Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

Purpose: In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Methods: Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7 days or 1 month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. Results: All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. Conclusions: The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

Systemic taurine treatment affords functional and morphological neuroprotection of photoreceptors and restores retinal pigment epithelium function in RCS rats.

The aim of our work was to study whether taurine administration has neuroprotective effects in dystrophic Royal College of Surgeons (RCS) rats, suffering retinal degeneration secondary to impaired retinal pigment epithelium phagocytosis caused by a MERTK mutation. Dystrophic RCS-p + female rats (n = 36) were divided into a non-treated group (n = 16) and a treated group (n = 20) that received taurine (0.2 M) in drinking water from postnatal day (P)21 to P45, when they were processed. Retinal function was assessed with electroretinogram. Retinal morphology was assessed in cross-sections using immunohistochemical techniques to label photoreceptors, retinal microglial and macroglial cells, active zones of conventional and ribbon synaptic connections, and oxidative stress. Retinal pigment epithelium function was examined using intraocular fluorogold injections. Our results document that taurine treatment increases taurine plasma levels and photoreceptor survival in dystrophic rats. The number of photoreceptor nuclei rows at P45 was 3-5 and 6-11 in untreated and treated animals, respectively. Electroretinograms showed increases of 70% in the rod response, 400% in the a-wave amplitude, 30% in the b-wave amplitude and 75% in the photopic b-wave response in treated animals. Treated animals also showed decreased numbers of microglial cells in the outer retinal layers, decreased glial fibrillary acidic protein (GFAP) expression in Müller cells, decreased oxidative stress in the outer and inner nuclear layers and improved maintenance of synaptic connections. Treated animals showed increased FG phagocytosis in the retinal pigment epithelium cells. In conclusion, systemic taurine treatment decreases photoreceptor degeneration and increases electroretinographic responses in dystrophic RCS rats and these effects may be mediated through various neuroprotective mechanisms.

Intravitreal and subretinal syngeneic bone marrow mononuclear stem cell transplantation improves photoreceptor survival but does not ameliorate retinal function in two rat models of retinal degeneration.

PURPOSE: To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal. METHODS: A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally. RESULTS: Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses. CONCLUSIONS: Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.

Glial Cell Activation and Oxidative Stress in Retinal Degeneration Induced by ß-Alanine Caused Taurine Depletion and Light Exposure.

We investigate glial cell activation and oxidative stress induced by taurine deficiency secondary to ß-alanine administration and light exposure. Two months old Sprague-Dawley rats were divided into a control group and three experimental groups that were treated with 3% ß-alanine in drinking water (taurine depleted) for two months, light exposed or both. Retinal and external thickness were measured in vivo at baseline and pre-processing with Spectral-Domain Optical Coherence Tomography (SD-OCT). Retinal cryostat cross sections were immunodetected with antibodies against various antigens to investigate microglial and macroglial cell reaction, photoreceptor outer segments, synaptic connections and oxidative stress. Taurine depletion caused a decrease in retinal thickness, shortening of photoreceptor outer segments, microglial cell activation, oxidative stress in the outer and inner nuclear layers and the ganglion cell layer and synaptic loss. These events were also observed in light exposed animals, which in addition showed photoreceptor death and macroglial cell reactivity. Light exposure under taurine depletion further increased glial cell reaction and oxidative stress. Finally, the retinal pigment epithelial cells were Fluorogold labeled and whole mounted, and we document that taurine depletion impairs their phagocytic capacity. We conclude that taurine depletion causes cell damage to various retinal layers including retinal pigment epithelial cells, photoreceptors and retinal ganglion cells, and increases the susceptibility of the photoreceptor outer segments to light damage. Thus, beta-alanine supplements should be used with caution.

Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

Purpose: To analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity. Methods: Two-month-old Sprague-Dawley rats were exposed to light and processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Müller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography. Results: Phototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Müller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Müller cell gliosis at the areas devoid of cones. Conclusions: Light-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Müller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Müller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.

ß-alanine supplementation induces taurine depletion and causes alterations of the retinal nerve fiber layer and axonal transport by retinal ganglion cells.

To study the effect of taurine depletion induced by ß-alanine supplementation in the retinal nerve fiber layer (RNFL), and retinal ganglion cell (RGC) survival and axonal transport. Albino Sprague-Dawley rats were divided into two groups: one group received ß-alanine supplementation (3%) in the drinking water during 2 months to induce taurine depletion, and the other group received regular water. After one month, half of the rats from each group were exposed to light. Retinas were analyzed in-vivo using Spectral-Domain Optical Coherence Tomography (SD-OCT). Prior to processing, RGCs were retrogradely traced with fluorogold (FG) applied to both superior colliculi, to assess the state of their retrograde axonal transport. Retinas were dissected as wholemounts, surviving RGCs were immunoidentified with Brn3a, and the RNFL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. ß-alanine supplementation decreases significantly taurine plasma levels and causes a significant reduction of the RNFL thickness that is increased after light exposure. An abnormal pNFH immunoreactivity in some RGC bodies, their proximal dendrites and axons, and a further diminution of the mean number of FG-traced RGCs compared with Brn3a+RGCs, indicate that their retrograde axonal transport is affected. In conclusion, taurine depletion causes RGC loss and axonal transport impairment. Finally, our results suggest that care should be taken when ingesting ß-alanine supplements due to the limited understanding of their potential adverse effects.