Comparison of retinal nerve fiber layer thinning and retinal ganglion cell loss after optic nerve transection in adult albino rats

We compared the time-course and magnitude of retinal nerve fiber layer (RNFL) thinning with that of retinal ganglion cell (RGC) loss after intraorbital optic nerve transection (IONT) in adult rats

In Situ Dividing and Phagocytosing Retinal Microglia Express Nestin, Vimentin, and NG2 In Vivo

BACKGROUND: Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin(+) cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin(+) microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin(+) microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU(+) and nestin(+), although no further local cell proliferation occurred. In addition, nestin(+) microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin(+)NG2(+) microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons. CONCLUSIONS/SIGNIFICANCE: The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed

Measurement of retinal injury in the rat after optic nerve transection: an RT-PCR study

PurposeIn the current study, a non-histological approach, namely semi-quantitative RT-PCR, was used to provide information on retinal ganglion cell (RGC) injury and survival after optic nerve transection (ONT). The levels of mRNAs synthesized by RGCs and glial components were initially measured at defined time points after ONT. Subsequently, a comparison was made between the levels of these mRNAs in the ONT retinas of rats treated with the neuroprotectant BDNF and in rats which received vehicle.MethodsWistar rats received an ONT in one eye, while the fellow eye served as a control. ONT was performed 1-2 mm from the optic disc without damaging the retinal blood supply. In the first experiment, rats were killed at 1, 3, 5, 7, and 21 days after ONT. In the second experiment, brain derived neurotrophic factor (BDNF; 5 microg) or vehicle was injected intravitreally at the same time as the ONT and animals were killed after 7 days.ResultsAfter ONT, mRNA levels of RGC markers (NF-L and Thy-1) decreased substantially, while levels of GFAP and certain trophic factors mRNAs increased significantly. Administration of BDNF resulted in a substantial, but not complete, preservation of the levels of the RGC specific mRNAs, while ONT induced increases in GFAP and trophic factor mRNAs were not reduced to any great extent by BDNF.ConclusionsThe present studies show that measurement of NF-L and Thy-1 mRNAs provides a sensitive and reliable index of RGC injury after ONT, while measurement of GFAP and trophic factors mRNAs provides more general information on the effect of the injury on the retina.Glyn Chidlow, Robert Casson, Paloma Sobrado-Calvo, Manuel Vidal-Sanz, Neville N. Osborn

Shared and Differential Retinal Responses against Optic Nerve Injury and Ocular Hypertension

Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion cells (RGCs) and their axons. The pathophysiology of glaucoma is not fully understood, but it is currently believed that damage to RGC axons at the optic nerve head plays a major role. Rodent models to study glaucoma include those that mimic either ocular hypertension or optic nerve injury. Here we review the anatomical loss of the general population of RGCs (that express Brn3a; Brn3a+RGCs) and of the intrinsically photosensitive RGCs (that express melanopsin; m+RGCs) after chronic (LP-OHT) or acute (A-OHT) ocular hypertension and after complete intraorbital optic nerve transection (ONT) or crush (ONC). Our studies show that all of these insults trigger RGC death. Compared to Brn3a+RGCs, m+RGCs are more resilient to ONT, ONC, and A-OHT but not to LP-OHT. There are differences in the course of RGC loss both between these RGC types and among injuries. An important difference between the damage caused by ocular hypertension or optic nerve injury appears in the outer retina. Both axotomy and LP-OHT induce selective loss of RGCs but LP-OHT also induces a protracted loss of cone photoreceptors. This review outlines our current understanding of the anatomical changes occurring in rodent models of glaucoma and discusses the advantages of each one and their translational value

Targeting KV channels rescues retinal ganglion cells in vivo directly and by reducing inflammation

Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment, and results in part from microglia-mediated inflammation. Numerous experimental studies have focused on identifying drug targets to rescue these neurons. We recently showed that KV1.1 and KV1.3 channels are expressed in adult rat RGCs and that siRNA -mediated knockdown of either channel reduces RGC death after optic nerve transection. Earlier we found that KV1.3 channels also contribute to microglial activation and neurotoxicity; raising the possibility that these channels contribute to neurodegeneration through direct roles in RGCs and through inflammatory mechanisms. Here, RGC survival was increased by combined siRNA-mediated knockdown of KV1.1 and KV1.3 in RGCs, but survival was much greater when knockdown of either channel was combined with intraocular injection of a KV1.3 channel blocker (agitoxin-2 or margatoxin). After axotomy, increased expression of several inflammation-related molecules preceded RGC loss and, consistent with a dual mechanism, their expression was differentially affected when channel knockdown in RGCs was combined with KV1.3 blocker injection. KV1.3 blockers reduced activation of retinal microglia and their tight apposition along RGC axon fascicles after axotomy, but did not prevent their migration from the inner plexiform to the damaged ganglion cell layer. Expression of several growth factors increased after axotomy; and again, there were differences following blocker injection compared with RGC-selective channel knockdown. These results provide evidence that KV1.3 channels play important roles in apoptotic degeneration of adult RGCs through cell-autonomous mechanisms mediated by channels in the neurons, and non-autonomous mechanisms mediated by microglia and inflammation

MIF Inhibitor ISO-1 Protects Photoreceptors and Reduces Gliosis in Experimental Retinal Detachment

Abstract Photoreceptor death and retinal gliosis underlie the majority of vision threatening retinal diseases including retinal detachment (RD). Although the underlying pathobiology of vision limiting processes in RD is not fully understood, inflammation is known to play a critical role. We conducted an iTRAQ proteomic screen of up- and down-regulated proteins in a murine model of RD to identify potential targetable candidates. Macrophage migration inhibitory factor (MIF) was identified and evaluated for neurotoxic and pro-gliotic effects during RD. Systemic administration of the MIF inhibitor ISO-1 significantly blocked photoreceptor apoptosis, outer nuclear layer (ONL) thinning, and retinal gliosis. ISO-1 and MIF knockout (MIFKO) had greater accumulation of Müller glia pERK expression in the detached retina, suggesting that Müller survival pathways might underlie the neuroprotective response. Our data show the feasibility of the MIF-inhibitor ISO-1 to block pathological damage responses in retinal detachment and provide a rationale to explore MIF inhibition as a potential therapeutic option for RD