Inner retinal mechanisms engaged by retinal electrical stimulation

PURPOSE: Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, little is known about retinal activity during such stimulation. METHODS: Whole cell patch-clamp recordings were obtained from ganglion and bipolar cells in the salamander retinal slice preparation. A stimulating electrode was positioned at the vitreal surface of the slice. RESULTS: Brief pulses of cathodic current evoked transient inward currents in ganglion cells arising from action potentials. Longer pulses (>5 milliseconds) also evoked sustained inward currents in ganglion cells that appeared synaptic in origin because, unlike transient currents, sustained currents were blocked by inhibiting synaptic transmission with Cd2+. These synaptic currents reversed around ECl and were blocked by picrotoxin, strychnine, or both, suggesting they were mediated by GABAa/c and glycine receptors. Synaptic currents were also blocked by the NMDA antagonist MK801 and the KA/AMPA antagonist NBQX, suggesting that epiretinal stimulation evoked glutamate release from bipolar cells, which in turn stimulated the release of GABA and glycine from amacrine cells. Sustained currents were also evoked by epiretinal stimulation in bipolar cells. These currents reversed near ECl and were blocked by picrotoxin, suggesting they arose from GABAa/c receptors. CONCLUSIONS: Pulse duration is an important parameter for effective activation of the inner retina by epiretinal stimulation. Brief pulses evoke action potentials only in ganglion cells. However, longer pulses also evoke sustained synaptic currents by stimulating glutamate release from bipolar cell terminals, which, in turn, evokes the release of GABA and glycine from amacrine cells.     

Electrical stimulation in normal and retinal degeneration (rd1) isolated mouse retina

Stimulus threshold and response latencies were measured for electrically elicited retinal ganglion cell responses in retina isolated from the eyes of normal and retinal degenerate (rd1) mice. Stimulation of the ganglion cell-side in normal retina yielded a significantly lower mean threshold and shorter latency when compared with stimulation of the photoreceptor side in normal retina. The latency of the ganglion cell-side stimulation in normal retina also proved to be significantly shorter than the latency for stimulation of the ganglion cell side in rd1 retina. Thus both the electrode positioning as well as the health of the retinal tissue play a role in the stimulating current required to elicit a retinal response.     

Activation of retinal ganglion cells in wild-type and rd1 mice through electrical stimulation of the retinal neural network

We compared the thresholds and response properties of extracellularly recorded retinal ganglion cells (RGCs) in wild-type and rd1 mouse retinas to electrical stimulation of the retinal neural network. Retinas were stimulated in vitro with biphasic current pulses (1 ms/phase) applied with a 400-microm diameter, subretinal electrode. Three types of responses were observed in both wild-type and rd1 RGCs. Type I cells elicited a single burst of spikes within 20 ms following application of the electrical stimulus, type II cells elicited a single burst of spikes with a latency greater than 37 ms, and type III cells elicited two and occasionally three bursts of spikes. For all ages examined, ranging from postnatal day (P) 25 to P186, the thresholds of RGCs were overall consistently higher in rd1 mice. Median threshold values were 14 and 50 muA in wild-type and rd1 mice, respectively. We propose that photoreceptors lower the thresholds for activation of RGCs whereas postreceptoral neurons determine the response properties of RGCs to electrical stimuli.     

Correlation between tangential distortion of the outer retinal layer and metamorphopsia in patients with epiretinal membrane

Graefe's Archive for Clinical and Experimental Ophthalmology - To evaluate tangential morphological changes in the outer retina and assess their correlation with the degree of metamorphopsia in...     

Survival and integration of neural retinal transplants in rd mice

PURPOSE: To examine the ultrastructure of the rd retina after transplantation of small micro-aggregates of neural retina in order to determine their survival and integration with the host retina and for sites of communication between transplant and host neurons. METHODS: Neonatal micro-aggregates from transgenic mice expressing a LacZ gene reporter gene in their rods were transplanted into the subretinal space of transgenic rd mice expressing a LacZ reporter gene in their rod bipolar cells. The mice were killed at various times after transplantation surgery and studied by light and electron microscopy. RESULTS: Retinal transplants survived well, as long as 8 months, without signs of rejection and were well integrated into the host retina. Cell bodies of transplanted rods made membrane-to-membrane contacts with rod bipolar cells of the host at areas where there were gaps in the host external plexiform layer. One synaptic process of a transplanted rod was found on the vitreal side of the host's external limiting membrane. In two cases, a postsynaptic process in a transplanted rod spherule contained an Xgal label, implying that it belonged to a host rod bipolar. There was evidence of extension of processes between host and transplant retinas involving astrocytic rather than neural structures. CONCLUSIONS: Retinal allografts to the subretinal space of rd mice survive indefinitely. Close but non-synaptic contacts occur between transplant and host neurons that could allow ephaptic communication between these two retinas. Evidence of synaptic contacts between transplant and host was difficult to find.     

Light-driven retinal ganglion cell responses in blind rd mice after neural retinal transplantation

PURPOSE: Light-elicited retinal ganglion cell (RGC) responses after fetal neural retinal transplantation have not been demonstrated in animal or human subjects blind from outer retinal degeneration, despite apparent morphologic success. This study was designed to test the hypothesis that the functional success of retinal transplantation may be enhanced by using a young host retina (13 days old). METHODS:At postnatal day (P)13 C3H/HeJ (rd/rd) retinal degenerate mice received a subretinal transplant, in one eye only, of neural retinal tissue isolated from newborn normal C57/BL6J mice. Between 33 and 35 days after transplantation, local electroretinograms (ERGs) and ganglion cell responses were recorded directly from the retinal surface using a differential bipolar surface electrode. Measurements were performed both with and without light stimulation. Similar recordings were also performed in age-matched eyes subjected to sham transplantation, in control eyes that were not subjected to surgery, and in animals eyes that underwent transplantation at 8 weeks of age. After the recordings, the eyes were processed for light and transmission electron microscopy. RESULTS:Three of 10 mice showed bursts of ganglion cell action potentials (ON response only) as well as recordable intraocular ERGs over the transplant in response to 1-second and 200-msec light stimuli. Light-driven ganglion cell responses could not be recorded in areas outside the transplant in all transplant-recipient eyes, age-matched control eyes, and sham-transplantation eyes. Light responses also could not be recorded in animal eyes that received transplants at an older age (8 weeks). Electron microscopic examination confirmed the presence of photoreceptor outer segments in the areas affected by transplantation. CONCLUSIONS: This study demonstrates the presence of light-driven ganglion cell responses after subretinal transplantation in a retinal degenerate model. This finding may reflect functional integration of the transplant with the host, but a rescue effect on remaining host photoreceptors cannot be ruled out. The findings suggest, however, that modification of host parameters, such as host age, may be important approaches for improving the functional success of retinal transplantation.     

Increased spontaneous retinal ganglion cell activity in rd mice after neural retinal transplantation

PURPOSE: To study the functional success of neural retinal transplantation by means of retinal surface ganglion cell recordings. METHODS: Eight-week-old C3H/HeJ (rd/rd) retinal degeneration mice received transplants (subretinal) in one eye only of neural retinal tissue isolated from newborn normal C57/BL6J mice. Four weeks after transplantation, ganglion cell responses were recorded directly from the retinal surface over the transplant, with a differential bipolar surface electrode. Measurements were performed, both with and without light stimulation. Similar recordings were performed in nontransplant areas of the transplant-recipient eyes, and in age-matched sham-treated and untreated control eyes. After the recordings, the eyes were processed for light and transmission electron microscopy. RESULTS: Histologic examination showed that in some areas, transplanted cells were organized into small sheets and differentiated into photoreceptors with outer segments in intimate contact with the host RPE. No light-driven ganglion cell responses were recordable from the transplant-recipient or control eyes. However, the spontaneous ganglion cell activity was higher in the transplant areas (mean: 10.8 +/-12.0 spikes/1.6 sec) compared with nontransplant areas of these recipient eyes (mean: 2.4 +/- 5.7spikes/1.6 sec; P < 0.001), sham-treated eyes (mean: 2.5 +/- 4.8 spikes/1.6 sec; P < 0.001), and the untreated control eyes (mean: 2.2 +/- 4.4 spikes/1.6 sec; P < 0.001). CONCLUSIONS: Subretinal transplantation of neural retinal tissue results in a local increase of spontaneous ganglion cell activity. The increased activity may be due to the release of neurochemically active substances as a result of the presence of the transplant. Although light responses were not recordable, the technique of retinal surface ganglion cell recording may be useful for assessing the functional success of transplantation.     

rd小鼠出生后5周内视网膜外核层细胞及其外段的光镜和电镜观察

目的观察遗传性视网膜色素变性rd小鼠视网膜外核层细胞层的病理和超微结构变化,为进一步应用rd小鼠进行实验研究提供数据。方法rd新生鼠42只,分别在第0、3、7、14、21、28、35d取眼球,立即经10%中性甲醛固定,光学显微镜下观察眼球水平位视网膜赤道部外核层细胞的厚度;另取眼球经2.5%戊二醛溶液固定,电镜观察。结果病理结果显示,rd小鼠视网膜外核层细胞层数或密度从第14d开始减少2.14层±0.60层,第21d减至近单层1.30层±0.37层,第35d减至0.57层±0.25层。电镜结果显示,rd小鼠视网膜光感受器细胞层生后第14d出现凋亡;外段盘膜部位线粒体从第7d开始出现部分溶解、减少,逐渐加重;外界膜也从第7d开始变为较连续,第21d后不连续。结论视网膜光感受器细胞外段的线粒体和外界膜对于视网膜盘膜的形成起重要作用。这一结果为进一步开展rd小鼠的研究提供了科学依据。     

小胶质细胞活化与rd小鼠遗传性视网膜变性

目的研究小胶质细胞活化与rd小鼠遗传性视网膜变性的关系。方法对出生后8、10、12、14、16及18d的rd小鼠及对照小鼠视网膜进行感光细胞凋亡TUNEL法检测及形态计量学分析。CD11b免疫组织化学染色标记视网膜小胶质细胞。结果rd小鼠出生后10d视网膜感光细胞层开始出现TUNEL染色阳性细胞,第16d达到高峰。视网膜小胶质细胞在rd小鼠出生后10d开始活化,第14d达到高峰。小胶质细胞向感光细胞层的迁移与感光细胞凋亡之间存在紧密的时间和空间关系。结论rd小鼠视网膜变性以感光细胞凋亡为主。小胶质细胞活化可能在视网膜变性过程中发挥重要作用。     

Rod outer segment lipid-opsin ratios in the developing normal and retinal dystrophic rat

The outer segment membrane lipid and opsin contents were determined in photoreceptor cell rods isolated from the eyes of developing normal rats reared in cyclic light or dark environments and dark-reared dystrophic rats. In cyclic light-reared normals rhodopsin/eye increased 49% during the period 20–60 days. Total ROS lipid content, a measure of ROS length, increased 50% while the polyunsaturated fatty acid docosahexaenoate increased from 42–51 mol % during the same period. The phospholipid/opsin ratio of cyclic light reared rat ROS membranes was 67 mol/mol at 20 days and 68 mol/mol at 60 days. In young dark-reared normals the phospholipid/opsin ratio was the same as for cyclic light-reared rats. Although 60 day-old dark-reared normals had 30% more rhodopsin/eye than their cyclic light-reared counterparts, non-significant changes in ROS length (14% longer) and in the phospholipid/opsin ratio (8% lower) were measured in these rats. In addition, light deprivation had no significant effect on the concentrations of polyunsaturated fatty acids or the lipid composition of the isolated ROS. The eyes of dark-reared rats with retinal dystrophy accumulated two times more rhodopsin than dark-reared normals during the 20–60-day period. The phospholipid/opsin ratio of mutant rat ROS was only 7% lower than dark normal at 20 days and 13% lower at 34 days. However, by 60 days of age, the phospholipid/opsin ratio in dystrophic rat ROS was significantly lower than in ROS from either cyclic light-or dark-reared normals. Docosahexaenoic acid in mutant rat ROS lipids averaged 40 mol% during the developmental period. These levels were significantly lower than the levels of docosahexaenoate measured in dark normals at both the 34- and 64-day periods. The glycerophospholipid composition of dystrophic rat ROS was the same as normal at all ages but the cholesterol/phospholipid ratio was higher than in normals.The data show: (1) that the retina accomodates changes in rhodopsin content induced by environmental light, age and genetic differences by alterations in ROS opsin density and length: (2) that the content of ROS membrane polyunsaturated fatty acids (fluidity) increases during development in normals but not in dystrophic rats. The data also suggest that basal membrane synthesis and/or post sythetic membrane modification of ROS lipid are impaired as a function of age in dystrophic rats.     

The kinase inhibitor PKC412 suppresses epiretinal membrane formation and retinal detachment in mice with proliferative retinopathies

PURPOSE: Platelet-derived growth factor (PDGF) is an important stimulatory factor for proliferative retinopathies. Expression of PDGF-B in the retinas of transgenic mice (hemizygous rho/PDGF-B mice) results in rapid-onset retinal detachment caused by proliferation of glial cells, endothelial cells, and pericytes, whereas expression of PDGF-AA (homozygous rho/PDGF-A or PDGF-AA mice) causes slowly progressive retinal detachment from proliferation of glial cells. In this study, we investigated the effect in rho/PDGF-B and rho/PDGF-AA mice of several different receptor kinase inhibitors. METHODS: Hemizygous rhoPDGF-B or homozygous rho/PDGF-A mice were treated orally with PKC412 (an inhibitor of PDGF, VEGF, and c-kit receptor kinases and several isoforms of PKC), PTK787 (an inhibitor of PDGF, VEGF, and c-kit receptor kinases), SU1498 (an inhibitor of VEGF receptor kinases), imatinib mesylate (an inhibitor of PDGF, c-kit, and v-abl receptor kinases), or vehicle, and at appropriate time points epiretinal membrane (ERM) formation and retinal detachment were quantified. RESULTS: In either rho/PDGF-B or rho/PDGF-A mice, oral administration of PKC412 or PTK787, but not SU1498 or imatinib mesylate, significantly reduced ERM formation. PKC412 reduced the incidence of severe retinal detachments in both models and PTK787 did so in homozygous rho/PDGF-A mice. CONCLUSIONS: These data indicate that PKC412 (and possibly PTK787) has appropriate activity and sufficient intraocular bioavailability after oral administration to prevent retinal detachment in models of proliferative retinopathy. PKC412 should be considered for treatment of vascular and nonvascular proliferative retinopathies in humans.     

Ganglion cell responses to retinal light stimulation in the absence of photoreceptor outer segments from retinal degenerate rodents

PURPOSE: To determine whether a severely degenerated retina without photoreceptor outer segments and a non-recordable electroretinogram (ERG) can still show retinal ganglion cell (RGC) responses to retinal light stimulation. METHODS: The authors measured ERGs and retinal surface RGC responses from six week old rd mice and three month old homozygous S334ter line3 rats. Animal eyes were also studied by light microscopy, transmission electron microscopy, and immunohistochemistry (rats). RESULTS: The corneal ERGs were non-recordable and no photoreceptor outer segments were found in either retinal degeneration model. A few cell bodies (without outer segments) that were immunoreactive for cone opsin and rhodopsin were found in the outer nuclear layer of the rats. Light-driven ON-RGC responses, however, were recordable from six week old rd mice. In addition, light-driven ON and OFF-RGC responses were recordable from three month old homozygous S334ter line 3 rats. CONCLUSIONS: This study suggests that despite the apparent absence of photoreceptor outer segments and a non-recordable ERG, ganglion cell responses to retinal light stimulation may remain preserved in some severe retinal degenerate transgenic rodents.