Evaluation of the protective effects of PACAP with cell-specific markers in ischemia-induced retinal degeneration

Brain Research Bulletin 81 (2010) 497–504

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Evaluation of the protective effects of PACAP with cell-specific markers in ischemia-induced retinal degeneration Tamas Atlasz a,b,∗ , Krisztina Szabadfi a,1 , Peter Kiss c,2 , Andrea Tamas c,3 , Gabor Toth d,4 , Dora Reglodi c,5 , Robert Gabriel a,1 a

Department of Experimental Zoology and Neurobiology, University of Pecs, Ifjusag str. 6, H-7624 Pecs, Hungary Department of Sportbiology, University of Pecs, Ifjusag str. 6, H-7624 Pecs, Hungary Department of Anatomy, University of Pecs, Ifjusag str. 6, H-7624 Pecs, Hungary d Department of Medical Chemistry, University of Szeged, Dugonics str. 13., H-6720 Szeged, Hungary b c

a r t i c l e

i n f o

Article history: Received 6 September 2009 Accepted 8 September 2009 Available online 12 September 2009 Keywords: Chronic hypoperfusion Immunohistochemistry VGLUT 1 VGAT Calbindin Calretinin Parvalbumin PKC␣ GFAP

a b s t r a c t Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as traumatic brain injury, models of neurodegenerative diseases and cerebral ischemia. We have provided evidence that PACAP is neuroprotective in several models of retinal degeneration in vivo. In our previous studies we showed that PACAP treatment significantly ameliorated the damaging effects of permanent bilateral common carotid artery occlusion (BCCAO). In the present study cell-type-specific markers were used in the same models in order to further specify the protective effects of PACAP. In rats BCCAO led to severe degeneration of all retinal layers that was attenuated by PACAP (100 pmol) administered unilaterally immediately following BCCAO into the vitreous body of one eye. Retinas were processed for immunohistochemistry after 3 weeks. Immunolabeling was executed for vesicular glutamate transporter 1 (VGLUT 1), vesicular ␥-aminobutyric acid transporter (VGAT), protein kinase C␣ (PKC␣), glial fibrillary acidic protein (GFAP) and calcium-binding proteins, such as calbindin, calretinin, parvalbumin. In BCCAO retinas, intensity of immunopositivity for all antisera was dramatically decreased, except in the case of GFAP. In PACAP-treated retinas, immunostaining was similar to that of the control animals. In summary, our study presented immunohistochemical identification of cell types sensitive to chronic retinal hypoperfusion and the protective effects of PACAP. This analysis revealed that the retinoprotective effects of PACAP are not phenotype-specific, but it rather influences general cytoprotective pathways irrespective of the neuronal subtypes in the retina subjected to chronic hypoperfusion. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) belongs to the VIP/glucagon/secretin neuropeptide family [62,72]. One of the well-known effects of PACAP is its neuroprotective action that has been proven in several in vitro and in vivo mod-

∗ Corresponding author at: Department of Sportbiology, University of Pecs, Ifjusag str. 6, H-7624 Pecs, Hungary; Department of Experimental Zoology and Neurobiology, University of Pecs, Pecs, Hungary. Tel.: +36 72 503600x4613; fax: +36 72 501517. E-mail addresses: [email protected] (T. Atlasz), kriszta.szabadfi@gmail.com (K. Szabadfi), [email protected] (P. Kiss), [email protected] (A. Tamas), [email protected] (G. Toth), [email protected] (D. Reglodi), [email protected] (R. Gabriel). 1 Tel.: +36 72503600x4613. 2 Tel.: +36 72503600x1407. 3 Tel.: +36 72503600x1805. 4 Tel: +36 62545139. 5 Tel.: +36 72503600x5398. 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.09.004

els of neuronal injury [63,66,72]. Among others, PACAP treatment results in smaller infarct size in focal cerebral ischemia, it leads to less extensive hippocampal damage in global cerebral ischemia and it decreases postischemic endothelial dysfunction [37,44,51,64]. Mice deficient in endogenous PACAP have larger infarct size [16,43]. These results provide evidence that PACAP is an effective neuroprotective agent in ischemic insults. Disorders of the human sensory systems can be mimicked in the rat in order to better understand the underlying mechanisms and to investigate potential protective substances [33,81]. Permanent bilateral common carotid artery occlusion (BCCAO) is a useful model to study chronic hypoperfusion in the nervous system [23]. Depending on the rat strain and technique used, carotid artery occlusion leads from moderate to severe degeneration of the retina [2,3,9,36,46,75,80]. We have applied this model, yielding reproducible severe retinal degeneration, to study several retinoprotective agents such as diazoxide [3], PARP inhibitor [42] and urocortin 2 [69]. In this model PACAP also was proven to effectively counteract the deleterious effects of chronic retinal ischemia

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[2]. We found that retinas from rats receiving intravitreal PACAP treatment had significantly thicker outer and inner nuclear layers, inner plexiform layer and the cell number of ganglion cell layer was also significantly higher than in ischemic retinas. All these effects were counteracted by the PACAP receptor antagonist PACAP6-38, showing that the protective effects were specific [2]. PACAP has been proven to be retinoprotective also in other models of retinal degeneration. The first study reporting on its retinoprotective effects was an in vitro study, where the authors showed that PACAP ameliorated glutamate-induced toxicity in retinal cell culture [65]. Subsequently, it has been shown that the in vitro effects can be reproduced in vivo, e.g. in optic nerve transection [59,57], kainic acid- and monosodium-glutamate-induced (MSG) toxicity [5–7,70]. Using molecular methods, we have shown that PACAP upregulates anti-apoptotic pathways, while downregulates pro-apoptotic signaling in injured retinas [48–50]. Immunohistological studies have shown that certain noxious stimuli, including retinal ischemia induced by different methods, lead to differential damage of neuronal subtypes [9,10,34]. Therefore, our interest turned to identifying the neuronal types protected by PACAP treatment, as this is a way to gain further insight into the degenerative mechanisms and neuroprotective actions of substances potentially rescuing retinal cells [32,38,78]. We have demonstrated cell types that degenerate in MSG-induced excitotoxic injury of the newborn rat retina, and we have shown that PACAP provided neuroprotection for VGLUT 1-, VGAT-, TH-, Ca-binding proteins- and PKC␣-immunoreactive neurons [4]. Identifying the neuronal cell types where PACAP exerts its protective effects may help to better understand the underlying mechanism. It may also provide help for future drug development, as it is becoming a relevant and urging issue to use PACAP (or analogs/fragments) for treating human diseases [14,35,67]. Therefore, the aim of the present study was to immunohistochemically identify retinal neuronal cell types that are protected by PACAP treatment following retinal ischemic lesion. 2. Materials and methods Adult male Wistar rats (n = 15) weighing 250–300 g were subjected to permanent BCCAO. Animals were fed and watered ad libitum, under light/dark cycles of 12/12 h. All procedures were performed in accordance with the ethical guidelines approved by the University of Pecs (BA02/2000-20/2006). Under isoflurane anesthesia, carotid region was exposed through a midline cervical incision. Both common carotid arteries were ligated with a 3-0 filament. Immediately following the operation, PACAP (100 pmol in 5 ␮l saline) was injected into the vitreous body of the right eye and the same volume of saline was injected into the left eyes, as described previously [2]. Thus, the left eyes served as bilateral carotid-occluded eyes and the right eyes of the same animals served as PACAP-treated retinas. PACAP was synthesized as previously described [27]. The selected dose of PACAP was based on earlier studies where this dose proved to be effective against glutamate toxicity and optic nerve transection in the retina [6,57,70]. A group of animals underwent anesthesia and all steps of the surgical procedure, except for ligation of the carotid arteries. These animals served as control animals (n = 4). Three weeks after the carotid occlusion, animals were sacrificed with an overdose of anesthetic (120 mg/kg pentobarbital, Nembutal, Sanofi-Phylaxia, Hungary), the eyes were immediately dissected in ice-cold phosphate-buffered saline and fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB, pH 7.4) for 4 h at room temperature. Tissue was then washed in 0.1 M PB, and cryoprotected in 10% sucrose for 60 min, 20% sucrose in phosphate-buffered saline (PBS, Sigma, Hungary) overnight at 4 ◦ C. For cryostat sectioning, retinas were embedded in tissue freezing medium (Tissue-Tek, OCT Compound, Sakura Finetech, NL), cut in a cryostat (Leica, Nussloch, Germany) at 10–12 ␮m to obtain radial sections. Central retinal areas within 2 mm from the optic nerve head were used for immunocytochemical analysis. Sections where the GCL appeared thicker than a single cell row were excluded from evaluation. Sections were mounted on chrome–alumn–gelatin coated subbed slides and stored at −20 ◦ C until use. A minimum of 48 sections eye were examined. Retinal sections were rinsed in PBS, permeabilized by incubation for 6 × 5 min in 0.1% Triton X-100 (Sigma, Hungary) in PBS and incubated with 0.1% bovine serum albumin, 1% normal goat serum and 0.1% Na-azide in PBS for 1 h to minimize nonspecific labeling. Sections were incubated with the primary monoclonal or polyclonal antibody overnight at room temperature. We used the following antibodies: antiVGLUT 1, anti-VGAT, anti-calbindin, anti-calretinin, anti-parvalbumin, anti-PKC␣

and anti-GFAP. After several washes in PBS, sections were incubated for 2 h at 37 ◦ C in the dark with the corresponding secondary (red or green fluorescence) antibodies. Sections were then washed in PBS and were coverslipped using Fluoromount (SouthernBiotech). For control experiments, primary antisera were omitted, resulting in no specific staining. Digital photographs were taken with an Olympus Fluoview FV-1000 laser scanning confocal imaging microscope. Photographs were further processed with the Adobe Photoshop 7.0 program. Images were adjusted for contrast only, aligned, arranged and labeled using the functions of the above program. Images were evaluated by an examiner blinded to the experimental treatment.

3. Results VGLUT 1-immunoreactivity was localized in the outer plexiform layer (OPL) and throughout the inner plexiform layer (IPL), consistently with the expected synaptic localization of the protein in rat retina [29]. In our control preparations, VGLUT 1-immunopositive structures were present in the OPL and IPL of the rat retina, were identified as the terminals of photoreceptors and bipolar cells, respectively (Fig. 1(A)). Retinal tissue from animals with BCCAO showed severe degeneration compared to control retinas and the IPL was reduced (Fig. 1(B)). Intraocular PACAP treatment following BCCAO led to a nearly intact appearance of VGLUT 1-immunoreactivity, showing a substantial protective effect (Fig. 1(C)). As shown in Fig. 2(A), we were able to verify the cellular localization of the VGAT in the OPL and IPL [19] in control retinas. Strong VGAT-immunoreactivity could be detected in the IPL (Fig. 2(A)). A decrease in the immunolabeling intensity of VGAT-positive structures was detected following BCCAO. The immunoreactivity in the OPL disappeared and the structures in the IPL were faintly labeled (Fig. 2(B)). PACAP treatment significantly ameliorated the ischemic effect of BCCAO (Fig. 2(C)). Calcium-binding proteins such as calbindin [53], calretinin [26], and parvalbumin [76] are abundant in various types of retinal cells. Calbindin-immunoreactivity was found in the cell bodies and processes of the horizontal cells (Fig. 3(A)). Calbindinimmunoreactivity disappeared following BCCAO (Fig. 3(B)). After PACAP injection, the calbindin-positive cells were retained as demonstrated by the similar immunoreactivity to the control level (Fig. 3(C)). In the control retinas, calretinin-immunopositive structures were found in a large number of somata located in the inner nuclear layer (INL) and the ganglion cell layer (GCL) and also their processes in the IPL (Fig. 4(A)). BCCAO induced reduction in the thickness of INL, IPL, GCL and also the number of the labeled retinal cells seemed to be decreased (Fig. 4(B)). PACAP treatment led to not only an increase in the width of the retinal layers, but also the density of the immunoreactive cells seemed to be similar to that of the untreated retinas (Fig. 4(C)). In normal retinas, specific parvalbumin-immunoreactivity was identified in the AII glycinergic amacrine cells and a subpopulation of ganglion cells (Fig. 5(A)). Difference of the immunopositivity and the number of labeled cells in the parvalbumin-immunoreactivity between the control and the BCCAO retinas seem to be decreased (Fig. 5(B)). After BCCAO and PACAP treatment, the structure was similar to, but the immunoreactivity was weaker than the normal tissue (Fig. 5(C)). The presence of PKC␣ was detected formally in the rod bipolar cell population in the rat retina [71]. The labeled structures were the cell bodies of the INL and cell processes extending into the IPL, close to the GCL (Fig. 6(A)). Differences between control and BCCAO were observed in the arborization of bipolar cells (Fig. 6(B)). In addition, in BCCAO retinas, the number of bipolar cells seems to be decreased compared to that of control and PACAP-treated retinas. PACAP treatment counteracted the BCCAO-induced changes in PKC␣ immunolabeling (Fig. 6(C)).

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GFAP filaments are normally present in the inner half of the Muller cells and their endfeet (Fig. 7(A)) [12]. Following BCCAO, GFAP was massively upregulated and immunopositivity was found throughout the cell from the outer limiting membrane (OLM) to the inner limiting membrane (ILM; Fig. 7(B)). In PACAP-treated retinas GFAP levels were reduced in Muller glial cells and the retina was similar to that of the control ones (Fig. 7(C)).

Fig. 1. Representative vertical retinal sections stained by VGLUT 1 transporter antibody, showing the effect of BCCAO (B) compared with control retina (A). In the control retina preparations VGLUT 1-immunoreactivity was localized in the synaptic region in the OPL and IPL (A). Retinal degeneration caused by BCCAO showed severe changes in this pattern of immunoreactivity (B). After PACAP treatment immunopositive structures and the appearance of the retinal layers were similar to the control retinas (C). Scale bar: 20 ␮m. Abbreviations: OLM: outer limiting membrane; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; ILM: inner limiting membrane.

Fig. 2. Representative fluorescent microphotographs taken from vertical sections of the control (A), BCCAO (B), BCCAO + PACAP-treated rat retinas (C), processed for VGAT immunohistochemistry. Immunostaining for VGAT in control retina was apparent in the OPL and IPL (A). After BCCAO, the intensity of the immunoreaction was reduced and loss of VGAT-immunoreactivity was observed in the OPL (B). PACAP diminished the effects of BCCAO (C). Scale bar: 20 ␮m.

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[61,58]. PACAP influences cytokine production by Muller glial cells [56], relaxes retinal pericytes [41], plays a role in retinal development [8,13] and is an important modulator in retinohypothalamic and limboretinal processing [30,74]. The protective effects of PACAP in retinal injuries caused by several procedures show that PACAP effectively counteracts the damaging effects of glutamate, kainate, ischemia, and optic nerve transection [2,6,59,57,70]. In the present immunohistological study we provided further evidence for the retinoprotective effects of PACAP. It has been shown that the degree of retinal degeneration shown by immunohistological or routine histological findings do not always match following retinal ischemia [9]. Our present observations support and are in concordance with our previous study, where similar degree of neuroprotection by PACAP in retinal

Fig. 3. Effect of PACAP treatment on calbindin-immunoreactivity in representative retina sections. In control conditions, the calbindin-immunoreactivity was present in cell bodies and processes of the horizontal cells localized in the INL and IPL (A). Loss of immunoreactivity was observed following BCCAO-induced retinal damage (B). After PACAP treatment the stuctures expressing calbindin were retained (C). Scale bar: 20 ␮m.

4. Discussion In the present study we provided immunohistochemical description of several retinal cell types that are damaged by chronic hypoperfusion and can be partially or fully rescued by intravitreal PACAP treatment. PACAP has been shown to have various effects in the retina and in the retinal pathways [2,4–7,48–50]. The peptide and its receptors have been shown in several distinct layers of the retina [8,61,58]. PACAP protected all inner retinal layers, in correlation with previous results showing the distribution of PAC1 receptor in the retina [60]. Strongest expression of the receptor was found in the GCL and INL, while weaker expressions were found in the ONL and OPL

Fig. 4. Representative vertical retinal sections showing the effects of BCCAO (B) compared with control (A) and BCCAO + PACAP-treated (C) retinal tissue labeled by anti-calretinin antibody. Calretinin is normally present in amacrine and ganglion cells in the INL and GCL (A). BCCAO treatment led to a decrease in the number of labeled retinal cells and immunoreactivity (B). PACAP treatment counteracted the BCCAO-induced changes in calretinin-immunolabeling (C). Scale bar: 20 ␮m.

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Fig. 5. Photographs of representative retinal sections processed for parvalbuminimmunohistochemistry in control (A), BCCAO (B) and BCCAO + PACAP-treated retinas (C). In the control retina numerous AII amacrine cells were identified in the INL (A), which were reduced in the BCCAO retinas (B). After PACAP injections the retinal structure was similar to, but the immunoreactivity was weaker than that of the untreated tissue (C). Scale bar: 20 ␮m.

ischemia was shown by routine histological staining [2]. Identifying the neuronal cell types where PACAP exerts its protective effects may help to better understand the underlying mechanisms. Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system, including the retina [21]. Vesicular glutamate transporters play an essential role in the packaging of glutamate for synaptic release [25]. Three types of glutamate transporters have been identified: VGLUT 1, 2 and 3. VGLUT 1 is predominantly expressed in the photoreceptor layer and in the bipolar neurons, while the other types of VGLUT have a more restricted occurrence in the retina [25,29,68,77]. In BCCAO-treated retinas substantial reduction was found in the size and the number of the terminals of photoreceptor cells

Fig. 6. Representative retinal sections stained with an antiserum against PKC␣. High intensity of PKC␣-immunoreactivity was displayed by subpopulations of inner retinal cells, especially rod bipolar cells under control conditions (A). In the INL the arborization of the rod bipolar cells was altered by BCCAO (B). PACAP treatment resulted in most cases not only in a protection of the retinal layers, but also the density of the immunoreactive cells, which resulted in a distribution of immunoreactivity similar to that of the untreated tissue (C). Scale bar: 20 ␮m.

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Fig. 7. GFAP-immunoreactivity in the rat retina. In control retinas the Muller glial cells showed weak GFAP-immunoreactivity in the GCL and ILM (A). GFAP immunopositivity of the Muller glial cells were observed through the whole width of the retina after BCCAO treatment (B). Muller radial fibers appear less strongly GFAP-positive after PACAP treatment (C). Scale bar: 20 ␮m.

in the IPL and OPL, which was counteracted by PACAP treatment. VGAT is a membrane protein that seems to be predominately, if not exclusively, localized to synaptic vesicles in neuronal processes based on several different experimental observations [15,22]. The VGAT-immunoreactivity pattern in the IPL is consistent with ultrastructural studies showing the presence of synaptic vesicles at chemical synapses in amacrine cell processes containing GAD,

GABA, or glycine-immunoreactivity [18,31,39,40,73]. As reported previously, prominent VGAT immunolabeling in adult retina was observed in the OPL and IPL, whereas weak immunostaining was observed in cell bodies in the INL and GCL. The pattern of VGAT immunostaining was identical in rat retina, confirming a previous report [20]. Many of the bipolar inputs to amacrine cells must have been eliminated by BCCAO because ionotropic glutamate receptors are present on OFF-bipolar cells. PACAP treatment significantly ameliorated the ischemic effects caused by BCCAO. Calcium-binding proteins play important roles in calcium buffering in the retina [34]. Retinal neurons expressing calciumbinding protein immunoreactivity are differentially susceptible for ischemic insult [9,17,34]. We found a resistance of the calbindinpositive neurons to glutamate-induced toxicity in our previous study [4]. However, in the present study, immunoreactivity to all three types of Ca2+ -binding proteins was significantly decreased in ischemic retinas, and the cells which contain these substances suffered less damage after PACAP treatment. The PKC family of serine/threonine kinase isoenzymes is universally expressed in vertebrate tissues, including the bipolar cells of the retina, where it has been shown to have important regulatory role in phototransduction [1,79]. PKC␣immunoreactivity has been reported to be strongly reduced in retinas exposed to ischemic damage [28,47]. In this study, PKC␣-immunoreactivity was observed in the OPL, INL and IPL. Following ischemia, a remarkable reduction in PKC␣ intensity was detected. However, in the PACAP administered retinas the immunoreactive intensity was similar to that of the control slides. GFAP, which is found in retinal Muller cells and astrocytes, is generally upregulated by mild insults to the retina [11,45,54]. Several types of retinal injuries are accompanied by elevated GFAP protein levels, such as kainate-induced retinal toxicity [52,55]. Ischemia has also been reported to cause elevation in GFAP levels, and protective measures, such as preconditioning, reduce the elevated GFAP [24]. Similarly to this latter study, we found elevated GFAP levels in BCCAO retinas and decreased expression after PACAP treatment. We have previously identified the cell types which were sensitive to MSG treatment and rescued by PACAP injections in newborn rat retina [4]. We found that in MSG-treated retinas, the cell bodies and processes in the INL, IPL, and GCL displayed less immunoreactivity for all cell- and tissue-specific antisera. However, calbindin-immunoreactive horizontal cells did not seem to be affected by MSG application. In the present study we did not find phenotype-specific neuroprotection by PACAP in the ischemic retina. Based on this observation, it is suggested that PACAP influences general cytoprotective pathways that are not specific to certain neuronal populations. Previous studies have shown that PACAP stimulates anti-apoptotic signaling and suppresses proapoptotic pathways in the retina exposed to MSG toxicity [48–50]. Whether similar common mechanisms play a role in the neuroprotective effects of PACAP in retinal ischemia requires further molecular biological analysis, which was beyond the scope of the present study. In summary, our study presented immunohistochemical identification of neuronal cell types sensitive to chronic retinal hypoperfusion and the protective effects of PACAP. The present results fully support our previous routine histological description of the retinoprotective effect of PACAP in ischemic injury and suggest that this effect is not neuronal phenotype-specific. Further in vitro and in vivo molecular methods are needed to reveal the exact protective mechanism observed in the retina, which may provide the basis for future clinical application of PACAP treatment in retinal degenerations.

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Conflict of interest The authors declare that they have no competing financial interests.

Acknowledgements This work was supported by OTKA T061766, K72592, CNK78480; F67830, ETT, Bolyai Scholarship and Gedeon Richter Centenary Foundation. The purchase of Olympus Fluoview FV-1000 laser scanning confocal microscope system was supported by grant GVOP-3.2.1-2004-04-0172/3.0 to University of Pecs.

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