Johnny Di Pierdomenico

Inherited Photoreceptor Degeneration Causes the Death of Melanopsin-Positive Retinal Ganglion Cells and Increases Their Coexpression of Brn3a.

PURPOSE To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. METHODS At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. RESULTS In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). CONCLUSIONS Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.

Early Events in Retinal Degeneration Caused by Rhodopsin Mutation or Pigment Epithelium Malfunction: Differences and Similarities

To study the course of photoreceptor cell death and macro and microglial reactivity in two rat models of retinal degeneration with different etiologies. Retinas from P23H-1 (rhodopsin mutation) and Royal College of Surgeon (RCS, pigment epithelium malfunction) rats and age-matched control animals (Sprague-Dawley and Pievald Viro Glaxo, respectively) were cross-sectioned at different postnatal ages (from P10 to P60) and rhodopsin, L/M- and S-opsin, ionized calcium-binding adapter molecule 1 (Iba1), glial fibrillary acid protein (GFAP), and proliferating cell nuclear antigen (PCNA) proteins were immunodetected. Photoreceptor nuclei rows and microglial cells in the different retinal layers were quantified. Photoreceptor degeneration starts earlier and progresses quicker in P23H-1 than in RCS rats. In both models, microglial cell activation occurs simultaneously with the initiation of photoreceptor death while GFAP over-expression starts later. As degeneration progresses, the numbers of microglial cells increase in the retina, but decreasing in the inner retina and increasing in the outer retina, more markedly in RCS rats. Interestingly, and in contrast with healthy animals, microglial cells reach the outer nuclei and outer segment layers. The higher number of microglial cells in dystrophic retinas cannot be fully accounted by intraretinal migration and PCNA immunodetection revealed microglial proliferation in both models but more importantly in RCS rats. The etiology of retinal degeneration determines the initiation and pattern of photoreceptor cell death and simultaneously there is microglial activation and migration, while the macroglial response is delayed. The actions of microglial cells in the degeneration cannot be explained only in the basis of photoreceptor death because they participate more actively in the RCS model. Thus, the retinal degeneration caused by pigment epithelium malfunction is more inflammatory and would probably respond better to interventions by inhibiting microglial cells.

Different Ipsi- and Contralateral Glial Responses to Anti-VEGF and Triamcinolone Intravitreal Injections in Rats

PURPOSE To investigate the glial response of the rat retina to single or repeated intravitreal injections (IVI). METHODS Albino Sprague-Dawley rats received one or three (one every 7 days) IVI of anti-rat VEGF (5 μL; 0.015 μg/μL), triamcinolone (2.5 or 5 μL; 40 μg/μL; Trigón Depot), bevacizumab (5 μL; 25 μg/μL; Avastin), or their vehicles (PBS and balanced salt solution) and were processed 7 days after the last injection. Retinas were dissected as whole mounts and incubated with antibodies against: Iba1 (Ionized Calcium-Binding Adapter Molecule 1) to label retinal microglia, GFAP (Glial Fibrillary Acidic Protein) to label macroglial cells, and vimentin to label Müller cells. The retinas were examined with fluorescence and confocal microscopy, and the numbers of microglial cells in the inner retinal layers were quantified using a semiautomatic method. RESULTS All the injected substances caused an important micro- and macroglial response locally at the injection site and all throughout the injected retina that was exacerbated by repeated injections. The microglial response was also observed but was milder in the contralateral noninjected eyes. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the ipsilateral retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-foot hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-foot hypertrophy was localized and more extensive with triamcinolone use or after repeated injections. CONCLUSIONS Intravitreal injections cause micro- and macroglial responses that vary depending on the injected agent but increase with repeated injections. This inflammatory glial response may influence the effects of the injected substances on the retina.

Retinal remodeling following photoreceptor degeneration causes retinal ganglion cell death

The retina is the extension of the central nervous system that senses light. Cones and rods, situated in the outer retina, convert light into electrical signals that travel through intermediate neurons where these are further processed until they finally reach retinal ganglion cells (RGCs). The afferent neurons of the retina, the RGCs, send the visual information through their axons in the optic nerve to the retinorecipient nuclei in the brain, where is further analysed. Until recently, it was thought that rods and cones (known as classical photoreceptors) were the only cells sensing light in the retina. However, we now know that light might be converted into electrical signals also in a specific subset of RGCs, the melanopsin-expressing RGCs (mRGCs). These cells account in the rat for 2% to 3% of the entire RGC population (García-Ayuso et al., 2015) and are involved in non-image forming visual functions such as the circadian photoentrainment or the pupillary reflex. RGCs not expressing melanopsin constitute approximately 98% of the RGC population and serve image-forming visual functions (García-Ayuso et al., 2015). RGCs not expressing melanopsin may be identified by their expression of Brn3a, a transcription factor which is, in turn, not expressed by mRGCs. Inherited or acquired photoreceptor degenerations are a group of pathologies that involve the outer retina but that, with time, reach the inner retina affecting RGCs. Here we will review the current knowledge of retinal remodeling and loss of RGCs in photoreceptor degenerations caused by different aetiologies. Age-related macular degeneration (AMD) and inherited retinal degenerations (RD) represent a major clinical problem (Benfenati and Lanzani, 2018; LaVail et al., 2018). AMD is at present the most frequent cause of irreversible blindness in developed countries. Inherited RD are less frequent but cause blindness at early ages, thus being an important cause of blindness at working ages. The most frequent inherited RD in humans is retinitis pigmentosa (RP). Both AMD and RP cause vision loss and irreversible blindness due to photoreceptor degeneration, but progress differently. While AMD triggers the loss of cones and retinal pigment epithelium at the macula, RP causes first rod degeneration and, secondarily, cone degeneration. Both diseases are due to intrinsic (genetic) and extrinsic (environmental) factors, and nutrition and light exposure have been proposed as predisposing risk factors. Indeed, light has been shown to cause photoreceptor death and to accelerate photoreceptor degenerations. Also, light-induced RD models have been documented to mimic some features of human AMD (Marco-Gomariz et al., 2006; Marc et al., 2008; García-Ayuso et al., 2011). The main aim of RD research is to develop therapies to slow or prevent photoreceptor loss and to replace lost photoreceptors, since a relative survival of the inner retinal cells is assumed after photoreceptor loss. However, there is increasing evidence that the inner retina becomes progressively disorganized and remodeled as the outer retinal degeneration progresses (Villegas-Pérez et al., 1998; Marco-Gomariz et al., 2006; Marc et al., 2007, 2008; García-Ayuso et al., 2010, 2011, 2014, 2015; Kalloniatis et al., 2016). Concretely, when photoreceptors are lost, a sequence of progressive events is initiated in the outer retina that culminate with cell death and remodelling of the inner neural retina (Marc et al., 2007, 2008; Kalloniatis et al., 2016; LaVail et al., 2018). The degenerating retina is dynamic and some reprogramming of the neural retina occurs during retinal degeneration (Marc et al., 2007; Kalloniatis et al., 2016). Indeed, anatomical and neurochemichal changes have been proposed to be responsible for the observed activation of amacrine and RGCs in the absence of bipolar cell activation (Marc et al., 2007) and these changes may be responsible for long-term RGC viability. The question is whether the RGCs are affected and die during the course of these diseases, and if so, when. This is important because therapies aimed to replace photoreceptors (i.e., photoreceptor transplantation or retinal prosthesis implantation) depend on the assumption that RGCs are alive and properly functioning (Benfenati and Lanzani, 2018). If they are not, these therapies will not restore vision because the retinorecipient areas of the brain will not be reached. RD has been widely studied using laboratory animals. The rodent models have been the most extensively used because of their many experimental advantages. In our lab, we have studied the effect of retinal remodeling following photoreceptor degeneration on RGC survival. For this purpose, we have investigated RGC survival in three different rodent models with different etiologies: two models of inherited photoreceptor degeneration, the Royal College of Surgeons (RCS; Villegas-Pérez et al., 1998; García-Ayuso et al., 2014) and the P23H-1 (García-Ayuso et al., 2010) rat strains, and one model of light-induced photoreceptor degeneration in albino (García-Ayuso et al., 2011) and pigmented (Marco-Gomariz et al., 2006) rats. RCS rats suffer a recessive mutation of the MERKT gene, an orthologous human gene defect that impairs the phagocytosis of the outer segments of photoreceptors by the retinal pigment epithelium cells (LaVail et al., 2018). P23H rats bare a proline to histidine substitution at codon 23 in the rhodopsin gene, one of the commonest mutations associated with human autosomal dominant RP (LaVail et al., 2018). Both mutations have been shown to occur in certain human diseases and thus, both models have been used to study the consequences of these mutations in the retina (Villegas-Pérez et al., 1998; García-Ayuso et al., 2010, 2014). One of the main differences between these two models is that RD in the P23H-1 rat is due to a rod genetic defect while in the RCS rat the mutant gene impedes retinal pigment epithelium phagocytosis. Light-induced RD or phototoxicity has also been employed as a

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.