Assessment of inner retina dysfunction and progressive ganglion cell loss in a mouse model of glaucoma.

The DBA/2J mouse is a model of ocular hypertension and retinal ganglion cell (RGC) degeneration, the main features of which are iris pigment dispersion (IPD) and iris stromal atrophy (ISA). These animals also experience glaucomatous changes, including an increase in intraocular pressure (IOP) beginning at about 9-12 months of age and sectorial RGC death in the retina. The aim of this study was to determine the onset of functional changes exhibited by DBA/2J mice in the inner retina. This was performed by means of electroretinographic recordings (scotopic threshold response, STR) and their correlation with morphological changes (loss of RGCs). To this end, we recorded the scotopic threshold response in control C57BL/6J and in DBA/2J mice at different ages. The RGCs, in both DBA/2J and C57BL/6J animals, were identified at 15 months of age by retrograde tracing with an analogue of fluorogold, hydroxystilbamidine methanesulfonate (OHSt), applied on the superior colliculi. Whole mount retinas were processed to quantify the population of RGCs identified by fluorogold tracing and Brn3a immunodetection, and were counted using image analysis software; an isodensity contour plot was generated for each retina. DBA/2J mice showed a significant reduction in the positive STR (pSTR) amplitudes at 12 months of age, as compared to control C57BL/6J mice of the same age. The pSTR mean amplitude decreased to approximately 27.82% of the values recorded in control mice (p = 0.0058). STR responses decreased in both strains as a result of the natural process of aging, but the decrease was more pronounced in DBA/2J mice. Furthermore, quantification of the total number of RGCs identified by OHSt and Brn3a expression showed a reduced population of RGCs in DBA/2J mice as compared to control mice. Regression analysis revealed significant correlations between the decrease in pSTR and a non-homogeneous reduction in the number of RGCs throughout the retina. Our results indicate the existence of a correlation between retinal function impairment and RGC loss. This functional and morphological analysis allows a reliable assessment of the progression of the disease.

Potential role of P2X7 receptor in neurodegenerative processes in a murine model of glaucoma.

Glaucoma is a common cause of visual impairment and blindness, characterized by retinal ganglion cell (RGC) death. The mechanisms that trigger the development of glaucoma remain unknown and have gained significant relevance in the study of this neurodegenerative disease. P2X7 purinergic receptors (P2X7R) could be involved in the regulation of the synaptic transmission and neuronal death in the retina through different pathways. The aim of this study was to characterize the molecular signals underlying glaucomatous retinal injury. The time-course of functional, morphological, and molecular changes in the glaucomatous retina of the DBA/2J mice were investigated. The expression and localization of P2X7R was analysed in relation with retinal markers. Caspase-3, JNK, and p38 were evaluated in control and glaucomatous mice by immunohistochemical and western-blot analysis. Furthermore, electroretinogram recordings (ERG) were performed to assess inner retina dysfunction. Glaucomatous mice exhibited changes in P2X7R expression as long as the pathology progressed. There was P2X7R overexpression in RGCs, the primary injured neurons, which correlated with the loss of function through ERG measurements. All analyzed MAPK and caspase-3 proteins were upregulated in the DBA/2J retinas suggesting a pro-apoptotic cell death. The increase in P2X7Rs presence may contribute, together with other factors, to the changes in retinal functionality and the concomitant death of RGCs. These findings provide evidence of possible intracellular pathways responsible for apoptosis regulation during glaucomatous degeneration.

Neuroprotective Effects of FGF2 and Minocycline in Two Animal Models of Inherited Retinal Degeneration.

Purpose: The purpose of this study was to study the effect of minocycline and several neurotrophic factors, alone or in combination, on photoreceptor survival and macro/microglial reactivity in two rat models of retinal degeneration. Methods: P23H-1 (rhodopsin mutation), Royal College of Surgeon (RCS, pigment epithelium malfunction), and age-matched control rats (Sprague-Dawley and Pievald Viro Glaxo, respectively) were divided into three groups that received at P10 for P23H-1 rats or P33 for RCS rats: (1) one intravitreal injection (IVI) of one of the following neurotrophic factors: ciliary neurotrophic factor (CNTF), pigment epithelium-derived factor (PEDF), or basic fibroblast growth factor (FGF2); (2) daily intraperitoneal administration of minocycline; or (3) a combination of IVI of FGF2 and intraperitoneal minocycline. All animals were processed 12 days after treatment initiation. Retinal microglial cells and cone photoreceptors were immunodetected and analyzed qualitatively in cross sections. The numbers of microglial cells in the different retinal layers and number of nuclei rows in the outer nuclear layer (ONL) were quantified. Results: IVI of CNTF, PEDF, or FGF2 improved the morphology of the photoreceptors outer segment, but only FGF2 rescued a significant number of photoreceptors. None of the trophic factors had qualitative or quantitative effects on microglial cells. Minocycline treatment reduced activation and migration of microglia and produced a significant rescue of photoreceptors. Combined treatment with minocycline and FGF2 had higher neuroprotective effects than each of the treatments alone. Conclusions: In two animal models of photoreceptor degeneration with different etiologies, minocycline reduces microglial activation and migration, and FGF2 and minocycline increase photoreceptor survival. The combination of FGF2 and minocycline show greater neuroprotective effects than their isolated effects.

Effect of brain-derived neurotrophic factor on mouse axotomized retinal ganglion cells and phagocytic microglia.

PURPOSE: To assess the effect of a single intravitreal injection of brain-derived neurotrophic factor (BDNF) on the survival of mouse retinal ganglion cells (RGCs) and on phagocytic microglia after intraorbital optic nerve transection (IONT). METHODS: One week before IONT or processing, RGCs from pigmented C57/BL6 and albino Swiss mice were traced by applying hydroxystilbamidine methanesulfonate (OHSt) to both superior colliculi. Right afterward unilateral IONT, BDNF or vehicle were intravitreally administered. At increasing time intervals postlesion retinas were dissected as flat-mounts and subjected to BRN3A and Iba1 immunodetection. BRN3A(+)RGCs were automatically quantified in all retinas and their distribution was assessed using isodensity maps. In all retinas, the Iba1-positive and OHSt-filled microglial cells present in the ganglion cell layer were manually quantified. Their distribution was observed by neighbor maps. RESULTS: When vehicle was administered, IONT-induced RGC death was significant at 3 days, while BDNF treatment delayed it to 5 days. At 14 days after BDNF or vehicle injection, 45% and 18% of RGCs had survived, respectively. There was a significant increase in OHSt-filled microglial cells in the right (contralateral) retinas after both treatments, without concurring with quantifiable RGC death. In the injured eye, the number of OHSt-filled microglial cells increased as the population of RGCs decreased and spread from central to peripheral areas. CONCLUSIONS: In axotomized mouse retinas, a single intravitreal injection of BDNF protects RGCs throughout the whole retina. There is a strong contralateral response that involves microglial activation and OHSt phagocytosis.

Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas.

Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory's previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.