Müller cell vulnerability in aging human retina: Implications on photoreceptor cell survival.

Müller glial cells (MC) support various metabolic functions of the retinal neurons, and maintain the homeostasis. Oxidative stress is intensified with aging, and in human retina, MC and photoreceptors undergo lipid peroxidation and protein nitration. Information on how MC respond to oxidative stress is vital to understand the fate of aging retinal neurons. This study examined age-related changes in MC of donor human retina (age: 35-98 years; N = 18 donors). Ultrastructural and immunohistochemical observations indicate that MC undergo gliosis and increased lipid peroxidation, and show osmotic changes with advanced aging (>80 years). Photoreceptor cells also undergo oxidative-nitrosative stress with aging, and their synapses also show clear osmotic swelling. MC respond to oxidative stress via proliferation of smooth endoplasmic reticulum in their processes, and increased expression of aquaporin-4 in endfeet and outer retina. In advanced aged retinas (81-98 years), they showed mitochondrial disorganisation, accumulation of lipids and autophagosomes, lipofuscin granules and axonal remnants in phagolysosomes in their inner processes, suggesting a reduced phagocytotic potential in them with aging. Glutamine synthetase expression does not alter until advanced aging, when the retinas show its increased expression in endfeet and Henle fiber layer. It is evident that MC are vulnerable with normal aging and this could be a reason for photoreceptor cell abnormalities reported with aging of the human retina.

Sesamol attenuates bleomycin-induced pulmonary toxicity and fibrosis in experimental animals.

Sesamol, a lignan obtained from roasted seeds of Sesamum indicum, has high antioxidant and anti-inflammatory activity. In this study, we have investigated the effect of sesamol on Bleomycin (BLM) induced pulmonary toxicity as well as fibrosis in Wistar rats. Lung toxicity was induced by administration of BLM, 0.015 U/g ip, twice weekly for 28 days whereas lung fibrosis was induced by BLM, 0.015 U/g ip, every 5th day for 49 days. Sesamol administration was started 7 days before first dose of BLM in both the models. It was observed that sesamol 50 mg/kg most effectively attenuated pulmonary toxicity by reducing oxidative stress, inflammation and apoptosis. This dose was further evaluated for its anti-fibrotic effect. It was observed that there was a significant reduction in fibrosis. Lung collagen content was markedly reduced. Furthermore, expression of pro-fibrotic proteins, TGF-ß/SMAD and α-SMA, was reduced and that of anti-fibrotic protein, AMPK, was markedly increased. Even though the combination of sesamol with pirfenidone exhibited no additional protection than either drug alone, it is evident from our study that our test drug, sesamol is comparable in efficacy to pirfenidone. Thus, sesamol has promising therapeutic potential in treatment of pulmonary toxicity and fibrosis.

Glycogen in retinal horizontal cells of the African mud catfish Clarias gariepinus (Burchell, 1822) and its physiological significance.

This paper reports on glycogen store in the retinal horizontal cells (HC) of the African mud catfish Clarias gariepinus, as seen by histochemical reaction with periodic acid Schiff (PAS) and transmission electron microscopy in light- as well as dark-adapted state. Glycogen is abundant in the large somata and less in their axons, characterised ultrastructurally by many microtubules and extensive gap junctions interconnecting them. There was no apparent difference in glycogen content in HC somata between light- and dark adaptation, but the axons clearly showed absence of glycogen in dark condition. The HC somata (presynaptic) make synapses with dendrites in the outer plexiform layer. Müller cell inner processes, which contain more densely packed glycogen, invest the HC. Other cells of the inner nuclear layer do not show any appreciable content of glycogen. Rods, but not cones, contain abundant glycogen in their inner segments and synaptic terminals. It is likely that glycogen is used as energy substrate in hypoxia for this species that dwell muddy aquatic environment with low oxygen content. They appear to have high energy demand, and a high glycogen content in HC could act as a ready source to fulfil physiological processes, like microtubule-based transport of cargo from the large somata to axons and the maintenance of electrical activities across the gap junctions between the axonal processes. It is also likely that they can supplement glucose to the neighbouring inner nuclear layer neurons, which are clearly devoid of glycogen.

Immunohistochemical features of cells in peripheral microcystoid retinal degeneration.

Peripheral microcystoid retinal degeneration (PMD) is an age-related, benign condition in which the peripheral retina develops small holes and undergoes cystic degeneration. This paper demonstrates neuronal alterations in PMD, as studied by immunohistochemistry in postmortem donor eyes (age: 76-89 years; N = 6 donors). In all cases, the degeneration was located in the inferior temporal quadrant, creating holes in the far peripheral retina. There was thinning of the inner retinal layers and the outer plexiform layer (OPL) was patchy or inconspicuous. As a response, Müller cell processes showed increased vimentin immunoreactivity. None of the retinas examined expressed glial fibrillary acidic protein. Cone photoreceptor cells were significantly altered: compared to the adjoining cones that were short, those located in the cystoid retina underwent significant elongation of their inner segments, evident from calbindin immunolabeling, to maintain synaptic contacts with the remnant OPL. The latter consisted of small photoreceptor terminals and scanty processes from shrunken bipolar cells. Besides, cones and ganglion cells undergo oxidative stress, they showed immunoreactivity to 4-hydroxy 2-nonenal and nitrotyrosine. The level of superoxide dismutase-2 was relatively low in the PMD region than in adjacent area, suggesting that the former suffers from oxidative stress.

Pathogenic mechanisms contributing to the vulnerability of aging human photoreceptor cells.

In human retina, photoreceptor cell death (PCD) is a slow but conspicuous event, which continues with aging. Rods die earlier than cones, the latter continue to alter in a subtle manner until advanced aging. This review summarizes the existing information on age-related changes in photoreceptor cells, especially cones and analyses the possible associated factors. Oxidative and nitrosative stress are involved in photoreceptor alterations, which may stem from light and iron toxicity and other sources. Lipid peroxidation in macular photoreceptor outer segments and mitochondrial aberrations are prominent in aging. It is important to understand how those changes ultimately trigger PCD. The redistribution of calbindin D-28K and long/middle-wavelength-sensitive opsin in the parafoveal and perifoveal cones, anomalies in their somata and axons are strong predictors of their increasing vulnerability with aging. Signs of reduced autophagy, with autophagosomes containing organelle remnants are seen in aging photoreceptor cells. Currently, mechanisms that lead to human PCD are unknown; some observations favour apoptosis as a pathway. Since cones appear to change slowly, there is an opportunity to reverse those changes before they die. Therefore, a full understanding of how cones alter and the molecular pathways they utilize for survival must be the future research goal. Recent approaches to prevent PCD in aging and diseases are highlighted.

Expression patterns of iron regulatory proteins after intense light exposure in a cone-dominated retina.

Iron is implicated in ocular diseases such as in age-related macular degeneration. Light is also considered as a pathological factor in this disease. Earlier, two studies reported the influence of constant light environment on the pattern of expressions of iron-handling proteins. Here, we aimed to see the influence of light in 12-h light-12-h dark (12L:12D) cycles on the expression of iron-handling proteins in chick retina. Chicks were exposed to 400 lx (control) and 5000 lx (experimental) light at 12L:12D cycles and sacrificed at variable timepoints. Retinal ferrous ion (Fe2+) level, ultrastructural changes, lipid peroxidation level, immunolocalization and expression patterns of iron-handling proteins were analysed after light exposure. Both total Fe2+ level (p = 0.0004) and lipid peroxidation (p = 0.002) significantly increased at 12-, 48- and 168-h timepoint (for Fe2+) and 48- and 168-h timepoint (for lipid peroxidation), and there were degenerative retinal changes after 168 h of light exposure. Intense light exposure led to an increase in the levels of transferrin and transferrin receptor-1 (at 168-h) and ferroportin-1, whereas the levels of ferritins, hephaestin, (at 24-, 48- and 168-h timepoint) and ceruloplasmin (at 168-h timepoint) were decreased. These changes in iron-handling proteins after light exposure are likely due to a disturbance in the iron storage pool evident from decreased ferritin levels, which would result in increased intracellular Fe2+ levels. To counteract this, Fe2+ is released into the extracellular space, an observation supported by increased expression of ferroportin-1. Ceruloplasmin was able to convert Fe2+ into Fe3+ until 48 h of light exposure, but its decreased expression with time (at 168-h timepoint) resulted in increased extracellular Fe2+ that might have caused oxidative stress and retinal cell damage.

Localization and hypersecretion of nerve growth factor in breast phyllodes tumors: Evidence from a preliminary study.

BACKGROUND: The pathophysiology of the breast phyllodes tumors is uncertain. Currently, wide surgical removal is the only available treatment option. The histopathological diagnosis of phyllodes tumors is often confused with that of fibroadenomas due to a striking histological resemblance. AIM: To identify a distinctive biomarker for phyllodes tumors of the breast. METHODS AND RESULTS: Fresh human breast tissue was obtained from surgically excised breast phyllodes and fibroadenoma tumors (test), breast cancer (positive control) and normal breast tissue (negative control). Immunohistochemistry and Sandwich ELISA were performed for the detection of nerve growth factor (NGF) in test and control tissues. A marked difference in NGF expression was detected in phyllodes tumors compared to fibroadenomas. The maximum NGF expression was observed in phyllodes tissue followed by cancer tissue, and the least expression in fibroadenomas (3-5 times less than in phyllodes; comparable with normal breast tissue). CONCLUSION: NGF secretion by a benign breast tumor is not known in literature. This study reports abundant NGF secretion by breast phyllodes, raising the possibility of its potential role in tumor pathogenesis and progression that can be exploited therapeutically. Additionally, NGF may be used as a distinct biomarker of phyllodes tumors, for differentiating them from fibroadenomas during histopathology.

Differences in outflow channels between two eyes of unilateral primary congenital glaucoma.

PURPOSE: Primary congenital glaucoma (PCG) occurs in only one eye in some patients. We aimed to characterize anatomical features of the angle and Schlemm's canal (SC) in vivo among fellow eyes of patients with unilateral primary congenital glaucoma. METHODS: Both eyes of 33 children with unilateral PCG and 30 healthy, age-matched children, old enough to co-operate were analysed using high-resolution anterior segment spectral domain (SD) OCT. Subgroup analysis was done for the presence/absence of angle dysgenesis as defined by the presence of abnormal tissue/hyper-reflective membrane within angle recess and/or the absence of SC. Other anatomical landmarks differentiating the fellow eyes from eyes with glaucoma were also evaluated and compared with healthy subjects. RESULTS: The presence of abnormal tissue at the angle and/or a hyper-reflective membranous structure covering the meshwork was seen in all affected PCG eyes (100%) and in 21 (63%) unaffected fellow eyes; p = 0.001. The SC could be seen in 8 (24%) affected in comparison with 29 (88%) fellow unaffected eyes; p = 0.001. The ASOCT scans of 54 (90%) healthy eyes and 3 (9%) fellow PCG eyes revealed a direct communication of anterior portion of the SC with the anterior chamber. Among the fellow eyes, a communication of the supraciliary space with anterior chamber could be discerned in 26 eyes (79%). CONCLUSIONS: Despite angle dysgenesis, outflow channels such as the uveoscleral or a direct communication of SC with the anterior chamber play a role in preventing the development of glaucoma in fellow eyes of unilateral PCG.

Surface quality and endothelial cell viability after femtosecond laser-assisted donor lenticule preparation for endothelial keratoplasty - An in-vitro study.

PURPOSE: To compare surface quality and endothelial cell viability of descemet stripping automated endothelial keratoplasty (DSAEK) donor lenticules prepared with femtosecond laser (FSL) or microkeratome (MK). METHODS: Experimental ex-vivo evaluation of 15 DSAEK donor lenticules prepared from optical quality donor corneas using 200 KHz FSL (9 eyes) or MK (6 eyes). Surface quality and smoothness of the cut were assessed using atomic force microscopy and endothelial cell viability was assessed using transmission electron microscopy. RESULTS: Mean lenticule thickness was 121.89 ± 17.13 µm in FSL group and 112.67 ± 5.89 µm in MK group (P = 0.33). Average roughness of stromal surface (RMSavg) [FSL- 30.51 ± 4.55 nm, MK-22.37 ± 1.83 nm; P = 0.02] and root mean square roughness (RMSrough) [FSL-31.39 ± 5.75 nm, MK-23.08 ± 0.40 nm; P = 0.012] was significantly more in FSL group. Increased granular and linear irregularities were observed in the FSL group. Endothelial cell disruption was more in FSL group (FSL- 29.49 ± 6.91% MK-13.28 ± 3.62%; P < 0.001) with decreased mean nucleus length (FSL-5.56 ± 0.17 µm, MK-7.52 ± 0.65 µm; P < 0.001). CONCLUSION: Automated MKs are still the standard of care for donor lenticule preparation and MK-assisted donor lenticules have smoother surface with less endothelial cell disruption than FSL. Further research is mandatory before FSL platforms can be considered a viable alternative to the MK.

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.

Changes in the Inner Retinal Cells after Intense and Constant Light Exposure in Sprague-Dawley Rats.

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7 days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48 h to induce significant damage. The same animals were reverted to 300 lux and reared for 15 days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48 h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to.

Genistein potentiates Centchroman induced antineoplasticity in breast cancer via PI3K/Akt deactivation and ROS dependent induction of apoptosis.

AIMS: Recently, strategies of cancer treatment using combination of agents with distinct molecular mechanism(s) of action are considered more promising due to its high efficacy and reduced systemic toxicity. The study is aimed to improve the efficacy of selective estrogen receptor modulator, Centchroman (CC) by combination with the phytoestrogen Genistein (GN). METHODS: Cytotoxicity was evaluated by Sulforhodamine B assay. Cell cycle analysis was done through flow cytometry. Further, Apoptosis was analyzed using Annexin V/PI staining, tunel assay and electron microscopic examination and verified using western blot analysis. In order to validate the in vitro results, in vivo analysis was performed using 4T1-syngeneic mouse model. KEY FINDINGS: In this study, we report that the dietary isoflavone genistein (GN) synergistically improved antineoplasticity of CC in breast cancer by arresting cells at G2/M phase culminating in ROS dependent apoptosis. The combination of CC plus GN caused dysregulation of Bax and Bcl-2 ratio inducing mitochondrial dysfunction, activation of Caspase-3/7, -9 and PARP cleavage. Further, combination significantly suppresses phosphorylation of PI3K/Akt/NF-κB, enhancing apoptosis. Additionally, combination markedly reduced tumor growth compared to CC and GN alone in mouse 4T1 breast tumor model. SIGNIFICANCE: Together, these studies suggest that GN represents a potential adjunct molecule whose role in CC induced apoptosis deserves attention.

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.

Mutational Analysis of the Mitochondrial DNA Displacement-Loop Region in Human Retinoblastoma with Patient Outcome.

Alteration in mitochondrial DNA plays an important role in the development and progression of cancer. The Displacement Loop (D-loop) region of mitochondrial DNA (mtDNA) is the regulatory region for its replication and transcription. Therefore, we aimed to characterize mutations in the D-loop region of mitochondrial DNA along with the morphological changes and analyzed their impact on survival in retinoblastoma patients. mtDNA D-loop region was amplified by Nested-Polymerase Chain Reaction (Nested-PCR) and mutations were analyzed in 60 tumor samples from retinoblastoma patients by DNA sequencing. Transmission electron microscopy was performed on 5 retinoblastoma specimens. Mutations were correlated with clinical, histopathological parameters and patient survival. D-loop mutations were found in total of 52/60 (86.6%) patients. The most common mutations were T to C and C to T followed by A to G. There were 5.81% mutations which were not previously reported in the MITOMAP database. A73G (83.33%) were the most frequent mutations found in our cases and it was statistically significant with poor tumor differentiation and age. In addition, this study was further analyzed for morphological changes in retinoblastoma that had disorganized, swollen and less numbers of mitochondria on electron microscopy. This is the first study showing high frequency of mtDNA mutation which might be due to abnormal morphology of mitochondria in retinoblastoma. Our results indicate that pathogenic mtDNA D-loop mutations may be involved in tumorigenesis of retinoblastoma tumor.

An experimental study to evaluate safety/toxicity of intravitreal natalizumab.

PURPOSE: The purpose of this prospective experimental study was to evaluate the safety/toxicity of α4ß1 integrin blockade in rabbit retina using its monoclonal antibody (Natalizumab). METHODS: Twelve New Zealand albino rabbits were divided into three groups (n = 4). Unilateral intravitreal injections of three different concentrations of natalizumab were performed in every rabbit of each group (Group A: 0.625 mg, Group B: 1.25 mg, and Group C: 2.5 mg). Baseline electroretinogram (ERG) and fundus photography were performed prior to injection. At days 1, 7, and 21 postinjection, ERG and fundus photography of each eye were performed. At last follow-up, Group C animals with highest drug concentration were sacrificed and the enucleated eyes were evaluated for retinal toxicity using transmission electron microscopy (TEM). RESULTS: No difference in ERG responses was observed in eyes injected with low and intermediate concentration of natalizumab between day 0 and day 21. Furthermore, rabbits injected intravitreally with highest dose showed reduction in amplitude of "a" wave (P = 0.0017) and a reduction in amplitude of "b" wave of ERG at day 21 (P = 0.0117). TEM revealed changes in the outer plexiform layer and inner nuclear layer, suggestive of toxicity primarily to the photoreceptor synaptic terminals and bipolar cells. CONCLUSION: Low-dose (0.625 mg) and intermediate-dose (1.25 mg) intravitreal injection of natalizumab appears safe for rabbit retina. However, functional and anatomical changes were observed in rabbit retina following a high-dose (2.5 mg) intravitreal injection of a monoclonal antibody blocking α4ß1 integrin.

Neuroregenerative Effects of Electromagnetic Field and Magnetic Nanoparticles on Spinal Cord Injury in Rats.

The present study aimed to evaluate the effect of iron oxide nanoparticles (IONPs) along with electromagnetic fields (MF) exposure on spontaneous and induced axonal sprouting after spinal cord injury (SCI). Adult male Wistar rats were subjected to spinal cord transection at the T13 segment. The IONP (25 µg/mL) embedded in 3% agarose gel was implanted at the injury site and subsequently exposed to MF (50 Hz, 17.96 µT, 2 hours/day for 5 weeks). Histological analysis of spinal cord tissue showed a significant increase in the expression of the growth-associated protein GAP-43 and it was found to be co-localized with neuronal nuclei marker and neurofilaments. The results show sprouting from mature neurons and axons, significantly less demyelination and more myelinated fibers were evident at the lesion site. However, no motor or somatosensory evoked potential response was observed, suggesting lack of long-distance functional connectivity. These findings highlight the therapeutic potential of IONPs along with MF exposure in promoting neuroregeneration after SCI.

Diagnostic Electron Microscopy of Retina.

The electron microscopy techniques were used in various fields as an analytical technique under in vitro conditions, which provides the sufficient resolution for better visualization and interpretation. This review gives a brief overview of the analytical application of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques and critical findings in different retinal pathologies. This review article aims to improvise understanding of retinal microstructures for clinicians which will help to improve the interpretation of the current advanced imaging techniques.

Corrigendum: Seabuckthorn Pulp Oil Protects against Myocardial Ischemia-Reperfusion Injury in Rats through Activation of Akt/eNOS.

[This corrects the article DOI: 10.3389/fphar.2016.00155.].

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.