Aging of the human choriocapillaris: Evidence that early pericyte damage can trigger endothelial changes.

The choriocapillaris (CC), the capillary bed in the choroid, essentially nourishes the photoreceptor cells. Its damage in aging and age-related diseases significantly influences the survival of the photoreceptor cells. Earlier reports implicated endothelial loss in aged and diseased CC; however, age-related pericyte changes and their contribution in CC death remain unknown. We examined human donor eyes (age: 56-94 years; N = 24), and found that CC pericyte damage preceded endothelial changes. With aging (>70 years), the sub-macular choroid accumulated debris in Bruch's membrane (BM). Of the debris content, the long-spaced collagens had a tendency to settle over the capillary basal lamina (BL), and this often resulted in endothelial projection into capillary lumen. Between 75 and 83 years, pericytes contained dark mitochondria, and their processes facing the BM debris showed partial loss of BL and intermediate filaments (IFs), when the endothelium remained unaltered. The endothelial changes appeared beyond 83 years, the abundance of IFs and autophagy reinforced their survival until late aging. TUNEL+ pericytes, and immunoreactivity to carboxymethyl lysine and 4-hydroxy 2-nonenal, but no nitro-tyrosine, was detected in aged CC walls. Iba-1+ dystrophic microglia were present in the vicinity of the CC. Our data indicate that (1) BM debris exerts pressure on the CC, leading to the damage of the capillary BL and pericyte processes (2) loss of IFs results in early pericyte destabilization (3) capillary wall undergoes lipid peroxidative and glycative damage, and (4) pericyte damage leads to late endothelial changes and ultimately CC loss. Future research should explore the normal ways of pericyte maintenance in the aging nervous system.

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.

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.

Post-transplant high-dose cyclophosphamide for the prevention of graft-versus-host disease.

Cyclophosphamide's lack of hematopoietic stem cell toxicity and its unique effects on the immune system have prompted several investigators to explore its potential for the prevention of graft-versus-host disease (GVHD). In haploidentical hematopoietic stem cell transplants, post-transplant cyclophosphamide together with standard prophylaxis reduces the incidence of GVHD to acceptable rates without the need for T cell depletion. In matched related and unrelated donor settings, cyclophosphamide alone has produced encouraging results. In particular, the low incidence of chronic GVHD is noteworthy. Here, we present a review of the current understanding of the mechanism of action of post-transplant cyclophosphamide and summarize the clinical data on its use for the prevention of GVHD.

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.

Duration of injury correlates with necrosis in caerulein-induced experimental acute pancreatitis: implications for pathophysiology.

Pancreatic acinar cell necrosis is indicative of severe pancreatitis and the degree of necrosis is an index of its outcome. We studied whether the dose and duration of injury correlates with severity, particularly in terms of necrosis, in caerulein-induced acute pancreatitis (AP) in Swiss albino mice. In addition to control group 1 (G1), groups 2 and 3 received four injections of caerulein every hour but were sacrificed at five hours (G2) and nine hours (G3) respectively, and group 4 received eight injections and was sacrificed at nine hours (G4). The severity of pancreatitis was assessed histopathologically and biochemically. The histopathological scores of pancreatitis in groups 3 and 4 were significantly higher than in groups 1 and 2 (4 vs. 1, 4 vs. 2, 3 vs. 1, 3 vs. 2; P < 0.05). TUNEL-positive apoptotic cells were significantly higher in groups 2 and 3 compared with groups 1 and 4 (P < 0.05). Necrosis was significantly more in group 4 than other groups (37.49% (4.68) vs. 19.97% (1.60) in G2; 20.36% (1.56) in G3; P = 0.006 for G 2 vs. 4 and P = 0.019 for G 3 vs. 4). Electron microscopy revealed numerous autophagosomes in groups 2 and 3 and mitochondrial damage and necrosis in group 4. The pancreatic and pulmonary myeloperoxidase activity in group 4 was significantly higher than that in the other groups (P < 0.01). Hence, severity of pancreatitis is a function of the dose of injurious agent, while inflammation is both dose and duration dependent, which may also explain the wide spectrum of severity of AP seen in clinical practice.

Electron-microscopic evidence of mitochondriae containing macroautophagy in experimental acute pancreatitis: implications for cell death.

BACKGROUND: Dysfunctional autophagy and necrosis are characteristic features of severe acute pancreatitis. OBJECTIVE: To unravel the cellular mechanisms underlying the pathogenesis of acute pancreatitis. METHODS: We studied the ultrastructural pancreatic morphology using electron microscopy in experimental acute pancreatitis. The control group of animals received intraperitoneal injections of normal saline. Different severity of acute pancreatitis was induced by low and high doses of caerulein in Swiss albino mice. In the low dose group, pancreatitis was induced by 4 injections of caerulein given hourly [50 µg/kg/dose - total of 200 µg/kg] and in the high dose group by 8 injections given hourly (total of 400 µg/kg). The experiments were repeated in Na-taurocholate model of acute pancreatitis in rats. The pancreatic tissue was processed and studied by transmission electron microscopy for ultrastructural changes. RESULTS: The acinar cells of the pancreatitis animals revealed autophagosomes that contained cellular organelles, including mitochondria. The animals that received a higher dose of caerulein had numerous cells showing a necrotic morphology, whereas the animals in the low dose group showed a predominantly apoptotic cell morphology. The Na-taurocholate model in rats also showed similar features of severe pancreatitis with cellular necrosis and macroautophagy. CONCLUSIONS: Dysfunctional mitochondria in the injured pancreatic acinar cells are degraded by macroautophagy. These observations are not model specific. Mitochondrial dysfunction and consequent energy deficit in the cells might be causally related to cellular necrosis.

Practical guidelines for setting up neurosurgery skills training cadaver laboratory in India.

Though the necessity of cadaver dissection is felt by the medical fraternity, and described as early as 600 BC, in India, there are no practical guidelines available in the world literature for setting up a basic cadaver dissection laboratory for neurosurgery skills training. Hands-on dissection practice on microscopic and endoscopic procedures is essential in technologically demanding modern neurosurgery training where ethical issues, cost constraints, medico-legal pitfalls, and resident duty time restrictions have resulted in lesser opportunities to learn. Collaboration of anatomy, forensic medicine, and neurosurgery is essential for development of a workflow of cadaver procurement, preservation, storage, dissection, and disposal along with setting up the guidelines for ethical and legal concerns.

Free-access open-source e-learning in comprehensive neurosurgery skills training.

BACKGROUND: Since the end of last century, technology has taken a front seat in dispersion of medical education. Advancements of technology in neurosurgery and traditional training methods are now being challenged by legal and ethical concerns of patient safety, resident work-hour restriction and cost of operating-room time. To supplement the existing neurosurgery education pattern, various e-learning platforms are introduced as structured, interactive learning system. MATERIALS AND METHODS: This study focuses on the concept, formulation, development and impact of web based learning platforms dedicated to neurosurgery discipline to disseminate education, supplement surgical knowledge and improve skills of neurosurgeons. 'Neurosurgery Education and Training School (NETS), e-learning platform' has integration of web-based technologies like 'Content Management System' for organizing the education material and 'Learning Management System' for updating neurosurgeons. NETS discussion forum networks neurosurgeons, neuroscientists and neuro-technologists across the globe facilitating collaborative translational research. RESULTS: Multi-authored neurosurgical e-learning material supplements the deficiencies of regular time-bound education. Interactive open-source, global, free-access e-learning platform of NETS has around 1) 425 visitors/month from 73 countries; ratio of new visitors to returning visitors 42.3; 57.7 (2); 64,380 views from 190 subscribers for surgical videos, 3-D animation, graphics based training modules (3); average 402 views per post. CONCLUSION: The e-Learning platforms provide updated educational content that make them "quick, surf, find and extract" resources. e-Learning tools like web-based education, social interactive platform and question-answer forum will save unnecessary expenditure of time and travel of neurosurgeons seeking knowledge. The need for free access platforms is more pronounced for the neurosurgeons and patients in developing nations.

Effect of "apical clearing" and "apical foramen widening" on apical ramifications and bacterial load in root canals.

The purpose of the study was to determine the effect of apical clearing and apical foramen widening in reducing apical ramifications and bacterial load in the apical third of root canals. The mesio-buccal roots of 21 maxillary first molar teeth were inoculated with Enterococcus faecalis suspension using a sterile pipette. Samples were incubated at 37°C for 72 hrs and divided into 3 groups: Group A, control group (n=5), no preparation; Group B (n=8) conventional preparation alone; and Group C (n=8), apical clearing and foramen widening in addition to conventional preparation. Bacterial counts were semi-quantitatively analyzed pre- and post-preparation. Samples were demineralized with 5% nitric acid after injection of India ink. Cross sections were obtained at every 0.5 mm from the apex to 3 mm of the root using a vibratome and viewed under a stereomicroscope at 64×magnification to locate any debris or apical ramifications. The Kruskal-Wallis and Mann-Whitney U tests were used for the statistical analysis. A statistically significant difference was observed (p value 0.006) in the number of ramifications among the 3 groups. Group C had a lower average number of ramifications (1) than Group B (2.5) or A (4). The debris score was analyzed at each level (0.5-3 mm). A statistically significant difference was observed at 0.5 mm and 1 mm between Group A and C (p=0.0041) and Group B and C (p=0.0050), whereas no difference was found between Group A and B (p>0.05). These results indicate that there was less debris and fewer apical ramifications in Group C. The microbiological study revealed a lower number of colony forming units (10(2) -10(3)) in Group B or C than in Group A (>10(5)). These results suggest that apical widening and clearing facilitates removal of apical ramifications and bacterial load within root canals.

Nerve fibre morphometry with transmission electron microscopy: Application of the nucleator probe in ImageJ.

Stereology and semiautomated binary image histomorphometry are two common methods used for morphometry of nerve fibres. Nucleator probe can be used for the estimation of morphometric parameters like diameter, perimeter, area and volume of a structure that is approximately either a circle or a sphere. In this study, we estimated these parameters with the help of ImageJ software on calibrated transmission electron micrographs. We procured samples of the cochlear nerve (CN) during winter months, within 6-12 hours of death, to reduce post-mortem autolytic changes. The temporal bones containing the CN were fixed by immersion in chilled paraformaldehyde. After dissecting out from the petrous part of the temporal bone, the CN were osmicated and processed for embedding in resin. From the resin blocks, silver coloured (70 nm) ultrathin sections were cut and picked on 300-mesh copper grids, stained with uranyl acetate and lead citrate and viewed under Tecnai G2-20 transmission electron microscope. The transmission electron micrographs had scale bars embedded into them by the software at the time of imaging, and the morphometric parameters of randomly selected nerve fibres were measured using the ImageJ software. The ImageJ software could become a low-cost and dependable tool for nerve fibre morphometry.•Nucleator probe is used for the estimation of morphometric parameters like diameter, perimeter, area or volume•Morphometric parameters were estimated by the ImageJ software on calibrated transmission electron micrographs•The ImageJ software could become a low-cost and dependable tool for nerve fibre morphometry.

The ultrastructural study of human cochlear nerve at different ages.

Ultrastructural and molecular changes in the myelin of the cochlear nerve (CN) have been associated with decreased hearing-acuity with increasing age. But most of these are animal studies or with very few human samples. Hence, we studied the ultrastructure of the human CN at different ages. We obtained samples of CN from persons, who at the time of death belonged to young, middle or old age-groups; defined as ≤ 30, 31 to 50, and ≥ 51 years of age, respectively. These were processed for viewing under a transmission electron microscope (TEM). Morphology and morphometry were assessed after blinding the observer. Measurements of diameter (whole nerve fibre, axon), myelin thickness and calculation of G-ratio were made on calibrated images using ImageJ software. K-Means cluster analysis was performed based on total and inner nerve fibre area. Middle and old age CN showed degenerating axons, splitting of myelin sheath and myelin balloons. Between the middle and old age groups there was significant decrease in axon diameter (p<0.001), inner nerve fibre area (p<0.001), myelin thickness (p<0.001), nerve fibre diameter (p<0.001), and G-ratio (p<0.001). By clustering, we identified three distinct populations of myelinated nerve fibres: large, medium and small. The large fibres (by size), seen in the young, disappeared in the old age-group. We were unable to find any unmyelinated nerve fibres in this study. The morphological deterioration CN fibres may be a visible sign of molecular degeneration and contribute to decreased hearing-acuity.

Mutation screening and genotype phenotype correlation of α-crystallin, γ-crystallin and GJA8 gene in congenital cataract.

PURPOSE: To screen α-crystallin (CRYAB), γ-crystallin (CRYGC and CRYGD), and Connexin 50 (Cx-50 or GJA8) genes in congenital cataract patients and controls. METHODS: Thirty clinically diagnosed congenital cataract cases below 3 years of age from northern India, presenting at Dr. R. P. Centre for Ophthalmic Sciences (AIIMS, New Delhi, India) were enrolled in this study. Genomic DNA was extracted from peripheral blood, all coding and exon/intron regions were amplified using PCR and direct sequencing was performed to detect any nucleotide variation. ProtScale and Discovery Studio programs were used for insilico and structural analysis of non-synonymous mutations. RESULTS: DNA sequencing analysis of CRYAB, CRYGC, CRYGD, and GJA8 showed a total of six variations of which two were novel (CRYGC:p.R48H and GJA8:p.L281C) and four have been previously reported (CRYAB: rs11603779T>G, GJA8: p.L268L, CRYGD: p.R95R, and c.T564C). Both the novel changes, in CRYGC and GJA8 were found in 16.6% of the patients. Previously reported nucleotide alterations (CRYGD:p.R95R and c.T564C) were found in 90% of the patients. Insilico and structural analysis data suggested that two novel non-synonymous mutations altered the stability and solvent accessibility of γC-crystallin and Cx-50 proteins which may lead to lens opacification. CONCLUSIONS: We observed two novel nonsynonymous variations and four reported variations in CRYAB, CRYGC, CRYGD, and GJA8. The p.R48H variation in γC-crystallin may disrupt the normal structure of lens and can cause cataract. Cx50 is responsible for joining the lens cells into a functional syncytium and a mutation (p.L281C) in GJA8 may lead to lens opacification resulting in cataract formation. This study further expands the mutation spectrum of congenital cataract and help understanding how mutant proteins lead to opacification of lens.

Co-administration of nalbuphine attenuates the morphine-induced anxiety and dopaminergic alterations in morphine-withdrawn rats.

INTRODUCTION: The classical effects of exogenous opioids, such as morphine, are predominantly mediated through µ-opioid receptors. The chronic use of morphine induces anxiety-like behavior causing functional changes in the mesolimbic dopaminergic system. The mixed µ/κ-agonist, nalbuphine, used either as an analgesic or as an adjuvant with morphine, produces different and opposite effects. However, whether nalbuphine can be used to antagonize morphine-induced anxiety and dopaminergic alterations is not fully known. OBJECTIVE: This study aimed to compare acute and chronic effects of nalbuphine on morphine-induced anxiety and dopaminergic alterations in rats. METHODS: Male adult Wistar albino rats were made opioid-dependent by administering increasing doses of morphine (5-25 mg/kg; i.p.; b.i.d.). Withdrawal was induced by naloxone (1 mg/kg, i.p.), 4 h after the last morphine injection. Anxiety-like behavior was measured using Activity Monitor (Coulbourn Instruments, Inc. USA). Thereafter, the animals were sacrificed and the brain dissected out and the level of cAMP and the transcriptional and translational expression of TH was measured. Nalbuphine was co-administered with morphine, acutely and chronically, at various doses (0.1, 0.3, 1.0, 3.0 mg/kg, i.p.). RESULTS: Morphine-dependent rats showed a significant higher anxiety and cAMP levels and a significant decrease in the expression of TH. Co-administration of chronic doses of nalbuphine attenuates the higher anxiety, cAMP levels, and upregulates the TH expressions; however, the acute nalbuphine treatment does not attenuate the morphine-induced side effects. CONCLUSION: Therefore, nalbuphine might have an important role in attenuating the anxiety and the effects of the dopaminergic pathway and may have potential in the treatment of opioid addiction.

Focal segmental glomerulosclerosis in idiopathic membranous glomerulonephritis: a clinico-pathological and stereological study.

BACKGROUND: The phenomenon of focal segmental glomerulosclerosis (FSGS) in idiopathic membranous glomerulonephritis (IMGN) has not been adequately studied. There is also a paucity of detailed glomerular morphometric and stereologic analyses data on renal biopsy in this association. METHODS: Twenty-three (23) patients with IMGN and superimposed FSGS were compared to 35 patients with IMGN alone with respect to the clinical and laboratory features, light microscopic findings and stereologic parameters (glomerular cross-sectional area and estimated glomerular volume). RESULTS: In the clinical parameters, patients with IMGN-FSGS had a significantly higher incidence of hypertension, raised serum creatinine and microscopic haematuria. The mean 24-h urinary protein excretion was higher in the group with IMGN-FSGS (7.4 +/- 1.36 g) as compared to IMGN alone (3.85 +/- 0.7 g, P < 0.001, Mann-Whitney test). On light microscopy, biopsies with IMGN-FSGS frequently had mesangial hypercellularity and more extensive tubulo-interstitial disease than those with IMGN alone. Stereological analysis showed that the non-sclerosed glomeruli in biopsies with IMGN-FSGS had a higher mean cross-sectional area (185466.7 +/- 32493.3 micro(2)) and higher estimated volume (855200 +/- 152640 micro(3)) as compared to glomeruli in cases with IMGN alone (76000 +/- 14719.2 micro(2) and 576666.7 +/- 131233.3 micro(3), respectively). CONCLUSION: The present study is probably the first systematic analysis of stereologic parameters in renal biopsies of IMGN with FSGS. Our results objectively demonstrate the glomerular enlargement in the non-sclerosed glomeruli in cases of IMGN with FSGS. This detection of enlarged glomeruli may serve to alert the renal pathologist to the possibility of coexisting FSGS, which is a poor prognostic factor in IMGN.

Age-related changes in the number of cresyl-violet-stained, parvalbumin and NMDAR 2B expressing neurons in the human spiral ganglion.

Animal-studies associate age-related hearing loss (presbycusis) with decreasing number of spiral ganglion neurons (SGNs) in Rosenthal's canal (RC) of cochlea. The excitatory neurotransmitter for SGNs is glutamate (through its receptor NMDAR 2B), which can be neurotoxic through Ca2+ overload. Neurotoxicity is balanced by calcium-binding proteins (CBPs) like Parvalbumin (PV), which is the predominant CBP of the SGNs. To estimate the volume of the RC and total number of SGNs that are immunoreactive to PV and NMDAR 2B, we used unbiased stereology in 35 human cochleae derived from cadavers of persons from 2nd to 8th decade of life (subsequently statistically divided into two groups) and compared them to the total number of cresyl violet (CV) stained SGNs. We also estimated the volume of individual neurons and their nuclei. Regression analysis was made on estimated parameters against age. Hierarchical-cluster analysis was done on the neuronal against neuronal nuclear volumes.The average volume of the RC did not change with increasing age (p = 0.4115). The total number of SGNs (CV-stained and those separately expressing PV and NMDAR 2B) significantly decreased with age (p < 0.001). We identified three distinct populations of neurons on the basis of their volumes among SGNs. Thus, there is significant age-related decline in the total number of SGNs, which starts early in life. It may be due to ambient noise and inadequate neutralisation of excitotoxicity.

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.

Development and Validation of Finite Element Analysis Model (FEM) of Craniovertebral Junction: Experimental Biomechanical Cadaveric Study.

STUDY DESIGN: Experimental Cadaveric Biomechanical Study. OBJECTIVE: To establish an experimental procedure in cadavers to estimate joint stiffness/stability at craniovertebral junction (CVJ) region with various implant systems and to develop/validate an indigenous cost effective 3D-FEM (three-dimensional finite element model) of CVJ region. SUMMARY OF BACKGROUND DATA: Finite element analysis (FEM) tools can provide estimates of internal stress and strain in response to external loading of various implant systems used in CVJ fixations. METHODS: Experimental setup for conducting biomechanical movements on CVJ region of cadaver was developed using cost effective innovative tools. A manually actuated seven- degrees of freedom parallel manipulator motion testing system (MA7DPM) was designed and developed to impart designed trajectories and to conduct various biomechanical motion studies at CVJ region for the present study. RESULTS: FEM model of CVJ region was developed and subsequently validated with CVJ morphometry data of 15 human subjects of Asian origin. Validated FEM was subjected to force motion studies at the CVJ region. The force-motion maps obtained from the FEM studies were subsequently validated against biomechanical experiment results from cadaveric experiment results obtained with three different implant fixations. CONCLUSIONS: A cost effective biomechanical tool (which did not require decapitation of cadaveric head) and a customised chair (to place cadaver in sitting position during conduct of biomechanical movements simulating real-life scenario) was indigenously designed and developed. Developed biomechanical tool (MA7DPM) for this study is likely to be useful for stress-testing analysis of various implant systems for individual patients undergoing surgery at CVJ region in near future. LEVEL OF EVIDENCE: 5.

Age-related changes of the human retinal vessels: Possible involvement of lipid peroxidation.

BACKGROUND: Aging of the human retina is accompanied by oxidative stress that exerts profound changes in the retinal neurons. It is unknown if oxidative stress influences the cellular components of the retinal vessels in some ways. METHODS: We examined changes in retinal vessels in human donor eyes (age: 35-94 years; N=18) by light and transmission electron microscopy, TUNEL and immunohistochemistry for biomarkers of vascular smooth muscle cells (SMC; actin), oxidative stress (4-hydroxy 2-nonenal [HNE] and nitrotyrosine), microglia (Iba-1) and vessels (isolectin B4). RESULTS: The earliest changes in the endothelium and pericytes of capillaries are apparent from the seventh decade. With aging, there is clear loss of organelles and cytoplasmic filaments, and a progressive thickening of the endothelial and pericyte basal lamina. Loss of filaments, accumulation of lipofuscin and autophagic vacuoles are significant events in aging pericytes and SMC. Actin immunolabelling reveals discontinuity in arterial SMC layers during eighth decade, indicating partial degeneration of SMC. This is followed by hyalinization, with degeneration of the endothelium and SMC in arteries and arterioles of the nerve fibre layer (NFL) and ganglion cell layer in ninth decade. Iba-1 positive microglia were in close contact with the damaged vessels in inner retina, and their cytoplasm was rich in lysosomes. HNE immunoreactivity, but not of nitrotyrosine, was detected in aged vessels from seventh decade onwards, suggesting that lipid peroxidation is a major problem of aged vessels. However, TUNEL positivity seen during this period was limited to few arteries and venules of NFL. CONCLUSION: This study shows prominent age-related alterations of the pericytes and SMC of retinal vessels. These changes may limit the energy supply to the neurons and be responsible for age-related loss of neurons of the inner retina.

Estimation of Volume of Stria Vascularis and the Length of Its Capillaries in the Human Cochlea.

BACKGROUND: The stria vascularis (SV) is a vascularized epithelium that secretes endolymph and is located on the lateral wall of the membranous cochlea. The capillaries of SV directly influence the composition of the endolymph and hence the generation of impulses by the hair-cells that are auditory receptors and thus affect hearing. Therefore, the real morphology of the SV would be very important for understanding the hearing system. There are few reliable reports of the morphology of the human SV. AIMS AND OBJECTIVES: In this research, we have estimated the volume of the SV and total length of strial capillaries in the apical, middle and basal turns of the human cochlea by updated stereological techniques. METHODS: The point-counting Cavalieri's method and hemispherical volume probes were applied on stained, 40 µm-thick serial sections of five celloidin-embedded, decalcified cochleae. RESULTS: The mean age of persons at the time of death was 51 ± 15.25 years, the mean volume of the SV was 0.56 ± 0.054 mm3 and the mean length of the SV capillaries was 289.08 ± 72.96 mm. We also estimated the same parameters with different stereological parameters, probes and in differently stained sections and checked the relationship and limits of agreement between different methods by paired t-test and Bland-Altman plot. We found agreement in our results. CONCLUSION: We provide reliable baseline data on the real morphology of the human SV.