Microtubule alterations may destabilize photoreceptor integrity: Age-related microtubule changes and pattern of expression of MAP-2, Tau and hyperphosphorylated Tau in aging human photoreceptor cells.

Photoreceptor cells undergo changes with aging. It is unknown if their microtubules are stable or not with aging. This study examined photoreceptor cell ultrastructure from 18 human donor retinas (32 eyes; age: 45-94 years) and quantified the photoreceptors with altered microtubules over six to ninth decades in four defined retinal regions. In addition, immunoreactivity (IR) to microtubule-associated protein-2 (MAP-2), tau and hyperphophorylated tau was performed in retinal sections from companion eyes. In young donor retinas below 75 years of age, microtubules appeared straight in photoreceptor inner segments and axons. With age, they appeared bent or misaligned in macular and mid-peripheral photoreceptors. In addition, dense granular materials were present in photoreceptor axons and synaptic terminals in advanced ages. In all decades, rod microtubules were affected more than their cone counterparts (28% vs 15%, p < 0.005). Both rods and cones were significantly affected in mid-peripheral retina (5-8 mm outside the macular border) in eighth decade, compared to other decades or retinal regions (parafoveal, perifoveal and nasal) examined (p < 0.005). IR showed a steady expression of MAP-2 in inner segments, and tau in inner segments to axons below 75 years of age, but was absent for both markers in scattered macular and mid-peripheral photoreceptors in advanced ages (>75 years). IR to hyperphosphorylated tau was present mainly in inner retina and increased with aging. Markers of oxidative stress, e.g., lipid peroxidation (4-hydroxy 2-nonenal) and nitrosative stress (nitrotyrosine) were immunopositive in aged photoreceptors. The sporadic loss of MAP-2 and tau-IR in photoreceptors may be due to microtubule changes; all these changes may affect intracellular transport and be partly responsible for photoreceptor death in aged human retina.

Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFß and Wnt signaling.

Cone photoreceptors are required for color discrimination and high-resolution central vision and are lost in macular degenerations, cone and cone/rod dystrophies. Cone transplantation could represent a therapeutic solution. However, an abundant source of human cones remains difficult to obtain. Work performed in model organisms suggests that anterior neural cell fate is induced 'by default' if BMP, TGFß and Wnt activities are blocked, and that photoreceptor genesis operates through an S-cone default pathway. We report here that Coco (Dand5), a member of the Cerberus gene family, is expressed in the developing and adult mouse retina. Upon exposure to recombinant COCO, human embryonic stem cells (hESCs) differentiated into S-cone photoreceptors, developed an inner segment-like protrusion, and could degrade cGMP when exposed to light. Addition of thyroid hormone resulted in a transition from a unique S-cone population toward a mixed M/S-cone population. When cultured at confluence for a prolonged period of time, COCO-exposed hESCs spontaneously developed into a cellular sheet composed of polarized cone photoreceptors. COCO showed dose-dependent and synergistic activity with IGF1 at blocking BMP/TGFß/Wnt signaling, while its cone-inducing activity was blocked in a dose-dependent manner by exposure to BMP, TGFß or Wnt-related proteins. Our work thus provides a unique platform to produce human cones for developmental, biochemical and therapeutic studies and supports the hypothesis that photoreceptor differentiation operates through an S-cone default pathway during human retinal development.

Age related distribution of 4-hydroxy 2-nonenal immunoreactivity in human retina.

The retina is prone to be damaged by oxidative stress (OS), owing to its constant exposure to light, high rate of oxygen consumption and high membrane lipid content. Lipid peroxidation in aging human retina has been shown by biochemical means. However, information on the cellular sites of OS and antioxidant responses in aging human retina remains limited. Here, we show distribution of immunoreactivity (IR) to a marker of lipid peroxidation (4-hydroxy 2-nonenal [HNE] and antioxidant enzymes involved in counteracting lipid peroxidation (glutathione S-transferase-π1 and glutarexoxin-1) in donor human retinas at different ages (35-91 years; N = 24). Initially, HNE-IR was present in few macular cone outer segments (COS, sixth decade). With aging, IR appeared in many COS and peaked at ninth decade (14 vs 62 per 3850 µm2 area between 6 and 9 decade; p < 0.001) in the parafovea then seen elsewhere (perifoveal, mid-peripheral and nasal). IR was seen in the parafovea of all retinas, whereas it was present in 8/24 of perifoveal and 6/24 of mid-peripheral retinas, indicating that the parafovea is susceptible to undergo lipid peroxidation. Foveolar COS were immunonegative until 81 years, which developed IR later (>83 years). IR to glutathione S-transferase-π1 was moderate until eight decade and then showed a decrease in photoreceptor cells between ninth and tenth decade, while glutaredoxin-1 maintained a steady expression with aging. Damaged COS were present in aged retinas, and inner segments and photoreceptor nuclei also showed some degree of alterations. Although there was increased lipid peroxidation with aging, cone death was minimal in those retinas. The two antioxidant enzymes studied here, may play a role in protecting photoreceptors against OS with advanced aging.

Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods.

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate-aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.

Differential Expression of AQP1 and AQP4 in Avascular Chick Retina Exposed to Moderate Light of Variable Photoperiods.

Aquaporins (AQPs) are integral membrane proteins which maintain cellular water and ion homeostasis. Alterations in AQP expression have been reported in rod-dominated rodent retinas exposed to light. In rodents and also in birds, light of moderate intensities (700-2000 lux) damages the retina, though detailed changes were not examined in birds. The aim of our study was to see if light affects cone dominated retinas, which would be reflected in expression levels of AQPs. We examined AQP1 and AQP4 expressions in chick retina exposed to 2000 lux under 12 h light:12 h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod) and 24L:0D (constant light). Additionally, morphological changes, apoptosis (by TUNEL) and levels of glutamate and GFAP (a marker of injury) in the retina were examined to correlate these with AQP expressions. Constant light caused damage in outer and inner nuclear layer (ONL, INL) and ganglion cell layer (GCL). Also, there were associated increases in GFAP and glutamate levels in retinal extracts. In normal photoperiod, AQP1 was expressed in GCL, outer part of INL and photoreceptor inner segments of. AQP4 was additionally expressed in nerve fiber layer. Immunohistochemistry and Western blotting revealed over all decreased AQP1 and AQP4 expression in constant light condition compared to those in other two groups. The elevated GFAP and glutamate levels might be involved in the reduction of AQPs in constant light group. Such decreases in AQP expressions are perhaps linked with retinal cell damage seen in constant light condition, while their relatively enhanced expression in two other conditions may help in maintaining a normal retinal architecture, indicating their neuroprotective potential.

Accumulation of lipid inclusions in astrocytes of aging human optic nerve.

We examined age-related changes in the human optic nerve (ON) from 10 postmortem donor eye samples (age: 21- to 94-year-old). In aged ON, many axons showed paucity of cytoskeleton, and possessed disorganized myelin that remained in the extracellular space. Lipid inclusions were detected in glia, as stained by oil red O, and these accumulated with aging. To identify and confirm which glial cell type possessed lipid inclusions, we performed immunohistochemistry (IHC) and transmission electron microscopy (TEM). Comparisons were made from TEM features and size of the glia immunolabeled with glial fibrillary acidic protein and glutamine synthetase (markers for astrocytes) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (a marker for oligodendrocytes). It was found that lipid inclusions were restricted to the astrocytes having larger perikarya than the oligodendrocytes (IHC) and possessing filaments in cytoplasm (TEM). These astrocytes also possessed myelin debris and it is thus likely that those inclusions originated from degenerated myelin of the ON axons. These data indicate that astrocytes play a role in phagocytosis and clearance of disorganized myelin in aging human ON.

Expression of synaptic proteins in the hippocampus and spatial learning in chicks following prenatal auditory stimulation.

Prenatal auditory stimulation by species-specific sound influences the expression and levels of calcium-binding proteins in the chick hippocampus, which is important to learning and memory. Stimulation by sitar music additionally produces structural changes in the hippocampus. Synapse density, which influences the synaptic plasticity, is also increased following both types of sound stimulation. Here we report the expression of mRNA as well as levels of synaptic proteins (synaptophysin, synapsin I and PSD-95) in the hippocampus of developing chicks subjected to prenatal auditory stimulation. Further, to evaluate the behavioral outcome following acoustic stimulation, posthatch day 1 (PH1) chicks were analyzed by T-maze test for spatial learning. Fertilized zero day eggs were incubated under normal conditions and subjected to patterned sounds of species-specific or sitar music at 65 dB levels for 15 min/h over 24 h at a frequency range of 100-6,300 Hz for a period of 11 days from embryonic day (E) 10 until hatching. Following both types of prenatal acoustic stimulation, a significant increase in the levels of synaptophysin mRNA and protein was found from E12, whereas that of synapsin I and PSD-95 was observed from E16, suggesting early maturation of the excitatory synapse. A significant decrease in the time taken to reach the target over the 3 trials in both sound-stimulated groups indicates improved spatial learning. In the music-stimulated group, however, the time taken to reach the target was reduced from the very first trial, which may point to an involvement of other behavioral attributes in facilitating spatial navigation.

Effect of prenatal auditory stimulation on numerical synaptic density and mean synaptic height in the posthatch Day 1 chick hippocampus.

Previous studies on prenatal auditory stimulation by species-specific sound or sitar music showed enhanced morphological and biochemical changes in chick hippocampus, which plays an important role in learning and memory. Changes in the efficiency of synapses, synaptic morphology and de novo synapse formation affects learning and memory. Therefore, in the present study, we set out to investigate the mean synaptic density and mean synaptic height at posthatch Day 1 in dorsal and ventral part of chick hippocampus following prenatal auditory stimulation. Fertilized 0 day eggs of domestic chick incubated under normal conditions were exposed to patterned sounds of species-specific and sitar music at 65 dB levels for 15 min/h round the clock (frequency range: 100-6300 Hz) from embryonic Day 10 till hatching. The synapses identified under transmission electron microscope were estimated for their numerical density by physical disector method and also the mean synaptic height calculated. Our results demonstrate a significant increase in mean synaptic density with no alterations in the mean synaptic height following both types of auditory stimulation in the dorsal as well as ventral part of the hippocampus. The observed increase in mean synaptic density suggests enhanced synaptic substrate to strengthen hippocampal function.

Localization of nitro-tyrosine immunoreactivity in human retina.

Oxidative stress (OS) is associated with retinal aging and age-related macular degeneration (AMD). In both cases there are reports for the presence of markers of lipid peroxidation in retinal cells. We investigated if nitrosative stress also occurs in the human retina with aging. We examined the cellular localization of nitro-tyrosine, a biomarker of protein tyrosine nitration, in human donor retina (17-91 years; N = 15) by immunohistochemistry. Immunoreactivity (IR) to nitro-tyrosine was present in ten retinas and absent in five retinas. It was predominant in photoreceptor inner segments, cell bodies and axons. In six retinas, IR was present in abnormal, swollen axons of macular and peripheral cones. In the inner retina, weak immunoreactivity was detected in the outer and inner plexiform layer. Transmission electron microscopy revealed a variable degree of microtubule disorganization, abnormal outgrowth from the swollen macular axons (as the fibers of Henle) and few dead axons. The present study adds further evidence to the presence of aberrant photoreceptor axonal changes in the human retina and that nitro-tyrosine immunoreactivity is associated with the photoreceptor cells in select human retina.

In ovo Sound Stimulation Mediated Regulation of BDNF in the Auditory Cortex and Hippocampus of Neonatal Chicks.

Brain-derived neurotrophic factor (BDNF) is known to mediate activity-dependent changes in the developing auditory system. Its expression in the brainstem auditory nuclei, auditory cortex and hippocampus of neonatal chicks (Gallus gallus domesticus) in response to in ovo high intensity sound exposure at 110 dB (arrhythmic sound: recorded traffic noise, 30-3000 Hz with peak at 2700 Hz, rhythmic sound: sitar music, 100-4000 Hz) was examined to understand the previously reported altered volume and neuronal number in these regions. In the brainstem auditory nuclei, no mature BDNF, but proBDNF at the protein level was detected, and no change in its levels was observed after in ovo sound stimulation (music and noise). Increased ProBDNF protein levels were found in the auditory cortex in response to arrhythmic sound, along with decreased levels of one of the BDNF mRNA transcripts, in response to both rhythmic and arrhythmic sound stimulation. In the hippocampus, increased levels of mature BDNF were found in response to music. Expression microarray analysis was performed to understand changes in gene expression in the hippocampus in response to music and noise, followed by gene ontology analysis showing enrichment of probable signaling pathways. Differentially expressed genes like CAMK1 and STAT1 were found to be involved in downstream signaling on comparing music versus noise-exposed chicks. In conclusion, we report that BDNF is differentially regulated in the auditory cortex at the transcriptional and post-translational level, and in the hippocampus at the post-translational level in response to in ovo sound stimulation.

Calbindin D-28K and parvalbumin expression in embryonic chick hippocampus is enhanced by prenatal auditory stimulation.

Calcium-binding proteins (CaBPs) buffer excess of cytosolic Ca(2+), which accompanies neuronal activity following external stimuli. Prenatal auditory stimulation by species-specific sound and music influences early maturation of the auditory pathway and the behavioral responses in chicks. In this study, we determined the volume, total number of neurons, proportion of calbindin D-28K and parvalbumin-positive neurons along with their levels of expression in the developing chick hippocampus following prenatal auditory stimulation. Fertilized eggs of domestic chicks were exposed to sounds of either species-specific calls or sitar music at 65 dB for 15 min/h round the clock from embryonic day (E) 10 until hatching. Hippocampi of developmental stages (E12, E16 and E20) were examined. With an increase in embryonic age during normal development, the hippocampus showed an increase in its volume, total number of neurons as well as in the neuron proportions and levels of expression of calbindin D-28K and parvalbumin. A significant increase of volume at E20 was noted only in the music-stimulated group compared to that of their age-matched control (p<0.05). On the other hand, both auditory-stimulated groups showed a significant increase in the proportion of immunopositive neurons and the levels of expression of calbindin D-28K and parvalbumin as compared to the control at all developmental stages studied (p<0.003). The increase in proportions of CaBP neurons during development and in the sound-enriched groups suggests an activity-dependent increase in Ca(2+) influx. The enhanced expression of CaBPs may help in cell survival by preventing excitotoxic death of neurons during development and may also be involved in long-term potentiation.

Nimodipine is more effective than nifedipine in attenuating morphine tolerance on chronic co-administration in the rat tail-flick test.

Opioids, when co-administered with L-type calcium channel blockers (L-CCBs) show morphine like higher antinociceptive effect. This antinociceptive effect has been further investigated using a different experimental paradigm. The effect of two different L-CCBs (nifedipine and nimodipine) on morphine-induced antinociception was studied by the tail-flick test (40 min after morphine administration) in adult Wistar rats. A fixed-dose of nimodipine or nifedipine (2 mg/kg, once daily) was combined with a fixed dose of morphine (10 mg/kg, twice daily) for 10 days. Co-administration of L-CCBs significantly increased the antinociceptive effect of morphine, even 12 hr after administration. Also, nimodipine was more effective than nifedipine. Nimodipine was further studied using a higher and escalating doses of morphine (20-30 mg/kg twice daily for 14 days). Nimodipine increased the antinociceptive effect of morphine in the latter part of the study (days nine to fourteen) though significant difference was observed on 11th evening and 12th morning. No obvious adverse effects were observed in the present study. The results show for the first time that nimodipine is more effective than nifedipine and that these L-CCBs continue to be effective, even 12 hr after administration in the tail-flick test.

Morphological and morphometrical maturation of ventral cochlear nucleus in human foetus.

Auditory impulses perceived by the hair cells of the organ of corti are relayed in the cochlear nucleus, the first relay station in the brainstem, by the cochlear nerve. The human foetus is well known to respond to sound during the last trimester of gestation. On the contrary, studies conducted in rat, cat and mouse have shown that these mammals have an immature auditory system at the time of birth. There are very few reports available regarding the morphological and functional maturation of the cochlear nucleus in human. Although the human cochlear nucleus neurons attain adult morphological characters by mid-gestation, there are hardly any studies discussing the functional maturation of the cochlear nucleus. Hence the present study was aimed at observing the morphological as well as functional maturation of the human foetal cochlear nuclei at various gestational ages. Morphological maturation was observed qualitatively while stereological estimation of the volume of well defined ventral cochlear nucleus (VCN) was calculated by the Cavalieri principle; neuronal count and density was estimated by dissector principle. The functional maturation was assessed by observing the expression of synaptophysin, a synaptic marker, at different gestational ages and by the presence of parvalbumin, a calcium binding functional neuronal marker by immunohistochemistry. Neurons showed coarse Nissl's substance and well developed cell processes and gradual increase in cell size by the 24th-30th gestational week. Synaptophysin labeling in the complete cochlear nucleus was observed at 20 weeks of gestation. Adult pattern of synaptophysin labeling was observed finally at37weeks of gestation. Earliest presence of parvalbumin expression was detected at 16 weeks of gestation and a distinct adult pattern was seen at 37 weeks of gestation. This study concluded that morphological and functional maturation of the human cochlear nuclei occurs simultaneously during mid-gestation which represents the critical period of development and continues up to term.

Intrathecal co-administration of morphine and nimodipine produces higher antinociceptive effect by synergistic interaction as evident by injecting different doses of each drug in rats.

Earlier, we reported that morphine-nimodipine combination produces significantly higher antinociception after intrathecal but not after systemic administration in mice. Different doses of morphine and nimodipine (5 microg of morphine, 5 microg of nimodipine, 5 microg each of morphine and nimodipine, 10 microg of morphine, 10 microg of nimodipine, 10 microg morphine with 5 microg nimodipine and 5 microg of morphine with 10 microg of nimodipine) were now injected intrathecally in Wistar rats to further characterise this antinociceptive effect. The acute antinociceptive effect was measured by the tail-flick test between 15 min to 7 h. The onset of maximum antinociception (100% MPE) was earlier (by 15 min) in nimodipine (5 microg) than in morphine (5 microg) treated group (by 30 min). Though earlier in onset, 5 microg nimodipine produced transient antinociception, which was significantly higher than saline treated controls for the initial 30 min only. Morphine (5 microg) produced significantly higher antinociception between 15 min to 3:30 h in comparison to control animals. However, co-administration of both morphine and nimodipine led to significantly higher antinociception than morphine alone at 4:00 h and also between 5:00 to 6:30 h. Interestingly, the combined antinociceptive action of morphine and nimodipine was not significantly different from 10 microg of morphine, which indicated synergistic interaction. Naloxone (5 mg/kg) could reverse this antinociceptive effect of morphine-nimodipine combination though it failed to reverse nimodipine (5 microg)-mediated antinociception at 15 min. Increasing the dose of either morphine or nimodipine to 10 mug did not increase antinociception except between 6:30-7:00 h. No obvious side effect was noted after administration of either morphine or nimodipine or both.

Experimental oral iron administration: Histological investigations and expressions of iron handling proteins in rat retina with aging.

Iron is implicated in age-related macular degeneration (AMD). The aim of this study was to see if long-term, experimental iron administration with aging modifies retinal and choroidal structures and expressions of iron handling proteins, to understand some aspects of iron homeostasis. Male Wistar rats were fed with ferrous sulphate heptahydrate (500mg/kg body weight/week, oral; elemental iron availability: 20%) from 2 months of age onward until they were 19.5 month-old. At 8, 14 and 20 months of age, they were sacrificed and serum and retinal iron levels were detected by HPLC. Oxidative stress was analyzed by TBARS method. The retinas were examined for cell death (TUNEL), histology (electron microscopy) and the expressions of transferrin, transferrin receptor-1 [TFR-1], H- and L-ferritin. In control animals, at any age, there was no difference in the serum and retinal iron levels, but the latter increased significantly in 14- and 20 month-old iron-fed rats, indicating that retinal iron accumulation proceeds with progression of aging (>14 months). The serum and retinal TBARS levels increased significantly with progression of aging in experimental but not in control rats. There was significant damage to choriocapillaris, accumulation of phagosomes in retinal pigment epithelium and increased incidence of TUNEL+ cells in outer nuclear layer and vacuolation in inner nuclear layer (INL) of 20 month-aged experimental rats, compared to those in age-matched controls. Vacuolations in INL could indicate a long-term effect of iron accumulation in the inner retina. These events paralleled the increased expression of ferritins and transferrin and a decrease in the expression of TFR-1 in iron-fed rats with aging, thereby maintaining iron homeostasis in the retina. As some of these changes mimic with those happening in eyes with AMD, this model can be utilized to understand iron-induced pathophysiological changes in AMD.

Prenatal acoustic stimulation influences neuronal size and the expression of calcium-binding proteins (calbindin D-28K and parvalbumin) in chick hippocampus.

Prenatal auditory enrichment by species-specific sounds and sitar music enhances the expression of immediate early genes, synaptic proteins and calcium binding proteins (CaBPs) as well as modifies the structural components of the brainstem auditory nuclei and auditory imprinting area in chicks. There is also facilitation of postnatal auditory preference of the chicks to maternal calls following both types of sound stimulation indicating prenatal perceptual learning. To examine whether the sound enrichment protocol also affects the areas related to learning and memory, we assessed morphological changes in the hippocampus at post-hatch day 1 of control and prenatally sound-stimulated chicks. Additionally, the proportions of neurons containing calbindin D-28K and parvalbumin immunoreactivity as well as their protein levels were determined. Fertilized eggs of domestic chick were incubated under normal conditions of temperature, humidity, forced draft of air as well as light and dark (12:12h) photoperiods. They were exposed to patterned sounds of species-specific and sitar music at 65 dB for 15 min per hour over a day/night cycle from day 10 of incubation till hatching. The hippocampal volume, neuronal nuclear size and total number of neurons showed a significant increase in the music-stimulated group as compared to the species-specific sound-stimulated and control groups. However, in both the auditory-stimulated groups the protein levels of calbindin and parvalbumin as well as the percentage of the immunopositive neurons were increased. The enhanced proportion of CaBPs in the sound-enriched groups suggests greater Ca(2+) influx, which may influence long-term potentiation and short-term memory.

Vascular changes of the retina and choroid in systemic lupus erythematosus: pathology and pathogenesis.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that affects multiple organ systems. When eyes are involved, the principle manifestations are hemorrhage, retinal cotton wool spots, microangiopathy and vaso-occlusion. Research in the past two decades has significantly contributed to our understanding about this disease in general and its therapeutic management, although knowledge about the mechanism of ocular involvement and pathogenesis in SLE is limited. This is an important issue, because the ocular symptoms in this disease could be potentially sight threatening in acute cases. Here, we present an overview of the clinical and histopathologic features of retinal and choroidal vascular changes, as seen in patients with SLE. We discuss the role of immune complex deposition in vascular pathogenesis in the eye. Reports indicated an involvement of antiphospholipid antibodies (APAs) in the retinal and choroidal vasculopathy in SLE, although their precise role in this process is uncertain. It is important to look for mechanisms of immune complex-mediated vasculopathy and role of inflammatory mediators in this process in SLE. For this, established animal models can be utilized in research to learn about the precise role of various autoantibodies and complements involved in disease pathogenesis. A clear knowledge about the immunopathogenesis is warranted, and the rationale for the future therapy should be based on reducing vascular inflammation as well as ameliorating autoimmunity in this disease.

The occurrence of cone inclusions in the ageing human retina and their possible effect upon vision: an electron microscope study.

During normal ageing, photoreceptors of the human retina undergo various structural changes. We examined retinas from 33 donors (56 eyes; age span 13-94 years) by electron microscopy to see morphological changes in the cones with ageing. We show mitochondrial alterations and occurrence of electron-dense globules in the cone inner segments from the fifth decade of life. The globules are more prevalent in the macular cones than those in the mid-peripheral or nasal retinas (p<0.05) and absent in peripheral retinal cones and rods. They peak in the sixth decade and then decline in the seventh decade (p<0.05), from seventh to ninth decade, however, there was no significant change in their occurrence in the cones. We also show a type of inclusion, made up of bundled microtubules, which occur exclusively in the macular cones at the eighth decade of life. Evidence suggests that altered cone mitochondria with cristae remnants and dense matrix participate in globule formation in the ageing retina. Such mitochondrial changes may cause energy depletion, and bundling of microtubules (to form filamentous inclusions) could result in decreasing intracellular transport, in which case cones may die in the long run. These factors may be responsible for reported cone loss in the human retina with ageing.

Immunolocalisation pattern of complex I-V in ageing human retina: Correlation with mitochondrial ultrastructure.

Earlier studies reported accumulation of mitochondrial DNA mutations in ageing and age-related macular degeneration. To know about the mitochondrial status with age, we examined immunoreactivity (IR) to markers of mitochondria (anti-mitochondrial antibody and voltage-dependent anion channel-1) and complex I-V (that mediate oxidative phosphorylation, OXPHOS) in donor human retinas (age: 19-94years; N=26; right eyes). In all samples, at all ages, IR to anti-mitochondrial antibody and voltage-dependent anion channel-1 was prominent in photoreceptor cells. Between second and seventh decade of life, strong IR to complex I-V was present in photoreceptors over macular to peripheral retina. With progressive ageing, the photoreceptors showed a decrease in complex I-IR (subunit NDUFB4) at eighth decade, and a weak or absence of IR in 10 retinas between ninth and tenth decade. Patchy IR to complex III and complex IV was detected at different ages. IR to ND1 (complex I) and complex II and V remained unaltered with ageing. Nitrosative stress (evaluated by IR to a nitro-tyrosine antibody) was found in photoreceptors. Superoxide dismutase-2 was found upregulated in photoreceptors with ageing. Mitochondrial ultrastructure was examined in two young retinas with intact complex IR and six aged retinas whose counterparts showed weak to absence of IR. Observations revealed irregular, photoreceptor inner segment mitochondria in aged maculae and mid-peripheral retina between eighth and ninth decade; many cones possessed autophagosomes with damaged mitochondria, indicating age-related alterations. A trend in age-dependent reduction of complex I-IR was evident in aged photoreceptors, whereas patchy complex IV-IR (subunits I and II) was age-independent, suggesting that the former is prone to damage with ageing perhaps due to oxidative stress. These changes in OXPHOS system may influence the energy budget of human photoreceptors, affecting their viability.

Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light of variable photoperiods.

Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration in human. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Although birds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences a cone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina of white Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7 days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 lux at 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed for transmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synaptic proteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in 24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantly decreased expressions of opsins and synaptic proteins, compared to those seen in 12L:12D and 18L:6D conditions. Also, there were ultrastructural changes in outer and inner plexiform layer (OPL, IPL) of the retinas exposed to 24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, with changes (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic protein expressions in OPL and IPL and death of degenerated axonal processes in IPL.