Long-Term Transplant Effects of iPSC-RPE Monolayer in Immunodeficient RCS Rats.

Retinal pigment epithelium (RPE) replacement therapy is evolving as a feasible approach to treat age-related macular degeneration (AMD). In many preclinical studies, RPE cells are transplanted as a cell suspension into immunosuppressed animal eyes and transplant effects have been monitored only short-term. We investigated the long-term effects of human Induced pluripotent stem-cell-derived RPE (iPSC-RPE) transplants in an immunodeficient Royal College of Surgeons (RCS) rat model, in which RPE dysfunction led to photoreceptor degeneration. iPSC-RPE cultured as a polarized monolayer on a nanoengineered ultrathin parylene C scaffold was transplanted into the subretinal space of 28-day-old immunodeficient RCS rat pups and evaluated after 1, 4, and 11 months. Assessment at early time points showed good iPSC-RPE survival. The transplants remained as a monolayer, expressed RPE-specific markers, performed phagocytic function, and contributed to vision preservation. At 11-months post-implantation, RPE survival was observed in only 50% of the eyes that were concomitant with vision preservation. Loss of RPE monolayer characteristics at the 11-month time point was associated with peri-membrane fibrosis, immune reaction through the activation of macrophages (CD 68 expression), and the transition of cell fate (expression of mesenchymal markers). The overall study outcome supports the therapeutic potential of RPE grafts despite the loss of some transplant benefits during long-term observations.

Critical Effects on Akt Signaling in Adult Zebrafish Brain Following Alterations in Light Exposure.

Evidence from human and animal studies indicate that disrupted light cycles leads to alterations of the sleep state, poor cognition, and the risk of developing neuroinflammatory and generalized health disorders. Zebrafish exhibit a diurnal circadian rhythm and are an increasingly popular model in studies of neurophysiology and neuropathophysiology. Here, we investigate the effect of alterations in light cycle on the adult zebrafish brain: we measured the effect of altered, unpredictable light exposure in adult zebrafish telencephalon, homologous to mammalian hippocampus, and the optic tectum, a significant visual processing center with extensive telencephalon connections. The expression of heat shock protein-70 (HSP70), an important cell stress mediator, was significantly decreased in optic tectum of adult zebrafish brain following four days of altered light exposure. Further, pSer473-Akt (protein kinase B) was significantly reduced in telencephalon following light cycle alteration, and pSer9-GSK3ß (glycogen synthase kinase-3ß) was significantly reduced in both the telencephalon and optic tectum of light-altered fish. Animals exposed to five minutes of environmental enrichment showed significant increase in pSer473Akt, which was significantly attenuated by four days of altered light exposure. These data show for the first time that unpredictable light exposure alters HSP70 expression and dysregulates Akt-GSK3ß signaling in the adult zebrafish brain.

Selective optical control of synaptic transmission in the subcortical visual pathway by activation of viral vector-expressed halorhodopsin.

The superficial layer of the superior colliculus (sSC) receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR), a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs) by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

Live imaging of radiation-induced apoptosis by yolk injection of Acridine Orange in the developing optic tectum of medaka.

To observe the sequential radiation-induced apoptosis in a living embryo, we injected Acridine Orange (AO) solution into the yolk of embryo and visualized radiation-induced apoptosis in developing optic tectum (OT). Medaka embryos at stage 28, when neural cells proliferate rapidly in the OT, were irradiated with 5 Gy X-rays which is a non-lethal dose for irradiated embryos at hatching. The irradiated embryos hatched normally without morphological abnormalities in their brains, even though a large number of apoptotic cells were induced transiently in OT. By yolk injection, apoptotic cells in OT were distinguished as AO-positive small nuclei at 3 h after irradiation. At 8-10 h after irradiation, AO-positive rosette-shaped clusters were obviously distinguished in marginal tectal regions of OT where cells are proliferating intensely. The AO-positive clusters became bigger and more obvious, but the number did not increase up to 24 h after irradiation and completely disappeared up to 49 h after irradiation. This characteristic appearance of the AO-positive nuclei/clusters is in good agreement with our previous results, based on the examination of fixed specimens stained with AO by injection into the peri-vitelline space, suggesting that the AO-yolk injection method is highly reliable for detecting apoptotic cells in living embryos. The live imaging of apoptotic cells in developing Medaka embryos by AO-yolk injection method is expected to reveal more of the details of the dynamics of apoptotic responses in the irradiated brain and other tissues.

Glaucoma alters the circadian timing system.

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (approximately 50-70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.

Nociceptive responses of midbrain dopaminergic neurones are modulated by the superior colliculus in the rat.

Midbrain dopaminergic neurones exhibit a short-latency phasic response to unexpected, biologically salient stimuli. In the rat, the superior colliculus is critical for relaying short-latency visual information to dopaminergic neurones. Since both collicular and dopaminergic neurones are also responsive to noxious stimuli, we examined whether the superior colliculus plays a more general role in the transmission of short-latency sensory information to the ventral midbrain. We therefore tested whether the superior colliculus is a critical relay for nociceptive input to midbrain dopaminergic neurones. Simultaneous recordings were made from collicular and dopaminergic neurones in the anesthetized rat, during the application of noxious stimuli (footshock). Most collicular neurones exhibited a short-latency, short duration excitation to footshock. The majority of dopaminergic neurones (92/110; 84%) also showed a short-latency phasic response to the stimulus. Of these, 79/92 (86%) responded with an initial inhibition and the remaining 14/92 (14%) responded with an excitation. Response latencies of dopaminergic neurones were reliably longer than those of collicular neurones. Tonic suppression of collicular activity by an intracollicular injection of the local anesthetic lidocaine reduced the latency, increased the duration but reduced the magnitude of the phasic inhibitory dopaminergic response. These changes were accompanied by a decrease in the baseline firing rate of dopaminergic neurones. Activation of the superior colliculus by the local injections of the GABA(A) antagonist bicuculline also reduced the latency of inhibitory nociceptive responses of dopaminergic neurones, which was accompanied by an increased in baseline dopaminergic firing. Aspiration of the ipsilateral superior colliculus failed to alter the nociceptive response characteristics of dopaminergic neurones although fewer nociceptive neurones were encountered after the lesions. Together these results suggest that the superior colliculus can modulate both the baseline activity of dopaminergic neurones and their phasic responses to noxious events. However, the superior colliculus is unlikely to be the primary source of nociceptive sensory input to the ventral midbrain.

Induction of the candidate-plasticity NGFI-A protein in the adult rat superior colliculus after visual stimulation.

In this work, we studied the visually driven expression of the plasticity-related transcription factor NFGI-A in the superficial layers of the rat superior colliculus (sSC) using immunohistochemistry. After dark adaptation, NGFI-A expression was completely down-regulated, indicating this protein is not constitutively expressed in the sSC. Light stimulation for 10 min after dark adaptation was insufficient to induce detectable levels of this protein. But after 30 min of light stimulation, few NGFI-A+ cells were observed in the superficial layers, indicating that the minimal time of stimulation that is sufficient to induce this protein is sometime between 10 and 30 min. The number of NGFI-A+ cells increased progressively, reaching a peak after 90 min. This peak is not reached if animals are returned to darkness after 30 min of stimulation, when a presumable peak in NGFI-A mRNA is reached. Light stimulation of animals in which the retinocollicular or corticocollicular projections were removed revealed that NGFI-A expression is mainly driven by retinal contralateral projections. Removal of corticocollicular projections did not cause any change in the NGFI-A expression in the ipsilateral sSC, in relation to the contralateral (control) sSC, suggesting that this pathway has a minor influence. Our results showed that NGFI-A protein expression in the sSC is entirely dependent on visual stimulation and suggests that the sSC visual circuitry is an interesting model for studies about the involvement of this transcription factor in synaptic plasticity.

Superior colliculus responses to light - preserved by transplantation in a slow degeneration rat model.

PURPOSE: To determine whether retinal transplantation can preserve visual responses in the superior colliculus (SC) of the S334ter-line-5 rat, a transgenic model for slow photoreceptor degeneration, which is more similar to human retinitis pigmentosa than the fast degeneration line 3 S334ter rat. METHODS: Visual responses to a light flash were recorded in the SC. Rats that had received embryonic day (E) 19-20 fetal retinal sheet transplants at the age of 26-30 days were tested at the ages of 200-254 days. Controls were age-matched rats without surgery and with sham surgery. As a baseline, in no-surgery line-5 rats, the temporal pattern of visual sensitivity loss was evaluated electrophysiologically in the SC from 60 days up to one year of age. RESULTS: In untreated S334ter-line-5 rats, decline in visual sensitivity in the SC was parallel to the photoreceptor loss. At 109 day of age, a relative scotoma developed in the area of the SC corresponding to the nasal retinal region. At 200-254 days of age, the majority of the SC was devoid of any light-driven responses. In contrast, at this time point, transplanted rats with 'good' retinal grafts with normal lamination had visual responses in the caudal region of the SC, the area corresponding topographically to the transplant location in the retina. In these rats, the various parameters of SC responses such as the latency of the onset of the visual response, the response peak amplitude and the consistency of the visual response were significantly different from the control groups (no-surgery, sham surgery, 'poor' transplants) and were more comparable to normal albino rats, however, with a slightly longer latency (70-90 vs. 30-50 msec). CONCLUSIONS: Fetal retinal sheet transplantation showed a long-term rescue effect on visual function in this animal model of slow photoreceptor degeneration.

Neuroendoscopic approach to tectal tumors: a consecutive series.

OBJECT: The authors report a consecutive series of 10 patients who presented with signs and symptoms caused by tectal tumors. Clinical findings, radiographic features, neuroendoscopic management strategies, and histological findings are reported and discussed. METHODS: Since January 1990, 11 neuroendoscopic procedures were performed in 10 patients who harbored tectal tumors. The patients were followed for an average of 5 years (range 2 months-11 years), and a retrospective study was conducted in which case notes, radiological findings, operative notes, and histopathological findings were assessed. Magnetic resonance (MR) imaging was performed, and the images were used to classify patients into three groups: those with hypertrophy of the tectum in whom isointensity appeared on T1-weighted images (Group 1); those with a tectal tumor occupying the cerebral aqueduct in whom decreased signal intensity appeared on T1-weighted images, as well as no enhancement after gadolinium administration (Group 2); and those with a tectal tumor in whom mixed signal intensity and conspicuous evidence of contrast enhancement appeared on T1-weighted images (Group 3). The results of histological examination were consistent with MR imaging features: in Group 1, glial tissue or gliosis; in Group 2, benign astrocytoma; and in Group 3, malignant astrocytoma. Cerebrospinal fluid diversion was the only surgical treatment that provided relief from obstructive hydrocephalus. One patient in Group 3 underwent radiotherapy and subsequent partial tumor removal under neuroendoscopic guidance. Thereafter, the tumor remained in decline. All patients had normal intellectual status after undergoing surgery in which a neuroendoscope was used. CONCLUSIONS: Neuroendoscopic procedures can provide histological diagnosis, define the tumor-midbrain interrelationship, and be highly effective in treating obstructive hydrocephalus and in removing tectal tumors. This procedure may receive clinical application as a new management strategy for tectal glioma.

The effects of simulated ischemia on the levels of adenosine and its metabolites in slices of cerebellum, superior colliculus and hippocampus in the guinea pig-in vitro study.

The purpose of this present study is to clarify adenosine (ADO) metabolism in guinea pig brain slices during simulated ischemia. In slice preparations after decapitation, ADO levels were lowest in slices of the cerebellum (1.2 nmol/mg protein), followed by the superior colliculus (3.4) and highest in the hippocampus (6.4), and the combined concentrations of inosine (Ino) and hypoxanthine (HX) were highest in the cerebellum (5.5), followed by the superior colliculus (3.5) and the hippocampus (1.5). After preincubation with standard medium with oxygen and glucose for 30 min, total ADO levels (tissue ADO plus ADO lost into medium during incubation) decreased to 0.3 in the cerebellum, to 1.3 in the superior colliculus and to 2. 9 in the hippocampus. On the other hand, levels of total Ino and HX increased to 21.1 in the cerebellum, to 14.3 in the hippocampus and to 12.0 in the superior colliculus. To investigate the effect of simulated ischemia on ADO metabolism, preincubated slices were exposed for 10 min in medium deprived of oxygen and glucose. The increases of ADO content after 10 min ischemia were 0.2 in the cerebellum, 1.0 in the superior colliculus and 1.3 in the hippocampus. In contrast, the increases of both Ino and HX concentrations were 2.9 in the cerebellum, 2.2 in the superior colliculus and 1.4 in the hippocampus. The total amount of the increase in ADO, Ino and HX was approximately 3 in all three regions. These results indicate that there are significant differences in the metabolic rate to degrade ADO into Ino and HX in various areas of brain possibly due to differences in adenosine deaminase activity in those areas.