Gema Esquiva

Safranal, a Saffron Constituent, Attenuates Retinal Degeneration in P23H Rats

Saffron, an extract from Crocus sativus, has been largely used in traditional medicine for its antiapoptotic and anticarcinogenic properties. In this work, we investigate the effects of safranal, a component of saffron stigmas, in attenuating retinal degeneration in the P23H rat model of autosomal dominant retinitis pigmentosa. We demonstrate that administration of safranal to homozygous P23H line-3 rats preserves both photoreceptor morphology and number. Electroretinographic recordings showed higher a- and b-wave amplitudes under both photopic and scotopic conditions in safranal-treated versus non-treated animals. Furthermore, the capillary network in safranal-treated animals was preserved, unlike that found in untreated animals. Our findings indicate that dietary supplementation with safranal slows photoreceptor cell degeneration and ameliorates the loss of retinal function and vascular network disruption in P23H rats. This work also suggests that safranal could be potentially useful to retard retinal degeneration in patients with retinitis pigmentosa.

Impairment of Intrinsically Photosensitive Retinal Ganglion Cells Associated With Late Stages of Retinal Degeneration

PURPOSE To evaluate quantitative and qualitative age-related changes in intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) in transgenic P23H rats, an animal model of autosomal dominant retinitis pigmentosa (RP) was examined. METHODS ipRGC density, morphology, and integrity were characterized by immunohistochemistry in retinas extracted from P23H and Sprague-Dawley (SD) rats aged 4, 12, and 18 months. Differences between SD and P23H rats throughout the experimental stages, as well as the interactions among them, were morphologically evaluated. RESULTS In rat retinas, we have identified ipRGCs with dendrites stratifying in either the outer margin (M1) or inner side (M2) of the inner plexiform layer, and in both the outer and inner plexuses (M3). A small group of M1 cells had their somas located in the inner nuclear layer (M1d). In SD rats, ipRGCs showed no significant changes associated with age, in terms of either mean cell density or the morphologic parameters analyzed. However, the mean density of ipRGCs in P23H rats fell by approximately 67% between 4 and 18 months of age. Moreover, ipRGCs in these animals showed a progressive age-dependent decrease in the dendritic area, the number of branch points and terminal neurite tips per cell, and the Sholl area. CONCLUSIONS In the P23H rat model of retinitis pigmentosa, density, wholeness, and dendritic arborization of melanopsin-containing ganglion cells decrease in advanced stages of the degenerative disease.

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.

Circadian dysfunction in P23H rhodopsin transgenic rats: effects of exogenous melatonin.

Abstract:  This study focuses on the effects of retinal degeneration on the circadian patterns of P23H rats, as well as on the effect of exogenous melatonin administration. To this end, the body temperature of P23H and Sprague–Dawley rats was continuously monitored and their retinas examined at different stages of degeneration, by means of histological labeling and electroretinogram recordings. Melatonin (2 mg/kg BW/day) was supplied ad libitum throughout the experiment to a subset of animals. The body temperature recordings from wild‐type and mutant animals showed no differences in the periodogram and the pattern of the mean waveform. However, a progressive decrease in the relative amplitude of the rhythm (RA), a decline in the coupling strength of the rhythm to environmental zeitgebers (interdaily stability, IS) and increased rhythm fragmentation (intradaily variability, IV) were observed in P23H rats, when compared to wild‐type animals. The P23H animals showed a progressive decrease in light‐induced retinal responses until reaching 18 months of age. By this age, all photoreceptors had already disappeared, and no responses were found in the EGRs. Exogenous administration of melatonin improved the visual response of P23H rats. In fact, the maximum b‐wave recorded at 14 months of age was significantly higher in melatonin‐treated P23H rats than in the control animals. Furthermore, the maximum b‐wave recorded for P23H rats at the age of 14 months significantly correlated with RA, IS, and IV. This leads us to conclude that vision loss in P23H rats is correlated with a progressive fragmentation of their circadian patterns. Both effects are partially reversed by melatonin administration.

Circadian dysfunction in a rotenone-induced parkinsonian rodent model.

Parkinsons disease (PD) is a neurodegenerative disorder that also involves circadian rhythm alterations. Modifications of circadian rhythm parameters have been shown to occur in both PD patients and toxin-induced PD animal models. In the latter case, rotenone, a potent inhibitor of mitochondrial complex I (nicotinamide adenine dinucleotide [NADH]-quinone reductase), has been used to elicit degeneration of dopaminergic neurons and development of parkinsonian syndrome. The present work addresses alterations induced by rotenone on both locomotor and body temperature circadian rhythms in rats. Rotenone-treated rats exhibited abnormalities in equilibrium, postural instability, and involuntary movements. Long-term subcutaneous administration of rotenone significantly reduced mean daily locomotor activity in most animals. During rotenone administration, mean body temperatures (BTs) and BT rhythm amplitudes were significantly lower than those observed in the control group. After long-term rotenone administration, the circadian rhythms of both locomotor activity (LA) and BT displayed decreased amplitudes, lower interdaily phase stability, and higher rhythm fragmentation, as compared to the control rats. The magnitude of the LA and BT circadian rhythm alterations induced by rotenone positively correlated with degree of motor impairment. These results indicate that rotenone induces circadian dysfunction in rats through some of the same mechanisms as those responsible for the development of motor disturbances. (Author correspondence: pedro.lax@ua.es)

Loss of Melanopsin-Expressing Ganglion Cell Subtypes and Dendritic Degeneration in the Aging Human Retina

In mammals, melanopsin-expressing retinal ganglion cells (mRGCs) are, among other things, involved in several non-image-forming visual functions, including light entrainment of circadian rhythms. Considering the profound impact of aging on visual function and ophthalmic diseases, here we evaluate changes in mRGCs throughout the life span in humans. In 24 post-mortem retinas from anonymous human donors aged 10–81 years, we assessed the distribution, number and morphology of mRGCs by immunostaining vertical retinal sections and whole-mount retinas with antibodies against melanopsin. Human retinas showed melanopsin immunoreactivity in the cell body, axon and dendrites of a subset of ganglion cells at all ages tested. Nearly half of the mRGCs (51%) were located within the ganglion cell layer (GCL), and stratified in the outer (M1, 12%) or inner (M2, 16%) margin of the inner plexiform layer (IPL) or in both plexuses (M3, 23%). M1 and M2 cells conformed fairly irregular mosaics, while M3 cell distribution was slightly more regular. The rest of the mRGCs were more regularly arranged in the inner nuclear layer (INL) and stratified in the outer margin of the IPL (M1d, 49%). The quantity of each cell type decrease after age 70, when the total number of mRGCs was 31% lower than in donors aged 30–50 years. Moreover, in retinas with an age greater than 50 years, mRGCs evidenced a decrease in the dendritic area that was both progressive and age-dependent, as well as fewer branch points and terminal neurite tips per cell and a smaller Sholl area. After 70 years of age, the distribution profile of the mRGCs was closer to a random pattern than was observed in younger retinas. We conclude that advanced age is associated with a loss in density and dendritic arborization of the mRGCs in human retinas, possibly accounting for the more frequent occurrence of circadian rhythm disorders in elderly persons.

Age-related changes in photosensitive melanopsin-expressing retinal ganglion cells correlate with circadian rhythm impairments in sighted and blind rats

ABSTRACT The melanopsin system consists of intrinsically photosensitive retinal ganglion cells containing the photopigment melanopsin (mRGCs). These mRGCs mediate several non-image-forming visual functions, including light entrainment of circadian rhythms. Here we evaluate age-related alterations of the melanopsin system and circadian rhythms in P23H line 1 (P23H-1) rats, a rodent model of retinitis pigmentosa (RP). In homozygous P23H-1 rats and wild-type control rats from the same genetic background (Sprague–Dawley), body temperature and locomotor activity were continuously monitored at 10-min intervals for 7 days, once every 4–5 weeks, between 2 and 24 months of age, using a telemetry transmitter. The distribution and number of mRGCs were assessed in control rats at 12, 18, and 24 months of age and in P23H-1 rats aged 12, 18, 24, and 30 months by immunostaining whole-mount retinas with antibodies against melanopsin. The mean density of mRGCs in control rats showed no significant variations when evaluated at 12 and 18 months of age, and fell by approximately 56% between 18 and 24 months of age. Meanwhile, a significant decrease in the mean number of mRGCs was found in 18-month-old P23H-1 rats as compared to 18-month-old control rats (81% decrease). Parametric and non-parametric analyses of the records showed a gradual age-dependent weakening of body temperature and locomotor activity circadian rhythms robustness in both control and P23H-1 rats from 2 to 24 months of age. However, body temperature and locomotor activity circadian patterns were less robust throughout the experiment in P23H-1 as compared to control rats, with lower amplitude, weaker coupling strength to environmental zeitgebers and higher fragmentation of the rhythms. The present study shows that the degeneration of photoreceptors and inner retinal neurons, characteristic of RP, has age-related degenerative effects on the melanopsin system and is associated with weaker circadian patterns.

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.

Retinal Vascular Degeneration in the Transgenic P23H Rat Model of Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases involving a progressive degeneration of photoreceptor cells. Following the loss of photoreceptors, retinal vascularization tends to decrease, which seems to play a role in the degenerative process of retinal cells. This study reports changes in retinal vascular network architecture in the P23H rat model of RP at different stages of retinal degeneration. Homozygous P23H line-3 rats of ages ranging from 18 days to 16 months were used in this study. Age-matched Sprague-Dawley (SD) rats were used as control animals. Vertical sections and wholemount retinas were immunolabeled for type IV collagen or stained using NADPH diaphorase histochemistry, and retinal vascular networks were drawn using a camera lucida. The superficial and deep capillary plexus (DCP) were fully developed at P18 in P23H rat retinas and showed no differences from the control animals. In 4-month-old P23H rat retinas, the superficial and intermediate capillary plexus were similar to those observed in age-matched SD rats, but a reduction in the DCP could be observed in these animals, with a significant decrease in both capillary density and capillary loops. At 16 months, the DCP was completely lost, and only vessels exhibiting an abnormal, tortuous dead-end could be observed. The middle capillary plexus had virtually disappeared at this age. Only perpendicular vessels connecting the superficial and DCP were found. The superficial plexus showed no changes in the vascular surface with age. In RP, photoreceptor loss is accompanied by degenerative changes in the retinal vascular network. The disruption of the capillary plexus, with loss of capillary density and capillary loops, can hamper the normal supply of oxygen and nutrients to retinal cells, thus accelerating retinal degeneration. Therefore, changes in retinal vascularization must be taken into account in the design of therapies targeting retinal degenerative diseases.

Degeneration of human photosensitive retinal ganglion cells may explain sleep and circadian rhythms disorders in Parkinson’s disease

Parkinson’s disease (PD) patients often suffer from non-motor symptoms like sleep dysregulation, mood disturbances or circadian rhythms dysfunction. The melanopsin-containing retinal ganglion cells are involved in the control and regulation of these processes and may be affected in PD, as other retinal and visual implications have been described in the disease. Number and morphology of human melanopsin-containing retinal ganglion cells were evaluated by immunohistochemistry in eyes from donors with PD or control. The Sholl number of intersections, the number of branches, and the number of terminals from the Sholl analysis were significantly reduced in PD melanopsin ganglion cells. Also, the density of these cells significantly decreased in PD compared to controls. Degeneration and impairment of the retinal melanopsin system may affect to sleep and circadian dysfunction reported in PD pathology, and its protection or stimulation may lead to better disease prospect and global quality of life of patients.