Rotenone induces degeneration of photoreceptors and impairs the dopaminergic system in the rat retina

Rotenone is a widely used pesticide and a potent inhibitor of mitochondrial complex I (NADH-quinone reductase) that elicits the degeneration of dopaminergic neurons and thereby the appearance of a parkinsonian syndrome. Here we have addressed the alterations induced by rotenone at the functional, morphological and molecular levels in the retina, including those involving both dopaminergic and non-dopaminergic retinal neurons. Rotenone-treated rats showed abnormalities in equilibrium, postural instability and involuntary movements. In their outer retina we observed a loss of photoreceptors, and a reduced synaptic connectivity between those remaining and their postsynaptic neurons. A dramatic loss of mitochondria was observed in the inner segments, as well as in the axon terminals of photoreceptors. In the inner retina we observed a decrease in the expression of dopaminergic cell molecular markers, including loss of tyrosine hydroxylase immunoreactivity, associated with a reduction of the dopaminergic plexus and cell bodies. An increase in immunoreactivity of AII amacrine cells for parvalbumin, a Ca2+-scavenging protein, was also detected. These abnormalities were accompanied by a decrease in the amplitude of scotopic and photopic a- and b-waves and an increase in the b-wave implicit time, as well as by a lower amplitude and greater latency in oscillatory potentials. These results indicate that rotenone induces loss of vision by promoting photoreceptor cell death and impairment of the dopaminergic retinal system.

Immunohistochemical evidence of synaptic retraction, cytoarchitectural remodeling, and cell death in the inner retina of the rat model of Oygen-Induced Retinopathy (OIR)

Purpose. Postnatal exposure to hyperoxia destroys the plexiform layers of the neonatal rat retina, resulting in significant electroretinographic anomalies. The purpose of this study was to identify the mechanisms at the origin of this loss. Methods. Sprague-Dawley (SD) and Long Evans (LE) rats were exposed to hyperoxia from birth to postnatal day (P) 6 or P14 and from P6 to P14, after which rats were euthanatized at P6, P14, or P60. Results. At P60, synaptophysin staining confirmed the lack of functional synaptic terminals in SD (outer plexiform layer [OPL]) and LE (OPL and inner plexiform layer [IPL]) rats. Uneven staining of ON-bipolar cell terminals with mGluR6 suggests that their loss could play a role in OPL thinning. Protein kinase C(PKC)-α and recoverin (rod and cone ON-bipolar cells, respectively) showed a lack of dendritic terminals in the OPL with disorganized axonal projections in the IPL. Although photoreceptor nuclei appeared intact, a decrease in bassoon staining (synaptic ribbon terminals) suggests limited communication to the inner retina. Findings were significantly more pronounced in LE rats. An increase in TUNEL-positive cells was observed in LE (inner nuclear layer [INL] and outer nuclear layer [ONL]) and SD (INL) rats after P0 to P14 exposure (425.3%, 102.2%, and 146.3% greater than control, respectively [P < 0.05]). Conclusions. Results suggest that cell death and synaptic retraction are at the root of OPL thinning. Increased TUNEL-positive cells in the INL confirm that cells die, at least in part, because of apoptosis. These findings propose a previously undescribed mechanism of cell death and synaptic retraction that are likely at the origin of the functional consequences of hyperoxia.

Expression and cellular localization of the voltage-gated calcium channel α2δ3 in the rodent retina

High-voltage-activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes, including the influx of extracellular Ca2+, neurotransmitter release, gene transcription, and synaptic plasticity. These channels consist of a primary α1 pore-forming subunit, which is associated with an extracellular α2δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α2δ3 subunit in the mouse and rat retina is unknown. In this study using RT-PCR, a single band at ∼305 bp corresponding to the predicted size of the α2δ3 subunit fragment was found in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123-kDa band. Immunohistochemistry with an affinity-purified antibody to the α2δ3 subunit revealed immunoreactive cell bodies in the ganglion cell layer and inner nuclear layer and immunoreactive processes in the inner plexiform layer and the outer plexiform layer. α2δ3 immunoreactivity was localized to multiple cell types, including ganglion, amacrine, and bipolar cells and photoreceptors, but not horizontal cells. The expression of the α2δ3 calcium channel subunit to multiple cell types suggests that this subunit participates widely in Ca-channel-mediated signaling in the retina.

Microglia activation in a model of retinal degeneration and TUDCA neuroprotective effects

Background: Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound. Methods: For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified. Results: Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space. Conclusions: These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.

Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa

Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi−) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

Astrocytes and Müller Cell Alterations During Retinal Degeneration in a Transgenic Rat Model of Retinitis Pigmentosa

Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes, and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer (GCL) of P23H vs. SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.