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Altered glutamate, GABA and glycine levels in streptozotocin-induced diabetic rat retinas
Friday, 5:30-7:00 P.M., Sep 25, 1992 Palazzo Dei Congressi
X ICER Abstracts 843
19
IN NORMAL
GABA AND CLYCIrS IMMuNoREAcrMTY ACIP’IWUTED RETINAS
d of Lund, S-221 85
Sweden
The distribution of GABA- and glycine- (GLY) like immunoreactivity (LIR) studied in the retina of adult rabbits. GABA-LIR was seen in untreated and in controi retinas h the 3 inxwnmost cell row (out of 5) of the inner nuclear layer (INL), with the majority of the cells located next to the inner plexiform layer (IPL). In addition, a small number of GABA-LIR cells were occasionally seen in the INL, next to the outer plexlform layer (OPL), and in the ganglion celllayer(GCL),bOr~~theIPLTheIPLan4 insomecases,theOPLwere labeled. Five hrs at& an intmoc&ar (i.0.) injection of 140 nmol kainic die1 acid ( 24 ), the number of positive cells was reduced by about 80%. The surviving cells were found mainly next to the IPL L&e ’ in the IPL was seen in distinct bands corresponding to sublaminas 1,23, and7 . No marked changes were seen after 24 hrs. At 2 and 6 days post-injection, a further decrease was noted. However, positive cells were still seen in the innermost cell row of the INL. Some diffiuxe labeling was again seen in the IPL after 6 days. GLY-LIR was found in untreated and control retinas in a far e number of cell bodies out the WI.., although most stained ce f ls were found in the inner ;otEftp II-it Proceses could be seen entering the. IPL or the OPL. A few stained cell bodies were seen in the GCL Both plexiform layers were strongly labeled. KA treatment (140 nmol i.o.) reduced by 80-90% the number of GLYLIR cells in the inner half of the INL and in GCL, and by 50% in the outer half of the INL, which was malntalned for up to 6 days post-injection. Staining was Seen in the IPL in 3 sublaminas (1, 3-4 and 5) for the first 24 brs postinjection, after which sublamina 5 could not be distinguished. The results with KA appear to indicate that, for up to 6 days post-injection, a subpopulation of GABA- and GLY-LIR retinal cells is resistant to KA-induced damage. was
844
20 ALTERED
Heat shock proteins are synthesized after stress, and may help protect neurons from injury. We investigated the expression of the 72 kDa heat shock protein (HSP72) in cultured retinal ganglion cells (RGCs) after injury from anoxia. Dissociated retinal cells from neonatal rats were plated on a Miiller cell monolayer after RGCs were retrogradely labelled with a fluorescent marker. Co-cultures of Miiller glia and retinal neurons were exposed were made anoxic for 6 hours and fixed for immunocytochemistry. We used a monoclonal antibody and the biotinstreptavidin-fluorescein DLthod to identify HSP72-like immunoreactivity (HSPLI). HSPLI appeared in retinal neurons after 15 minutes and was maximal after 1 hour of anoxia; 20% of all neurons and 54% of labelled RGCs demonstrated HSPLI. HSPLI appeared in Miiller cells after 30 minutes and was maximal after 6 hours of kainate exposure: 58% of Miiller cells demonstrated immunoreactivity. The results suggest that RGCs, non-RGC retinal neurons, and Miiller glia can synthesize HSP72 in response to anoxia. The relative abilities of these cells to express HSP may relate to their differential susceptibility to ischemia conditions in culture. metabolic
847
23 GLIA PROTECTS RETINAL CELLS PROM EXCITOTOXIC AND NYPOXIC DANAGE Kiteno. 8.. Caurioli. .J.. Cummoins. D. Yale University Department of Ophthalmology
GABA AND GLYCINE LEVELS IN STREPTOZOTOCIN-INDUCED DIABETIC RAT RETINAS Ishikawa,A., Ishiguro,S., Tanei,M. Department of Ophthalmology, Tohoku University, School of Medicine, Sendai, Japan
GLUTAMATE,
CO-CULTURE
WITH Mi.kLER
Excitatory
amino
neuron
It is well known that diabetic patients show reduced amplitudes and prolonged peak latencies of oscillatory potentials(OPs) of ERG eve" in the early stage without appearent retinopathy. Their underlying neurochemical abnormalities, however, are not fully investigated. Other in vitro electrophysioloqical studies have show" that some amino acid naurotransmitters applied exogenously alter OPs of EkG. In this study, we analyzed free amino acid levels in the retinas to assess the effect of diabetes on the amino acid metabolism, by using streptozotocin-induced diabetic rats. streptozotocin(60ng/kg) was injected intraperitoneally to 12 Sprague-Dawley rats. Six survived rats showed blood glucose level “ore than 500mg/dl 4 months later. At this stage, scotopic single frash ERG showed reduced amplitudes and prolonged peak Ietencies of OPs and b wave. Retinal free amino acid levels were measured by "Pica-Tag" HPLC amino acid analizer. In the diabetic rat retinas, 10% increase in excitatory neurotransmitter glutamate was observed as compared with normal control. Inhibitory neurotransnitter, glycine end GAFJA were also increased by 21% and 32%. respectively. It was considered that these metabolic changes involving amino acid neurotransmitters, probably induced by diabetes. seemed to be related to the abnormalities observed in ERG.
acid
@AA)
death from ischemia
toxicity
and other
GANGLION
may contribute
to
CNS diseases.
We
studied cultured labeled
the neurotoxic effects of RAAs and hypoxia on rat retinal ganglion cells (RGCs). RGCs were by prior injection of DiI into the superior COlliCUli. RGCs were co-cultured with cortical astrocytes or with Mtiller glia. The survival rates of RGCs exposed to kainate, glutamate and NNBA (SO UN to 500 UM) were timeand concentration-depenr-.lt. RGCs cultured with Miiller glia showed lower mortality with E&4 exposure (at 200 uM for 6 hours) than RGCs cultured with astrocytes. RGCs exposed to hypoxia demonstrated a timeand 02 concentration-dependent survival rate, and lower mortality with hypoxia (10% 02for 6 hours) when cultured with Mtiller glia. The presence of Miiller glia, compared to astroglia, confers protection against excitotoxic and hypoxic damage. RGC SURVIVAL with NNDA GLUTAMATE KAINATE HYPOXIA
Nillles 84.2 80.6 76.3 70.0
+ 2 + +
RATE (% of control, cells 4.6 3.9 5.0 7.4
+ SEN)
with asrronlia 68.6 2 4.8 60.5 + 4.0 40.8 + 3.6 53.6 + 7.8
845
21 ALTERATION
OF RETINAL
S'~.~EFPOZOT02It~-II~U~~~ I;aJlurs Y., IIlC"^ II., 3,cpartmnt of Ophthalmoloqy,
y-Al4INOBUTY9IC DIABETIC R4T Kobe
ACID
IN
University,
FILAlB#NT Japan
In patients with diabetes mellitus (DM), decrease 0.: oscillatory potential of tha ercctroretlnoyran (E?G) can be found at an early stage rui'chout re:inonatnv. The oscillatory potential are i
eyes since
848
22 HYPOXIA INDUCES SYNTHESIS OF HEAT SHOCK PROTEIN . * IN CULTURED RETINAL GANGLION CELLS. v~oll a nd o Xltw Yale University School of Medicine, Department of Ophthalmology and Visual Science, New Haven, CT.
AND RAINIC
were enucleated occurrence of
after 1, ~114. Those
2,
3 weeks
and
1 inontil
retinas were stained wit,1 ABC method iasing anti GAllA antibodyr. ';AJA irm~lunoreactivity could be found in inner nuclear lover, inner pleviforn layer an3 ,qaniJlio,l cell layer tile retina in normal and i)il rats. In 1 veek-DM -ats, SADA immunoreactivity in the inner nuclear layer were iliq!l an;l deqree of staininq for GABA vere mai~~tained 1 nonth. The resuics su~~~ested that the increase of GABA in diatietlc retina may produce the change of oscillatory ~"rell~~al of ERG.
of to
S.245
K
X ICER Abstracts 843
19
IN NORMAL
GABA AND CLYCIrS IMMuNoREAcrMTY ACIP’IWUTED RETINAS
d of Lund, S-221 85
Sweden
The distribution of GABA- and glycine- (GLY) like immunoreactivity (LIR) studied in the retina of adult rabbits. GABA-LIR was seen in untreated and in controi retinas h the 3 inxwnmost cell row (out of 5) of the inner nuclear layer (INL), with the majority of the cells located next to the inner plexiform layer (IPL). In addition, a small number of GABA-LIR cells were occasionally seen in the INL, next to the outer plexlform layer (OPL), and in the ganglion celllayer(GCL),bOr~~theIPLTheIPLan4 insomecases,theOPLwere labeled. Five hrs at& an intmoc&ar (i.0.) injection of 140 nmol kainic die1 acid ( 24 ), the number of positive cells was reduced by about 80%. The surviving cells were found mainly next to the IPL L&e ’ in the IPL was seen in distinct bands corresponding to sublaminas 1,23, and7 . No marked changes were seen after 24 hrs. At 2 and 6 days post-injection, a further decrease was noted. However, positive cells were still seen in the innermost cell row of the INL. Some diffiuxe labeling was again seen in the IPL after 6 days. GLY-LIR was found in untreated and control retinas in a far e number of cell bodies out the WI.., although most stained ce f ls were found in the inner ;otEftp II-it Proceses could be seen entering the. IPL or the OPL. A few stained cell bodies were seen in the GCL Both plexiform layers were strongly labeled. KA treatment (140 nmol i.o.) reduced by 80-90% the number of GLYLIR cells in the inner half of the INL and in GCL, and by 50% in the outer half of the INL, which was malntalned for up to 6 days post-injection. Staining was Seen in the IPL in 3 sublaminas (1, 3-4 and 5) for the first 24 brs postinjection, after which sublamina 5 could not be distinguished. The results with KA appear to indicate that, for up to 6 days post-injection, a subpopulation of GABA- and GLY-LIR retinal cells is resistant to KA-induced damage. was
844
20 ALTERED
Heat shock proteins are synthesized after stress, and may help protect neurons from injury. We investigated the expression of the 72 kDa heat shock protein (HSP72) in cultured retinal ganglion cells (RGCs) after injury from anoxia. Dissociated retinal cells from neonatal rats were plated on a Miiller cell monolayer after RGCs were retrogradely labelled with a fluorescent marker. Co-cultures of Miiller glia and retinal neurons were exposed were made anoxic for 6 hours and fixed for immunocytochemistry. We used a monoclonal antibody and the biotinstreptavidin-fluorescein DLthod to identify HSP72-like immunoreactivity (HSPLI). HSPLI appeared in retinal neurons after 15 minutes and was maximal after 1 hour of anoxia; 20% of all neurons and 54% of labelled RGCs demonstrated HSPLI. HSPLI appeared in Miiller cells after 30 minutes and was maximal after 6 hours of kainate exposure: 58% of Miiller cells demonstrated immunoreactivity. The results suggest that RGCs, non-RGC retinal neurons, and Miiller glia can synthesize HSP72 in response to anoxia. The relative abilities of these cells to express HSP may relate to their differential susceptibility to ischemia conditions in culture. metabolic
847
23 GLIA PROTECTS RETINAL CELLS PROM EXCITOTOXIC AND NYPOXIC DANAGE Kiteno. 8.. Caurioli. .J.. Cummoins. D. Yale University Department of Ophthalmology
GABA AND GLYCINE LEVELS IN STREPTOZOTOCIN-INDUCED DIABETIC RAT RETINAS Ishikawa,A., Ishiguro,S., Tanei,M. Department of Ophthalmology, Tohoku University, School of Medicine, Sendai, Japan
GLUTAMATE,
CO-CULTURE
WITH Mi.kLER
Excitatory
amino
neuron
It is well known that diabetic patients show reduced amplitudes and prolonged peak latencies of oscillatory potentials(OPs) of ERG eve" in the early stage without appearent retinopathy. Their underlying neurochemical abnormalities, however, are not fully investigated. Other in vitro electrophysioloqical studies have show" that some amino acid naurotransmitters applied exogenously alter OPs of EkG. In this study, we analyzed free amino acid levels in the retinas to assess the effect of diabetes on the amino acid metabolism, by using streptozotocin-induced diabetic rats. streptozotocin(60ng/kg) was injected intraperitoneally to 12 Sprague-Dawley rats. Six survived rats showed blood glucose level “ore than 500mg/dl 4 months later. At this stage, scotopic single frash ERG showed reduced amplitudes and prolonged peak Ietencies of OPs and b wave. Retinal free amino acid levels were measured by "Pica-Tag" HPLC amino acid analizer. In the diabetic rat retinas, 10% increase in excitatory neurotransmitter glutamate was observed as compared with normal control. Inhibitory neurotransnitter, glycine end GAFJA were also increased by 21% and 32%. respectively. It was considered that these metabolic changes involving amino acid neurotransmitters, probably induced by diabetes. seemed to be related to the abnormalities observed in ERG.
acid
@AA)
death from ischemia
toxicity
and other
GANGLION
may contribute
to
CNS diseases.
We
studied cultured labeled
the neurotoxic effects of RAAs and hypoxia on rat retinal ganglion cells (RGCs). RGCs were by prior injection of DiI into the superior COlliCUli. RGCs were co-cultured with cortical astrocytes or with Mtiller glia. The survival rates of RGCs exposed to kainate, glutamate and NNBA (SO UN to 500 UM) were timeand concentration-depenr-.lt. RGCs cultured with Miiller glia showed lower mortality with E&4 exposure (at 200 uM for 6 hours) than RGCs cultured with astrocytes. RGCs exposed to hypoxia demonstrated a timeand 02 concentration-dependent survival rate, and lower mortality with hypoxia (10% 02for 6 hours) when cultured with Mtiller glia. The presence of Miiller glia, compared to astroglia, confers protection against excitotoxic and hypoxic damage. RGC SURVIVAL with NNDA GLUTAMATE KAINATE HYPOXIA
Nillles 84.2 80.6 76.3 70.0
+ 2 + +
RATE (% of control, cells 4.6 3.9 5.0 7.4
+ SEN)
with asrronlia 68.6 2 4.8 60.5 + 4.0 40.8 + 3.6 53.6 + 7.8
845
21 ALTERATION
OF RETINAL
S'~.~EFPOZOT02It~-II~U~~~ I;aJlurs Y., IIlC"^ II., 3,cpartmnt of Ophthalmoloqy,
y-Al4INOBUTY9IC DIABETIC R4T Kobe
ACID
IN
University,
FILAlB#NT Japan
In patients with diabetes mellitus (DM), decrease 0.: oscillatory potential of tha ercctroretlnoyran (E?G) can be found at an early stage rui'chout re:inonatnv. The oscillatory potential are i
eyes since
848
22 HYPOXIA INDUCES SYNTHESIS OF HEAT SHOCK PROTEIN . * IN CULTURED RETINAL GANGLION CELLS. v~oll a nd o Xltw Yale University School of Medicine, Department of Ophthalmology and Visual Science, New Haven, CT.
AND RAINIC
were enucleated occurrence of
after 1, ~114. Those
2,
3 weeks
and
1 inontil
retinas were stained wit,1 ABC method iasing anti GAllA antibodyr. ';AJA irm~lunoreactivity could be found in inner nuclear lover, inner pleviforn layer an3 ,qaniJlio,l cell layer tile retina in normal and i)il rats. In 1 veek-DM -ats, SADA immunoreactivity in the inner nuclear layer were iliq!l an;l deqree of staininq for GABA vere mai~~tained 1 nonth. The resuics su~~~ested that the increase of GABA in diatietlc retina may produce the change of oscillatory ~"rell~~al of ERG.
of to
S.245
K