perfusion. The blood
618
sample was centrifuged at 15000xg for 5 min at 4°C, and the supernatant was frozen at -20°C
619
until measurements. Blood sera were diluted 1:100 and 1:1000. Immediately after perfusion,
620
retinas were dissected, weighed, and homogenized in 100 µl ice-cold 1x PBS. The extracts
621
were centrifuged at 10000xg for 15 min at 4°C. The supernatant was kept undiluted or was
622
diluted 1:100. The fluorescence in each 100 µl sample was measured (excitation, 492 nm;
623
emission, 521 nm) using a spectrofluorometer (SpectraMax M5; Molecular Devices, LLC,
624
San José, California, USA) with 1x PBS as a blank. Retinal or serum autofluorescence was
625
corrected based on not-injected controls. For normalization, the corrected retinal fluorescence
626
was divided by the retinal weight and by the corrected serum fluorescence, with the results
627
being expressed in relative fluorescence unit (RFU).
628
For extravasation analysis of albumin or IgG via western blotting 30, animals were euthanized
629
as described above, followed by a transcardial perfusion with 10 ml ice-cold 1x PBS to
630
reduce blood contaminants. Eyes were enucleated, retinas were immediately dissected under
631
a stereomicroscope and shock-frozen in liquid nitrogen. Retinas were processed for standard
632
Western blot analysis. Western blotting was performed as described above. As controls,
633
recombinant albumin (45µg, 8076, Roth, Karlsruhe, Germany), mouse IgG (9 µg; sc-2025,
634
Santa Cruz Biotechnology), and retinal lysates from non-perfused animals were loaded. The
635
following primary and secondary antibodies were used to detect endogenous, extravasated
636
albumin and IgG: goat anti-mouse serum albumin (1:1000; ab19194, Abcam), Peroxidase
26
637
AffiniPure goat anti-mouse IgG (H+L) (1:5000 in 5% milk; 705-035-147, Jackson Immuno
638
Research Laboratories Inc.). For protein expression analysis, relative protein band densities
639
were calculated by normalizing to the loading control (GAPDH) using ImageJ/Fiji. For each
640
time point d1 and d7, relative protein levels of the NMDA-treated eyes were normalized to
641
relative protein levels of the PBS control eye of the same animal. For all image analyses,
642
backgrounds were subtracted. For each treatment, 3 animals were analyzed.
643 644
Image acquisition
645
For each analysis, images were acquired with constant microscopy settings (exposure time,
646
pixel size, bit depth, binning) across experiments. For fluorescence intensity analysis, all
647
images were acquired on the same day to avoid possible variations in fluorescence lamp
648
excitation. Images of sagittal retina sections and primary astrocytes were acquired with Leica
649
DM IRBE inverted fluorescent microscope with a 40x oil immersion objective (Leica) and a
650
Spot Insight FireWire 2 camera with VisiView software (Visitron Systems GmbH,
651
Diagnostic Instruments, Puchheim, Germany). Images of mouse and human retina whole
652
mounts were acquired with a Nikon A1R confocal laser scanning microscope (at the Nikon
653
Imaging Center of Heidelberg University, Germany) with a 63x oil immersion objective
654
(Nikon, Minato, Tokio, Japan) or with a Leica SP8 confocal laser scanning microscope with a
655
63x oil immersion objective (Leica).
656 657
Quantification of RGCs
658
For RGC number quantification in retinal whole mounts, Brn3a-positive cells were manually
659
counted in 8 images per retina equally acquired in the central and peripheral part of the retina
660
using ImageJ/Fiji (see schema Fig. 1B). For each eye, RGC number was summed. Relative
661
RGC numbers were calculated by normalizing RGC number in the NMDA-treated eye to the
27
662
number of RGCs in the PBS-treated eye of the same animal. For each treatment, 6 animals
663
were analyzed.
664 665
INL Analysis
666
For INL analysis, the thickness of the INL was measured at 5 different positions across the
667
INL in 3 sagittal retinal sections of 10 µm thickness using ImageJ/Fiji. For each section,
668
values were averaged and converted into µm taking the microscope resolution into account.
669
For each treatment, 3 animals were analyzed.
670 671
Quantification of axonal damage
672
For axonal damage analysis, the area of SMI-32-positive clusters was quantified in 3-4
673
sagittal optic nerve sections of 10 µm thickness using ImageJ/Fiji. For each optic nerve,
674
cluster area was summed and normalized to the analyzed area. Relative cluster areas were
675
calculated by normalizing cluster area in the NMDA-treated optic nerve to the cluster area in
676
the PBS-treated optic nerve of the same animal. For each treatment, 6 animals were analyzed.
677 678
Quantification of vascular injury
679
For vascular injury analysis in retinal whole mounts, the number of regressed vessels – so
680
called-empty sleeves, which represent collapsed Collagen-IV-positive / Isolectin-B4-negative
681
vessels – was quantified using ImageJ/Fiji. For each eye, empty sleeves number was
682
summed. Relative empty sleeves numbers were calculated by normalizing number of empty
683
sleeves in the NMDA-treated eye to number of empty sleeves in the PBS-treated eye of the
684
same animal. For each treatment, 6 animals were analyzed.
685 686
Quantification of vessel architecture
28
687
For vessel architecture in retinal whole mounts, the isosurface of Collagen-IV
688
immunolabeled vessels was measured with ImageJ/Fiji and the plugin BoneJ. Vessel
689
diameter and length were measured with ImageJ/Fiji and the plugin DiameterJ. Relative
690
values were calculated by normalizing the mean values in the NMDA-treated eye to mean
691
values in the PBS-treated eye of the same animal. For each treatment, 6 animals were
692
analyzed.
693 694
Analysis of VEGFD expression
695
Somatic levels of VEGFD in sagittal retinal sections were analyzed as previously published
696
92
697
drawing regions of interests around the ganglion cell layer in 4-5 images, each corresponding
698
to one individual 16 µm sagittal retina section using ImageJ/Fiji. For each eye/condition,
699
mean integrated densities were calculated out of the single fluorescence values within each
700
sagittal retinal section. The mean integrated density of theNMDA-treated eye was then
701
normalized to the mean integrated densityof the PBS-treated eye of the same animal.
702
Background subtraction was performed prior to all analyses. For negative controls, a
703
blocking peptide (sc-373866, Santa Cruz Biotechnology) was pre-absorbed with the primary
704
antibody in a five-fold excess (by weight) resulting in absence of specific immunolabeling of
705
the primary antibody. For each treatment, n=3-4 animals were analyzed.
. Fluorescent signals were measured in cells located in the ganglion cell layer by manually
706 707
Electroretinography
708
Mice were anaesthetized with ketamine (150 mg/kg; Atarost GmbH and Co., Twistringen,
709
Germany) and xylazine (10 mg/kg; Albrecht, Aulendorf, Germany). ERG recordings were
710
recorded using the UTAS Visual Diagnostic System (LKC Technologies, Gaithersburg, MD,
711
USA) using a contact lens electrode with gold contact (LKC Technologies). The electrical
29
712
contact, as well as prevention of eye desiccation, was facilitated through application of
713
Liquifilm O.K. (Allergen, Westport, Ireland). Reference and ground needle electrode were
714
placed subcutaneously in the neck and tail, respectively. A heating pad controlled by rectal
715
temperature probe was maintained at 37oC. Mice were centered at a distance of 15 cm in
716
front of a 17-inch cathode ray tube monitor. In order to achieve maximum focus of the
717
pattern stimulation, pupils were not dilated. Vertical square wave grating stimulations (66%
718
contrast, temporal frequency of 1 Hz – equivalent to 2 pattern reversals per second) were
719
presented at different spatial frequencies (0.05, 0.10, and 0.20 cyc/deg), and noise levels were
720
measured with the monitor turned off. Due to the small amplitudes of pattern ERGs, 250
721
sweeps were averaged to increase the signal-to-noise ratio. Responses were sampled at
722
1000x, band-pass filtered between 0.3 and 300 Hz, and amplified (64 x gain). The amplitude
723
of the second harmonic (2 Hz) was analyzed with the fast Fourier Transformation function
724
using the EMWin software provided with the UTAS visual diagnostic system.
725 726
Statistical analysis
727
Mean values and standard error of the mean (SEM) were calculated in Excel (Microsoft),
728
plotted and analyzed in GraphPad Prism 7 software (GraphPad Software, Inc.). Values were
729
tested for normal distribution by performing Shapiro-Wilk normality test. Unpaired t-test,
730
one-way or two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test for
731
multiple comparisons were used. Details on the test used and N of each experiment are
732
indicated in the respective figure legends. Bars represent mean values. Error bars indicate
733
SEM. Asterisks indicate significant differences (****p<0.0001, ***p<0.001, **p<0.01,
734
*p<0.05).
735 736
30
737
Author Contributions
738
Conceptualization, D.M.; Methodology, A.S., D.M.; Formal Analysis, A.S., B.A., R.F.,
739
D.M.; Investigation, A.S., B.A., R.F, D.M.; Writing – Original Draft, A.S., D.M.; Writing –
740
Review & Editing, A.S., B.A., R.F., R.D., D.M.; Visualization, A.S., D.M.; Supervision,
741
A.S., D.M.; Project Administration, D.M., Funding Acquisition, D.M.
742 743
Acknowledgments
744
The authors wish to thank Dr. Patrick Merz from the Department of Ophthalmology, Lions
745
Eye Bank, Heidelberg University Hospital, for providing human retina samples. The authors
746
are extremely grateful towards Andrea Hellwig for providing the electron microscopy images
747
shown in Figure 2. The authors additionally thank Eva Bindewald and Viktoria Greeck for
748
technical assistance, Iris Bünzli-Ehret and Dr. Anna M. Hagenston for their help with the
749
preparation of astrocytic primary cultures, Dr. Carmen Ruiz de Almodovar for kindly
750
providing the TER119 antibody.
751
DM is a member of the Excellence Cluster CellNetworks at Heidelberg University. Part of the
752
pictures was acquired at the Nikon Imaging Center at Heidelberg University. We also
753
acknowledge support from the Interdisciplinary Neurobehavioral Core (INBC) at Heidelberg
754
University. This work was supported by the FOR2325 grant (project 2) and SFB1158 (project
755
A08) of the Deutsche Forschungsgemeinschaft (DFG) to DM, the FRONTIER grant of
756
Heidelberg University to DM.
757 758
Conflicts of interest statement
759
DM is one of the founders and shareholders of FundaMental Pharma GmbH.
760 761
31
762
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763 764
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38
1022
Figure legends
1023
Figure 1: NMDA-triggered excitotoxicity induces RGC death.
1024
A) Representative images of retinal whole mounts at d1 and d7 after intravitreal injections of
1025
NMDA or PBS. RGCs were immunolabeled using an antibody against Brn3a (green), nuclei
1026
were labeled with Hoechst (blue). Scale bar=20 µm. B) Schematic of the fields used for
1027
quantification in retinal whole mounts in central and peripheral parts of the retina. C)
1028
Quantification of RGCs in retinas injected as indicated. Unpaired t-test. n=6. PBS vs NMDA
1029
(d1), p=0.0274; PBS vs NMDA (d7), p=0.0038. D) Quantification of RGCs in retinas
1030
injected as indicated. RGC numbers in the NMDA-treated eye were normalized to the PBS
1031
control eye from the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1), p=0.0318; PBS
1032
vs NMDA (d7), p=0.0051. E) QRT-PCR analysis of thy1 and nefl expression in retinas one
1033
day after intravitreal injection of NMDA. Unpaired t-test. n=6. Thy1, p=0.0002; nefl,
1034
p=0.0002. F) Quantification of INL thickness in sagittal retina sections injected as indicated.
1035
Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.4015; PBS vs NMDA (d7), p=0.8741. Bars
1036
represent mean ± SEM. Dots represent single values. ***p<0.001, **p<0.01, *p<0.05.
1037 1038
Figure 2: Intravitreal injection of NMDA induces formation of empty sleeves via an
1039
indirect mechanism.
1040
A) Representative images of retinal whole mounts at d7 after intravitreal injections of NMDA
1041
or PBS. Vessels were immunolabeled with antibodies against Collagen-IV (green) and
1042
Isolectin-B4 (red). Arrows indicate empty sleeves. Scale bar=20 µm. B) Quantification of
1043
empty sleeves in retinas injected as indicated. Unpaired t-test. n=6. PBS vs NMDA (d1),
1044
p=0.0129; PBS vs NMDA (d7), p<0.0001. C) Quantification of empty sleeves in retinas
1045
injected as indicated. Number of empty sleeves in the NMDA-treated eye were normalized to
1046
the PBS control eye from the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1),
39
1047
p<0.0001; PBS vs NMDA (d7), p=0.0002. D) Representative images of electron microscopy
1048
of retinas injected as indicated. Left: Normal capillary in retinas of PBS-injected eyes with
1049
ECs (e), tight junctions (tj), and pericyte (p). Scale bar=5000 nm. Right: Capillary with
1050
swollen EC (e) in retinas of NMDA-injected eyes. Asterisks indicate occluded lumen. Tight
1051
junctions (tj) and pericytes (p) were still intact. Scale bar=500 nm. E) Quantification of vessel
1052
surface area in retinas injected as indicated. Surface area in the NMDA-treated eye was
1053
normalized to the PBS control eye from the same animal. Unpaired t-test. n=6. PBS vs
1054
NMDA (d1), p=0.0626; PBS vs NMDA (d7), p=0.5847. F) Quantification of vessel diameter
1055
in retinas injected as indicated. Diameter in the NMDA-treated eye was normalized to the
1056
PBS control eye from the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1), p=0.5313;
1057
PBS vs NMDA (d7), p=0.3695. G) Quantification of vessel fiber length in retinas injected as
1058
indicated. Fiber length in the NMDA-treated eye was normalized to the PBS control eye from
1059
the same animal. Unpaired t-test. n=6. PBS vs NMDA (d1), p=0.2375; PBS vs NMDA (d7),
1060
p=0.6235. H) QRT-PCR analysis of ocld, cld5 and tjp1 expression in retinas of mice seven
1061
days after intravitreal injection of PBS and NMDA. Unpaired t-test. n=6. ocld, p=0.2803;
1062
cld5, p=0.8959; tjp1, p=0.1733. I) Representative images of retinal whole mounts at d7 after
1063
intravitreal injections of NMDA or PBS. Vessels were immunolabeled with antibodies
1064
against Collagen-IV (red) and erythrocytes with antibodies against TER119 (green). Scale
1065
bar=50 µm. J) Quantification of vascular erythrocytes in retinas injected as indicated and
1066
expressed as percentage of the total N of erythrocytes present in the analyzed images (within
1067
and outside vessels). Unpaired t-test. n=4. PBS vs NMDA (d7), p=0.088. K) Quantification
1068
of dextran-fluorescein (3 kDa) extravasation in retinal extracts one and seven days after
1069
intravitreal PBS and NMDA injections. Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.3799;
1070
PBS vs NMDA (d7), p=0.6891. L) Representative immunoblots of albumin, IgG and
1071
GAPDH in retinas of unperfused mice (control) or in retinas of perfused mice one and seven
40
1072
days after intravitreal PBS and NMDA injections. M) Quantification of albumin levels in
1073
retinal lysates injected as indicated. GAPDH was used for control of the amount of protein.
1074
Samples from NMDA-treated eye were normalized to the PBS control eye from the same
1075
animal. Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.4845; PBS vs NMDA (d7), p=0.5231.
1076
N) Quantification of IgG levels in retinal lysates injected as indicated. GAPDH was used for
1077
control of the amount of protein. Samples from NMDA-treated eye were normalized to the
1078
PBS control eye from the same animal. Unpaired t-test. n=3. PBS vs NMDA (d1), p=0.7794;
1079
PBS vs NMDA (d7), p=0.5372. O) Representative immunoblots of different NMDAR
1080
(GluN) subunits and GAPDH in brain ECs (bEnd.3, left lane) and primary hippocampal
1081
neurons (right lane). P) Quantification of cultured brain ECs (bEnd.3) survival after 20 µM
1082
NMDA exposure in vitro for 10 min or 24 hrs. The number of living bEnd.3 cells was
1083
normalized to the number of total bEnd.3 cells as well as to the ratio of living/total cells in
1084
the control (untreated). One-way ANOVA, Bonferroni. n=3. Untreated vs NMDA 10 min,
1085
p>0.9999. Untreated vs NMDA 24 hrs, p>0.9999. NMDA 10 min vs NMDA 24 hrs,
1086
p>0.9999. Q) QRT-PCR analysis of ocld, cld5 and tjp1 expression in bEnd.3 with or without
1087
NMDA treatment. Unpaired t-test. n=6. ocld, p=0.7118; cld5, p=0.1998, tjp1, p=0.4434. R)
1088
Quantification of cultured primary astrocyte survival after 20 µM NMDA exposure for 24
1089
hrs. The number of living astrocytes was normalized to the number of total astrocytes as well
1090
as to the ratio of living/total astrocytes in the control (untreated). Unpaired t-test. n=3.
1091
Untreated vs NMDA 24 hrs, p=0.7016. Bars represent mean ± SEM. Dots represent single
1092
values. ****p<0.0001, *p<0.05.
1093 1094
Figure 3: NMDA-triggered excitotoxicity reduces VEGFD expression in RGCs.
1095
A) QRT-PCR analysis of vegfd, vegfc, vegfa, flt4, flt1, and kdr expression in retinal
1096
homogenates after intravitreal injection of NMDA. Unpaired t-test. n=6. vegfd, p=0.0085;
41
1097
vegfc, p=0.2052; vegfa, p=0.8524; flt4, p=0.9414; flt1, p=0.6074; kdr, p=0.2016. B)
1098
Representative images of human retinal whole mounts. Retinas were immunolabeled with
1099
antibodies against Bn3a (red) and VEGFD (green). Scale bars=10 µm. Negative controls of
1100
human retinas were immunolabeled only with secondary antibodies. C) Representative bands
1101
of VEGFD, VEGFA, and ß-actin cDNA products following qRT-PCR analysis of human
1102
retinal extracts obtained from two donors (subject 1, subject 2). D) Quantification of VEGFD
1103
protein levels in cells in the ganglion cell layer of retinas of mice injected as indicated.
1104
Unpaired t-test. n=3. PBS vs NMDA, p=0.0192. E) Representative images of mouse sagittal
1105
retina sections at d7 after intravitreal injections of NMDA or PBS. Retinas were
1106
immunolabeled for VEGFD (green). Nuclei were labeled with Hoechst (blue). GCL:
1107
Ganglion cell layer. Scale bar=20 µm. F) QRT-PCR analysis of vegfd, vegfc, vegfa, and flt4
1108
expression in brain endothelial cells (b.END3) 24 hrs after 20 µM NMDA treatment.
1109
Unpaired t-test. n=3. vegfd, p=0.1744; vegfc, p=0.7598; vegfa, p=0.8998; flt4, p=0.4457. G)
1110
QRT-PCR analysis of vegfd, vegfc, vegfa, and flt4 expression in primary astrocytes 24 hrs
1111
after 20 µM NMDA treatment. Unpaired t-test. n=3. vegfd, p=0.8716; vegfc, p=0.4918; vegfa,
1112
p=0.9902; flt4, p=0.9331. Bars represent mean ± SEM. Dots represent single values.
1113
**p<0.01.
1114 1115
Figure 4: VEGFD overexpression in RGCs protects them from NMDA-mediated
1116
damage. A) Representative images of retinal whole mounts at d7 after intravitreal injections
1117
of NMDA or PBS from mice injected either with rAAV-LacZ or rAAV-VEGFD as indicated.
1118
RGCs were immunolabeled against Brn3a (green). Scale bar=15 µm. B) Representative
1119
images of sagittal optic nerve sections injected as indicated. Stressed axons were
1120
immunolabeled using SMI-32 (green). Nuclei were labeled using Hoechst (blue). Scale
1121
bar=50 µm. C) Quantification of RGCs injected as indicated. Two-way ANOVA, Bonferroni.
42
1122
n=9. rAAV-LacZ+PBS vs rAAV-LacZ+NMDA, p<0.0001; rAAV-VEGFD+PBS vs rAAV-
1123
VEGFD+NMDA, p=0.5035. rAAV-LacZ+PBS vs rAAV-VEGFD+PBS, p>0.9999 rAAV-
1124
LacZ+NMDA vs rAAV-VEGFD+NMDA, p=0.0006. D) Quantification of RGCs injected as
1125
indicated. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye
1126
from the same animal. n=9. E) Quantification of SMI-32+ clusters in optic nerves from eyes
1127
injected as indicated. Two-way ANOVA, Bonferroni. n=6. rAAV-LacZ+PBS vs rAAV-
1128
LacZ+NMDA, p<0.0001; rAAV-VEGFD+PBS vs rAAV-VEGFD+NMDA, p=0.7794;
1129
rAAV-LacZ+PBS vs rAAV-VEGFD+PBS, p>0.9999; rAAV-LacZ+NMDA vs rAAV-
1130
VEGFD+NMDA, p<0.0001. F) Quantification of SMI-32+ clusters in optic nerves from eyes
1131
injected as indicated. SMI-32+ clusters in the NMDA-treated eye were normalized to the PBS
1132
control eye from the same animal. n=6. Bars represent mean ± SEM. Dots represent single
1133
values. ****p<0.0001, ***p<0.001. See also Figure S1.
1134 1135
Figure 5: Recombinant VEGFD protects RGCs from NMDA-mediated damage.
1136
A) Representative images of retinal whole mounts at d7 after intravitreal injections of PBS,
1137
NMDA, rVEGFD or rVEGFD+NMDA as indicated. RGCs were immunolabeled against
1138
Brn3a (green). Nuclei were immunolabeled with Hoechst (blue). Scale bar=40 µm. B)
1139
Representative images of sagittal optic nerve sections injected as indicated in A. Stressed
1140
axons were immunolabeled using SMI-32 (green). Nuclei were labeled using Hoechst (blue).
1141
Scale bar=50 µm. C) Quantification of RGCs at d1 after intravitreal injections, injected as
1142
indicated in A. Two-way ANOVA, Bonferroni. n=6. PBS vs NMDA (sham), p=0.0002; PBS
1143
vs NMDA (rVEGFD), p>0.9999; sham vs rVEGFD (PBS), p>0.9999; sham vs rVEGFD
1144
(NMDA), p=00013. D) Quantification of RGCs at d7 after intravitreal injections, injected as
1145
indicated in A. Two-way ANOVA, Bonferroni. n=6. PBS vs NMDA (sham), p=0.0012; PBS
1146
vs NMDA (rVEGFD), p=0.0973; sham vs rVEGFD (PBS), p>0.9999; sham vs rVEGFD
43
1147
(NMDA), p=0.1102. E) Quantification of RGCs at d7 after intravitreal injections, injected as
1148
indicated in A. RGC numbers in the NMDA-treated eye were normalized to the PBS control
1149
eye from the same animal. n=6. F) Quantification of SMI-32+ clusters in optic nerves at d7
1150
after intravitreal injections, injected as indicated in A. Two-way ANOVA, Bonferroni. n=6.
1151
PBS vs NMDA (sham), p=0.0012; PBS vs NMDA (rVEGFD), p=0.6742; sham vs rVEGFD
1152
(PBS), p>0.9999; sham vs rVEGFD (NMDA), p=0.0047. G) Quantification of SMI-32+
1153
clusters in optic nerves from eyes at d7 after intravitreal injections, injected as indicated in A.
1154
SMI-32+ clusters in the NMDA-treated eye were normalized to the PBS control eye from the
1155
same animal. n=6. H) Quantification of RGCs at d1 after intravitreal injections of PBS,
1156
NMDA, rGFP or rGFP+NMDA. Two-way ANOVA, Bonferroni. n=6. PBS vs NMDA
1157
(sham), p=0.0013; PBS vs NMDA (rVEGFD), p=0.0059; sham vs rVEGFD (PBS),
1158
p>0.9999; sham vs rVEGFD (NMDA), p=0.8657. I) Quantification of RGCs at d7 after
1159
intravitreal injections, injected as indicated in H. Two-way ANOVA, Bonferroni. n=6. PBS
1160
vs NMDA (sham), p<0.0001; PBS vs NMDA (rGFP), p<0.0001; sham vs rGFP (PBS),
1161
p>0.9999; sham vs rGFP (NMDA), p>0.9999. J) Quantification of RGCs at d7 after
1162
intravitreal injections, injected as indicated in H. RGC numbers in the NMDA-treated eye
1163
were normalized to the PBS control eye from the same animal. n=6. K) Quantification of
1164
SMI-32+ clusters in optic nerves at d7 after intravitreal injections, injected as indicated in H.
1165
Two-way ANOVA, Bonferroni. n=5. PBS vs NMDA (sham), p=0.0036; PBS vs NMDA
1166
(rGFP), p=0.0041; sham vs rGFP (PBS), p>0.9999; sham vs rGFP (NMDA), p>0.9999. L)
1167
Quantification of SMI-32+ clusters in optic nerves from eyes at d7 after intravitreal injections,
1168
injected as indicated in H. SMI-32+ clusters in the NMDA-treated eye were normalized to the
1169
PBS control eye from the same animal. n=6. Bars represent mean ± SEM. Dots represent
1170
single values. ****p<0.0001, ***p<0.001, **p<0.01. See also Figure S2.
1171
44
1172
Figure 6: Recombinant VEGFD prevents decrease of pattern electroretinography
1173
amplitudes after NMDA-triggered damage to RGCs.
1174
A) Representative images of retinal whole mounts at d7 after intravitreal injections of PBS,
1175
NMDA, rVEGFD or rVEGFD+NMDA. RGCs were immunolabeled against Brn3a. Scale
1176
bar=20 µm. B) Amplitude of the second harmonic of the Fast Fourier Transformation plotted
1177
against spatial frequency of stimulation. Asterisks indicate statistical significance between
1178
NMDA- and rVEGFD+NMDA-treated animals. Two-way ANOVA, Bonferroni. n=4. PBS vs
1179
NMDA (0.05 cyc/deg), p=0.6877; PBS vs NMDA (0.10 cyc/deg), p=0.0076; PBS vs NMDA
1180
(0.20 cyc/deg), p=0.0964; PBS vs NMDA (noise), p=0.8182; PBS vs rVEGFD (0.05
1181
cyc/deg), p=0.9669; PBS vs rVEGFD (0.10 cyc/deg), p=0.9140; PBS vs rVEGFD (0.20
1182
cyc/deg), p=0.5811; PBS vs rVEGFD (noise), p=0.9509; rVEGFD vs rVEGFD+NMDA (0.05
1183
cyc/deg), p=0.4433; rVEGFD vs rVEGFD+NMDA (0.10 cyc/deg), p=0.9976; rVEGFD vs
1184
rVEGFD+NMDA (0.20 cyc/deg), p=0.8797; rVEGFD vs rVEGFD+NMDA (noise),
1185
p=0.9980;
1186
rVEGFD+NMDA (0.10 cyc/deg), p=0.0296; NMDA vs rVEGFD+NMDA (0.20 cyc/deg),
1187
p=0.2776; NMDA vs rVEGFD+NMDA (noise), p=0.9980. C) Representative pattern
1188
electroretinography waveforms recorded from mice injected as indicated in A. Bars represent
1189
mean ± SEM. Dots represent single values. **p<0.01, *p<0.05.
NMDA
vs
rVEGFD+NMDA
(0.05
cyc/deg),
p=0.0144;
NMDA
vs
1190 1191
Figure 7: VEGFD reduces formation of empty sleeves triggered by intravitreal NMDA
1192
injection
1193
A) Representative images of retinal whole mounts at d7 after intravitreal injections of NMDA
1194
or PBS from mice injected either with rAAV-LacZ or rAAV-VEGFD as indicated. Vessels
1195
were immunolabeled against CollagenIV (Coll-IV, green) and using Isolectin-B4 (IB4, red).
1196
Scale bar=30 µm. B) Representative images of retinal whole mounts at d7 after intravitreal
45
1197
injections of PBS, NMDA, rVEGFD or rVEGFD+NMDA. Vessels were immunolabeled
1198
against CollagenIV (Coll-IV, green) and using Isolectin-B4 (IB4, red). Scale bar=30 µm. C)
1199
Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in A.
1200
Two-way ANOVA, Bonferroni. n=6. rAAV-LacZ+PBS vs rAAV-LacZ+NMDA, p=0.0008;
1201
rAAV-VEGFD+PBS vs rAAV-VEGFD+NMDA, p=0.6565; rAAV-LacZ+PBS vs rAAV-
1202
VEGFD+PBS, p=0.2950; rAAV-LacZ+NMDA vs rAAV-VEGFD+NMDA, p=0.1484. D)
1203
Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in A.
1204
Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye
1205
from the same animal. n=6. E) Quantification of empty sleeves at d1 after intravitreal
1206
injections with PBS, NMDA, rVEGFD, or rVEGFD+NMDA. Two-way ANOVA,
1207
Bonferroni. n=6. PBS vs NMDA (sham), p=0.0003; PBS vs NMDA (rVEGFD), p=0.5067;
1208
sham vs rVEGFD (PBS), p>0.9999; sham vs rVEGFD (NMDA), p=0.0022. F) Quantification
1209
of empty sleeves at d7 after intravitreal injections, injected as indicated in E. Two-way
1210
ANOVA, Bonferroni. n=6. PBS vs NMDA (sham), p=0.0001; PBS vs NMDA (rVEGFD),
1211
p=0.1673; sham vs rVEGFD (PBS), p=0.8438; sham vs rVEGFD (NMDA), p=0.0329. G)
1212
Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in E.
1213
Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye
1214
from the same animal. n=6. H) Quantification of empty sleeves at d1 after intravitreal
1215
injections of PBS, NMDA, rGFP or rGFP+NMDA. Two-way ANOVA, Bonferroni. n=6.
1216
PBS vs NMDA (sham), p=0.0019; PBS vs NMDA (rGFP), p=0.0004; sham vs rGFP (PBS),
1217
p>0.9999; sham vs rGFP (NMDA), p=0.6279. I) Quantification of empty sleeves at d7 after
1218
intravitreal injections, injected as indicated in H. Two-way ANOVA, Bonferroni. n=6. PBS
1219
vs NMDA (sham), p=0.0143; PBS vs NMDA (rGFP), p=0.0050; sham vs rGFP (PBS),
1220
p=0.5439; sham vs rGFP (NMDA), p=0.1376. J) Quantification of empty sleeves at d7 after
1221
intravitreal injections, injected as indicated in H. Number of empty sleeves in the NMDA-
46
1222
treated eye was normalized to the PBS control eye from the same animal. n=6. Graphs
1223
represent mean ± SEM. ***p<0.001, **p<0.01, *p<0.05. See also Figure S1.
1224 1225
Figure 8: VEGFD protection against NMDA-triggered excitotoxic retinal damage is
1226
mediated by neuronal VEGFR3 expression.
1227
A) Representative confocal scans of retinal whole mounts at d7 after intravitreal injections of
1228
PBS, NMDA, rVEGFD or rVEGFD+NMDA from mice intravitreally injected either with
1229
rAAV-shSCR- (control) or rAAV-shVEGFR3. RGCs were immunolabeled using Brn3a.
1230
Scale bar=20 µm. B) Quantification of RGCs injected as indicated in A. Two-way ANOVA,
1231
Bonferroni.
1232
shSCR+PBS vs rAAV-shSCR+rVEGFD, p=0.9583; rAAV-shSCR+PBS vs rAAV-
1233
shSCR+rVEGFD+NMDA, p=0.4943; shVEGFR3+PBS vs rAAV-shVEGFR3+NMDA,
1234
p=0.0046; rAAV-shVEGFR3+PBS vs rAAV-shVEGFR3r+VEGFD, p=0.7359; rAAV-
1235
shVEGFR3+PBS vs rAAV-shVEGFR3+rVEGFD+NMDA, p=0.0017. C) Quantification of
1236
RGCs at d7 after intravitreal injections, injected as indicated in A, in rAAV-shSCR-injected
1237
eyes. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye from
1238
the same animal. n=8. D) Quantification of RGCs at d7 after intravitreal injections injected as
1239
indicated in A, in rAAV-shVEGFR3-injected eyes. RGC numbers in the NMDA-treated eye
1240
were normalized to the PBS control eye from the same animal. n=8. E) Representative
1241
confocal scans of retinal whole mounts at d7 after intravitreal injections of PBS, NMDA,
1242
rVEGFD or rVEGFD+NMDA from mice injected either with rAAV-shSCR or rAAV-
1243
shVEGFR3 as indicated. Vessels were immunolabeled using Collagen-IV (CollIV, green) and
1244
Isolectin-B4 (IB4, red). Scale bar=30 µm. F) Quantification of empty sleeves injected as
1245
indicated in A. Two-way ANOVA, Bonferroni. n=6. rAAV-shSCR+PBS vs rAAV-
1246
shSCR+NMDA, p=0.0018; rAAV-shSCR+PBS vs rAAV-shSCR+rVEGFD, p=0.5622;
n=8.
rAAV-shSCR+PBS
vs
rAAV-shSCR+NMDA,
47
p=0.0067;
rAAV-
1247
rAAV-shSCR+PBS vs rAAV-shSCR+rVEGFD+NMDA, p=0.1073; rAAV-shVEGFR3+PBS
1248
vs
1249
shVEGFR3r+VEGFD,
1250
shVEGFR3+rVEGFD+NMDA, p=0.0001. G) Quantification of empty sleeves at d7 after
1251
intravitreal injections, injected as indicated in A, in rAAV-shSCR-injected eyes. Number of
1252
empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the
1253
same animal. n=8. H) Quantification of empty sleeves at d7 after intravitreal injections,
1254
injected as indicated in A, in rAAV-shVEGFR3-injected eyes. Number of empty sleeves in
1255
the NMDA-treated eye was normalized to the PBS control eye from the same animal. n=8. I)
1256
Schema of neuronal-mediated VEGFD protection against excitotoxic retinal damage. Upper
1257
row, left box: NMDA triggers RGC and EC degeneration in rAAV-shSCR-injected mice by
1258
direct action on RGCs (red arrows). RGCs and ECs express VEGFR3 (VEGFR3+). Lower
1259
row, left box: VEGFD prevents RGCs and ECs from NMDA-triggered degeneration in
1260
rAAV-shSCR-injected mice. RGCs and ECs express VEGFR3 (VEGFR3+). VEGFD can act
1261
on VEGFR3 in both RGCs and ECs (green arrows). Upper row, right box: NMDA triggers
1262
RGC and EC degeneration in rAAV-shVEGFR3-injected mice by direct action on RGCs (red
1263
arrows). RGCs do not express VEGFR3 (VEGFR3-), ECs express VEGFR3 (VEGFR3+).
1264
Lower row, right box: In rAAV-shVEGFR3-injected mice, VEGFD does not prevent RGCs
1265
and ECs from NMDA-triggered degeneration. RGCs do not express VEGFR3 (VEGFR3-),
1266
ECs express VEGFR3 (VEGFR3+). VEGFD cannot act on VEGFR3 in RGCs, but on
1267
VEGFR3 in ECs (green arrows). Graphs represent mean ± SEM. ***p<0.001, **p<0.01,
1268
*p<0.05. See also Figure S1.
rAAV-shVEGFR3+NMDA,
p=0.0009;
p=0.2623;
rAAV-shVEGFR3+PBS
rAAV-shVEGFR3+PBS
48
vs vs
rAAVrAAV-
Figure 1 d1
PBS
NMDA
PBS
d7
NMDA
Hoechst
Brn3a
A
Brn3a Hoechst
Brn3a Hoechst
Brn3a Hoechst
merge
Brn3a Hoechst
800 600 400
1.0
0.6 0.4
0
0.0
d1 d7
0.2
E 1.2
PBS NMDA
0.8
200
d1 d7
**
time after injection time after injection
*** *** PBS
1.0 0.8 0.6 0.4 0.2 0.0
F 60
NMDA
50
PBS NMDA
40 30 20 10 0
d1 d7
1000
*
INL thickness (µm)
1200
D 1.2
PBS NMDA
relative mRNA level
**
th y1 ne fl
*
relative N RGC
C 1400 N RGC
B
time after injection
PBS NMDA
rel. vessel surface area
1.2
PBS NMDA
1.0
d7
d1
0.6 0.4 0.2 0.0
d7
d7
d1
d7
time after injection
time after injection
J
100
PBS NMDA
vascular TER119+ cells (% of total)
Coll-IV TER119
80
K
60
0.5 0.0
GAPDH
36 kDa
1.0
1.0
0.0
0.0
Q 1.5
untreat. NMDA
1.0
0.8 0.6
0.5
0.2
un tre a 2 0 t. µM
0.0
NMDA
0.0
oc cl l d d tjp5 1
0.4
time after injection
R relative mRNA level
1.0
d7
GluN1
120 kDa
GluN2A
170 kDa
GluN2B
180 kDa
GluN2C
140 kDa
d7
d7
rel. ratio living/total Bend.3 cells
1.2
O
0.5
0.5
time after injection 10 min 24 h
time after injection
d1
25 kDa
PBS NMDA
un rel. ratio living/total astrocytes tre 2 0 at. µM
IgG
2.0 1.5
d1
69 kDa
N
rel. IgG level
rel. albumin level
albumin
PBS NMDA
1.5
NMDA
NMDA
PBS
PBS
d7
d1 conrol
d1
d7
oc cl ld d tjp5 1
time after injection 2.0
PBS NMDA
1.0
0
0.0
2.0 1.5
20
M
PBS NMDA
1.2 0.8
40
L
1.4
0.0
NMDA
* p
1.0
0.2
0.5
P
G
0.4
d1
relative mRNA level
Coll-IV TER119
1.0
PBS NMDA
1.2
0.0
PBS
1.4
0.6
0.2
d7
tj
d1
merge
F
0.8
0.6
I
*
*
time after injection
1.0
0.8
0.4
PBS NMDA
e
*
rel. vessel diameter
d7
d1
Isolectin-B4
time after injection
1.5
e
0
time after injection
H
NMDA
p
tj
1
50 0
Coll-IV IB4
PBS
2
100
Coll-IV IB4
PBS NMDA
3
150
E
D
* ****
4
bEND.3
Collagen-IV
200
C
neurons
* ****
250
rel. vessel length
B
NMDA
N empty sleeves
PBS
rel. fluorescence unit
d7
rel. N empty sleeves
Figure 2 A
1.2
24 h
1.0 0.8 0.6 0.4 0.2 0.0
NMDA
GluN2D GAPDH
170 kDa 36 kDa
Figure 3 2.5
relative mRNA level
A
B
PBS NMDA
2.0
Brn3a
VEGFD
1.5
**
neg. control
neg. control
human
1.0
merge
Brn3a VEGFD
0.5
0.0
E
PBS
NMDA
relative m RNA level
VEGFD
2.0
0.5
0.8
0.4 0.2 0.0
merge
0.6
VEGFD Hoechst
VEGFD Hoechst
GCL
GCL
2.0
untreated NMDA
1.5 1.0 0.5
0.0
0.0
ve v e g fd ve g fc gf a f lt 4
PBS NMDA
2.5
ve v e g fd ve g fc gf a f lt 4
rel. VEGFD protein level
1.0
**
1.0
Hoechst
Beta-actin
untreated NMDA
1.5
VEGF
1.2
G 2.5
VEGFD
D
F
relative m RNA level
subject 2
subject 1
C
d c a 4 1 r g f e gfegf f lt f lt kd e v v v
Figure 4
rAAV-LacZ 2000
N RGC
1500
**** ***
1000 500
rAAV-LacZ +PBS rAAV-LacZ +NMDA rAAV-VEGFD +PBS rAAV-VEGFD +NMDA
D 1.2
SMI-32 Hoechst
SMI-32 Hoechst
SMI-32 Hoechst
rAAV-VEGFD rAAV-LacZ rAAV-VEGFD
1.0 0.8 0.6 0.4 0.2
NM DA
time after injection
0.0
PB S
d7
0
E
Area SMI-32+ cluster (µm2)
C
Brn3a
NMDA
SMI-32 Hoechst
10000
**** ****
5000 1500 1000 500 400
rAAV-LacZ +PBS rAAV-LacZ +NMDA rAAV-VEGFD +PBS rAAV-VEGFD +NMDA
200 0
d7
Brn3a
relative N RGC
rAAV-VEGFD
PBS
time after injection
F
rAAV-LacZ rAAV-VEGFD
120 100 80 60 40 20 0
NM DA
NMDA
Brn3a
d7
PB S
PBS
B rAAV-LacZ
Brn3a
d7
rel. area SMI32+ cluster
A
Figure 5 A
d7
PBS
B
NMDA
Brn3a Hoechst
Brn3a Hoechst
Brn3a Hoechst
SMI-32 Hoechst
rVEGFD 500
E 1.2
PBS/NMDA rVEGFD/NMDA
1.0 0.8 0.6 0.4 0.2
0
1000
I
**** 2000 **** **** 1500 1000 500
0
0
d1
500
time after injection
PBS NMDA rGFP NMDA+ rGFP
J 1.2
treatm.
PBS/NMDA rGFP/NMDA
1.0 0.8 0.6 0.4 0.2 0.0
d7
N RGC
1500
PBS NMDA rGFP NMDA+ rGFP
N RGC
2000 ** **
time after injection
ctrl.
relative N RGC
time after injection
H
0.0
d7
d1
0
time after injection
ctrl.
treatm.
F
12000 ** 10000 8000 6000 4000 2000
**
G
PBS NMDA rVEGFD NMDA+ rVEGFD
200 100 0
rel. area SMI-32+ cluster
1000
PBS NMDA rVEGFD NMDA+ rVEGFD
PBS/NMDA rVEGFD/NMDA
110 100 90 40 20 0
time after injection
K
12000 ** ** 10000 8000 6000 4000 2000
L
PBS NMDA rGFP NMDA+ rGFP
rel. area SMI-32+ cluster
**
d7
1500
40
ctrl.
treatm.
PBS/NMDA rGFP/NMDA
30 20 10
200 100 0
d7
500
D
Area SMI-32+ cluster (µm2)
N RGC
1000
PBS NMDA rVEGFD NMDA+ rVEGFD
Area SMI-32+ cluster (µm2)
**
relative N RGC
**
N RGC
1500
NMDA
SMI-32 Hoechst
PBS SMI-32 Hoechst
rVEGFD
C
PBS
SMI-32 Hoechst
PBS
Brn3a Hoechst
d7
time after injection
0
ctrl.
treatm.
Figure 6 A
PBS
NMDA
B
Brn3a
PBS
Brn3a
d7
60
* **
40
Brn3a
30
rVEGFD
Brn3a
Amplitude (µV)
50
PBS NMDA rVEGFD rVEGFD+NMDA
20 10 0
0.05
0.10
0.20
noise
Spatial frequency (cyc/deg)
C
PBS cyc/deg 0.05
0.10
0.20
noise
25 µV 500 ms
NMDA
rVEGFD
rVEGFD+NMDA
Figure 7 A
PBS
d7
B
NMDA
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
NMDA
PBS
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
rVEGFD
rAAV-VEGFD
100
1.0
time after injection 3.0
PBS/NMDA rVEGFD/NMDA
H 400 300 **
2.5 2.0
***
200
1.5 1.0
NMDA
PBS I NMDA rGFP NMDA+ rGFP
100
0.5 0.0
PBS
ctrl.
treatm.
0
50 0
150
PBS NMDA rVEGFD NMDA+ rVEGFD
50 0
time after injection 400 300
*
**
200
time after injection
PBS J NMDA rGFP NMDA+ rGFP
3.0
PBS/NMDA rGFP/NMDA
2.5 2.0 1.5 1.0 0.5 0.0
0
time after injection
*
100
100
d1
G
N empty sleeves
d7
0.0
200
***
d7
1.5
250
N empty sleeves
2.0
0.5
0
rel. N empty sleeves
150
F PBS NMDA rVEGFD NMDA+ rVEGFD
rel. N empty sleeves
50
200 **
**
d1
100
2.5
250
d7
150
3.0
rAAV-LacZ rAAV-VEGFD
E
N empty sleeves
200
rAAV-LacZ D +PBS rAAV-LacZ +NMDA rAAV-VEGFD +PBS rAAV-VEGFD +NMDA
N empty sleeves
**
rel. N empty sleeves
C 250 N empty sleeves
PBS
Coll-IV IB4
rAAV-LacZ
Coll-IV IB4
d7
time after injection
ctrl.
treatm.
NMDA
rVEGFD
Brn3a
Brn3a
Brn3a
Brn3a
Brn3a
** **
N RGC
1000
PBS NMDA rVEGFD NMDA+ rVEGFD
500
C
rAAV-shSCR: 1.2
PBS/NMDA rVEGFD/NMDA
D
0.6
0.6
0.4 0.2
0.2
PBS
NMDA
0.0
treatm.
rVEGFD
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
Coll-IV IB4
400
**
300 200
*** ****
PBS NMDA rVEGFD NMDA+ rVEGFD
100 0
I
G rAAV-shSCR: rel. N empty sleeves
F
ctrl.
Coll-IV IB4
rAAV-shVEGFR3 rAAV-shSCR
E
shSCR shVEGFR3
2.0
PBS/NMDA rVEGFD/NMDA
1.5
VEGFR3+ NMDA
rVEGFD+NMDA Coll-IV IB4
Coll-IV IB4
H
rAAV-shVEGFR3: 2.0
PBS/NMDA rVEGFD/NMDA
0.5
ctrl.
0.0
treatm.
rAAV-shSCR
ctrl.
treatm.
rAAV-shVEGFR3
VEGFR3NMDA
VEGFR3+
VEGFR3+
VEGFR3+
VEGFR3-
NMDA
NMDA
VEGFD
VEGFD VEGFR3+
treatm.
1.0
1.0
0.0
ctrl.
1.5
0.5
shSCR shVEGFR3
PBS/NMDA rVEGFD/NMDA
0.8
0.8
0.0
1.2 1.0
1.0
0.4
0
rAAV-shVEGFR3:
relative N RGC
1500 **
Brn3a
Brn3a
relative N RGC
B
rVEGFD+NMDA
r el. N empty sleeves
PBS
Brn3a
rAAV-shVEGFR3 rAAV-shSCR
A
N empty sleeves
Figure 8
VEGFR3+