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.

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.

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 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.

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.

Morphological development of the human cochlear nucleus.

Morphological studies in developing brain determine critical periods of proliferation, neurogenesis, gliogenesis, and apoptosis. During these periods both intrinsic and extrinsic pathological factors can hamper development. These time points are not available for the human cochlear nucleus (CN). We have used design-based stereology and determined that 18-22 weeks of gestation (WG) are critical in the development of the human CN. Twenty-three fetuses and seven postnatal brainstems were processed for cresyl violet (CV) staining and immunoexpression of NeuN (neurons), GFAP (astrocytes), Ki-67 (proliferation) and TUNEL (apoptosis) and 3-D reconstruction. The volume of CN, total number of neurons selected profiles and the volume of neurons and their nuclei were estimated. Data were grouped (G) into: G1:18-20 WG, G2: 21-24 WG, G3: 25-28 WG and G4 >29 WG. The dimensions of morphologically identified neurons were also measured. The CN primordium was first identifiable at 10WG. Definitive DCN (Dorsal cochlear nucleus) and VCN (ventral cochlear nucleus) were identifiable at 16 WG. There was a sudden growth spurt in total volume of CN, number of neurons and astrocytes from 18 WG. We also observed an increase in proliferation and apoptosis after 22 WG. The number of neurons identifiable by CV was significantly lower than that by NeuN-immunostaining till 25 WG (p = 0.020), after which, both methods were equivalent. Eight morphological types of neurons were identifiable by 26 WG and could be resolved into four clusters by volume and diameter. The CN changed orientation from small, flat and horizontal at 10-16 WG to larger and oblique from 18WG onwards. Prevention of exposure to noxious factors at 18-22 WG may be important in preventing congenital deafness.

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.

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.

Morphological and neurochemical changes in GABAergic neurons of the aging human inferior colliculus.

It is well known that quality of hearing decreases with increasing age due to changes in the peripheral or central auditory pathway. Along with the decrease in the number of neurons the neurotransmitter profile is also affected in the various parts of the auditory system. Particularly, changes in the inhibitory neurons in the inferior colliculus (IC) are known to affect quality of hearing with aging. To date, there is no information about the status of the inhibitory neurotransmitter GABA in the human IC during aging. We have collected and processed inferior colliculi of persons aged 11-97 years at the time of death for morphometry and immunohistochemical expression of glutamic acid decarboxylase (GAD67) and parvalbumin. We used unbiased stereology to estimate the number of cresyl-violet and immunostained neurons. Quantitative real-time PCR was used to measure the relative expression of the GAD67 mRNA. We found that the number of total, GABAergic and PV-positive neurons significantly decreased with increasing age (p < 0.05). The proportion of GAD67-ir neurons to total number of neurons was also negatively associated with increasing age (p = 0.004), but there was no change observed in the proportion of PV-ir neurons relative to GABAergic neurons (p = 0.25). Further, the fold change in the levels of GAD67 mRNA was negatively correlated to age (p = 0.024). We conclude that the poorer quality of hearing with increasing age may be due to decreased expression of inhibitory neurotransmitters and the decline in the number of inhibitory neurons in the IC.

Ultrastructural Study of Rat Testis Following Conventional Phototherapy during Neonatal Period.

INTRODUCTION: Phototherapy is the most common treatment for neonatal jaundice. This study sought to determine ultrastructural changes in testis, at different time-points, after 48 hours of conventional phototherapy was given to newborn rats. METHODS: Newborn male Wistar rats (n = 36) were divided into two groups as follows - group 1 (G1), control (without phototherapy) and group 2 (G2), exposure to conventional phototherapy for 48 h. Six animals from each group were sacrificed on postnatal days (PND) 70, 100 and 130. The testes were dissected out and processed for Transmission Electron Microscopy (TEM). RESULTS: TEM showed that G2 on PND 70 and 100 showed damaged organelles, including nuclei, mitochondria, endoplasmic reticulum, vacuoles and electron dense bodies in the testes. Seminiferous Tubule on PND130 showed lesser damage. On PND70 ST wall thickness (STWT) of G2 was significantly higher (P < 0.001) than G1 STWT of G2 was significantly lower than G1 on PND100 (P = 0.047) and on PND130 (P < 0.001). Mitochondrial diameter in spermatogonia was significantly higher in G2 on PND70 (P = 0.001), PND100 (P = 0.031) and PND130 (P = 0.028). Primary spermatocytes in G2 also had larger mitochondria on PND70 (P < 0.001), PND100 (P = 0.007) and PND130 (P = 0.008). Further, spermatids had larger mitochondria in G2 on PND70 (P < 0.001), PND100 (P = 0.044) and PND130 (P < 0.001). CONCLUSION: Phototherapy causes degenerative changes in rat testis on PND70 and 100 that partially recover by PND 130.

Fluoride induced tissue hypercalcemia, IL-17 mediated inflammation and apoptosis lead to cardiomyopathy: Ultrastructural and biochemical findings.

An increased prevalence of cardiac complications has been observed in residents of fluorosis endemic areas chronically exposed to fluoride. Fluoride induces soft tissue injury due to oxidative stress, lipid peroxidation (LPO) and mitochondriopathy. It was hypothesized that chronic fluoride exposure induces apoptosis in cardiomyocytes due to inflammation, lysis of extra cellular matrix and altered calcium metabolism. This study was planned to evaluate the effects of chronic fluoride exposure and the mechanism of action in the cardiac muscle. Fifteen week old male Wistar rats were administered a human equivalent dose of fluoride (50 and 100 ppm ad-libitum, HED = 5 & 10 ppm in human) for 75-days. After 75-days of fluoride exposure, the animals were euthanized and fluoride, oxidative stress (SOD, GPX, Catalase activities) and LPO were measured. Histopathological and ultrastructural pathological examinations were conducted on the cardiac tissues using light, atomic force and electron microscopies. The cardiac tissues were also assessed for apoptosis (TUNEL/Caspase assays), and tissue calcium levels (Alizarin-assay and SEM-EDX). Tissue inflammation and expression of IL-17, MMP-9, Caspase-3 and Bcl-2 were evaluated. In the fluoride exposed groups, a significant (≤0.05) increase in levels of oxidative stress, LPO and apoptosis were observed. The IL-17, MMP-9 and Caspase-3 were significantly (≤0.05) higher in the cardiac muscle after chronic fluoride exposure. The fluoride seems to have induced inflammation in the cardiac tissues, as well as an increase in tissue calcium (≤0.05). There was significant damage to cardiac muscle fibres including, thinning, distortion and neo-vasculogenesis following chronic fluoride exposure. Mitochondriopathy, lysis of ground substance, oedema, and hyper-vacuolation was seen in fluoride treated groups. Remarkable levels of distortion and bending in Z band were observed under the AFM. Many of these observed changes mimic those occurring in cardiomegaly, cardiac hypertrophy and cardiomyopathies.

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.

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.

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.

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.

Quantitative analysis of the Kawase versus the modified Dolenc-Kawase approach for middle cranial fossa lesions with variable anteroposterior extension.

OBJECT: The surgical corridor to the upper third of the clivus and ventral brainstem is hindered by critical neurovascular structures, such as the cavernous sinus, petrous apex, and tentorium. The traditional Kawase approach provides a 10 × 5-mm fenestration at the petrous apex of the temporal bone between the 5th cranial nerve and internal auditory canal. Due to interindividual variability, sometimes this area proves to be insufficient as a corridor to the posterior cranial fossa. The authors describe a modification to the technique of the extradural anterior petrosectomy consisting of additional transcavernous exploration and medial mobilization of the cisternal component of the trigeminal nerve. This approach is termed the modified Dolenc-Kawase (MDK) approach. METHODS: The authors describe a volumetric analysis of temporal bones with 3D laser scanning of dry and drilled bones for respective triangles and rhomboid areas, and they compare the difference of exposure with traditional versus modified approaches on cadaver dissection. Twelve dry temporal bones were laser scanned, and mesh-based volumetric analysis was done followed by drilling of the Kawase triangle and MDK rhomboid. Five cadaveric heads were drilled on alternate sides with both approaches for evaluation of the area exposed, surgical freedom, and angle of approach. RESULTS: The MDK approach provides an approximately 1.5 times larger area and 2.0 times greater volume of bone at the anterior petrous apex compared with the Kawase's approach. Cadaver dissection objectified the technical feasibility of the MDK approach, providing nearly 1.5-2 times larger fenestration with improved view and angulation to the posterior cranial fossa. Practical application in 6 patients with different lesions proves clinical applicability of the MDK approach. CONCLUSIONS: The larger fenestration at the petrous apex achieved with the MDK approach provides greater surgical freedom at the Dorello canal, gasserian ganglion, and prepontine area and better anteroposterior angulation than the traditional Kawase approach. Additional anterior clinoidectomy and transcavernous exposure helps in dealing with basilar artery aneurysms.

The human trochlear and abducens nerves at different ages - a morphometric study.

The trochlear and abducens nerves (TN and AN) control the movement of the superior oblique and lateral rectus muscles of the eyeball, respectively. Despite their immense clinical and radiological importance no morphometric data was available from a wide spectrum of age groups for comparison with either pathological or other conditions involving these nerves. In the present study, morphometry of the TN and AN was performed on twenty post-mortem samples ranging from 12-90 years of age. The nerve samples were processed for resin embedding and toluidine blue stained thin (1µm) sections were used for estimating the total number of myelinated axons by fractionator and the cross sectional area of the nerve and the axons by point counting methods. We observed that the TN was covered by a well-defined epineurium and had ill-defined fascicles, whereas the AN had multiple fascicles with scanty epineurium. Both nerves contained myelinated and unmyelinated fibers of various sizes intermingled with each other. Out of the four age groups (12-20y, 21-40y, 41-60y and >61y) the younger groups revealed isolated bundles of small thinly myelinated axons. The total number of myelinated fibers in the TN and AN at various ages ranged from 1100-3000 and 1600-7000, respectively. There was no significant change in the cross-sectional area of the nerves or the axonal area of the myelinated nerves across the age groups. However, myelin thickness increased significantly in the AN with aging (one way ANOVA). The present study provides baseline morphometric data on the human TN and AN at various ages.

Quantitative analysis of variable extent of anterior clinoidectomy with intradural and extradural approaches: 3-dimensional analysis and cadaver dissection.

BACKGROUND: Drilling of the anterior clinoid process (ACP) is an integral component of surgical approaches for central and paracentral skull base lesions. The technique to drill ACP has evolved from pure intradural to extradural and combined techniques. OBJECTIVE: To describe the computerized morphometric evaluation of exposure of optic nerve and internal carotid artery with proposed tailored intradural (IDAC) and complete extradural (EDAC) anterior clinoidectomy. METHODS: We describe a morphometric subdivision of ACP into 4 quadrangles and 1 triangle on the basis of fixed bony landmarks. Computerized volumetric analysis with 3-dimensional laser scanning of dry-drilled bones for respective tailored IDAC and EDAC was performed. Both approaches were compared for the area and length of the optic nerve and internal carotid artery. Five cadaver heads were dissected on alternate sides with intradural and extradural techniques to evaluate exposure, surgical freedom, and angulation of approach. RESULTS: Complete anterior clinoidectomy provides a 2.5-times larger area and 2.7-times larger volume of ACP. Complete clinoidectomy deroofed the optic nerve to an equal extent as by proposed the partial tailored clinoidectomy approach. Tailored IDAC exposes only the distal dural ring, whereas complete EDAC exposes both the proximal and distal dural rings with complete exposure of the carotid cave. CONCLUSION: Quantitative comparative evaluation provides details of exposure and surgical ease with both techniques. We promote hybrid/EDAC technique for vascular pathologies because of better anatomic orientation. Extradural clinoidectomy is the preferred technique for midline cranial neoplasia. An awareness of different variations of clinoidectomy can prevent dependency on any particular approach and facilitate flexibility.