Trolox aids coenzyme Q10 in neuroprotection against NMDA induced damage via upregulation of VEGF in rat model of glutamate excitotoxicity.

Glutamate induced damage to retinal ganglion cells (RGCs) requires tight physiological regulation of the N-methyl-D-aspartate (NMDA) receptors. Previously, studies have demonstrated the neuroprotective abilities of antioxidants like coenzyme Q10 (CoQ10) and vitamin E analogs like α-tocopherol against neuropathies resulting from NMDA insult, but have failed to shed light on the effect of CoQ10 and trolox, a hydrophilic analog of vitamin E, on glaucomatous neurodegeneration. In the current study, we wanted to investigate whether the combined effect of trolox with CoQ10 could alleviate NMDA-induced death of retinal cells while also trying to elucidate the underlying mechanism in relation to the yet unexplained role of vascular endothelial growth factor (VEGF) in NMDA-mediated excitotoxicity. After successful NMDA-induced degeneration, we followed it up with the treatment of combination of Trolox and CoQ10. The structural damage by NMDA was repaired significantly and retina retained structural integrity comparable to levels of control in the treatment group of Trolox and CoQ10. Detection of ROS generation after NMDA insult showed that together, Trolox and CoQ10 could significantly bring down the high levels of free radicals while also rescuing mitochondrial membrane potential (MMP). A significant increase in NMDA receptor Grin2A by CoQ10 alone as well as by CoQ10 and trolox was accompanied by a lowered Grin2B receptor expression, suggesting neuroprotective action of Trolox and CoQ10. Subsequently, lowered VEGFR1 and VEGFR2 receptor expression by NMDA treatment also recovered when subjected to combined treatment of Trolox and CoQ10. Western blot analyses also indicated the same whereby Trolox and CoQ10 could increase the diminished levels of phosphorylated VEGFR2. Immunofluorescence studies also indicated a positive correlation between recovered VEGFR2 and NMDAR2A levels and diminished levels of NMDAR2D, confirming the results obtained by RT-PCR analysis. This is the first report in our knowledge that demonstrates the efficacy of trolox in combination with CoQ10 highlighting the importance of maintaining VEGF levels that are lowered in ocular diseases due to NMDA-related toxicities.

A comparative study of smooth muscle cell characteristics and myofibroblasts in processus vaginalis of pediatric inguinal hernia, hydrocele and undescended testis.

BACKGROUND: Congenital inguinal hernia, hydrocele and undescended testis (UDT) are associated with patent processus vaginalis. The smooth muscles present in the processus vaginalis aid in the descent of the testis and undergo programmed cell death after testicular descent leading to obliteration. The persisting amount of smooth muscle in the processus vaginalis influences the clinical outcome as inguinal hernia, hydrocele or UDT. Therefore, a study was conducted to evaluate the processus vaginalis in these three conditions to observe the presence and phenotype of smooth muscle cells and the presence of myofibroblasts. MATERIALS AND METHODS: The processus vaginalis sacs in patients with inguinal hernia, hydrocele and UDT were examined using light microscopy for the presence and distribution of smooth muscle cells and immunohistochemical staining for vimentin, desmin, and α-smooth muscle actin (SMA) to identify the smooth muscle phenotype. Transmission electron microscopy was also performed in all the sacs to observe the presence of myofibroblasts. RESULTS: Seventy-eight specimens of processus vaginalis (from seventy-four patients), distributed as 47%, 27%, and 26% as inguinal hernia, hydrocele and UDT respectively, were included in the study. The sacs from inguinal hernia and hydrocele had significantly more presence of smooth muscles distributed as multiple smooth muscle bundles (p < 0.001). Desmin and SMA staining of smooth muscle cells was observed in significantly more sacs from hydrocele, followed by inguinal hernia and UDT (p < 0.001). The sacs from UDT had a significant presence of striated muscles (p = 0.028). The sacs from inguinal hernia had a significant presence of myofibroblasts, followed by hydrocele and UDT (p < 0.001) and this significantly correlated with the light microscopy and immunohistochemical features. The processus vaginalis sacs from four female patients did not differ statistically from the male inguinal hernia sacs in any of the above parameters. CONCLUSION: The processus vaginalis sacs in pediatric inguinal hernia, hydrocele and undescended testis differ in the presence, distribution and phenotype of smooth muscles and the presence of myofibroblasts. The clinical presentations in these entities reflect these differences.

Obesity aggravates neuroinflammatory and neurodegenerative effects of bisphenol A in female rats.

Bisphenol A (BPA), a common plasticizer, is categorized as a neurotoxic compound. Its impact on individuals exhibits sex-linked variations. Several biological and environmental factors impact the degree of toxicity. Moreover, nutritional factors have profound influence on toxicity outcome. BPA has been demonstrated to be an obesogen. However, research on the potential role of obesity as a confounding factor in BPA toxicity is lacking. We studied the neurodegenerative effects in high-fat diet (HFD)-induced obese female rats after exposure to BPA (10 mg/L via drinking water for 90 days). Four groups were taken in this study - Control, HFD, HFD + BPA and BPA. Cognitive function was evaluated through novel object recognition (NOR) test. Inflammatory changes in brain, and changes in hormonal level, lipid profile, glucose tolerance, oxidative stress, and antioxidants were also determined. HFD + BPA group rats showed a significant decline in memory function in NOR test. The cerebral cortex (CC) of the brain showed increased neurodegenerative changes as measured by microtubule-associated protein-2 (MAP-2) accompanied by histopathological confirmation. The increased level of neuroinflammation was demonstrated by microglial activation (Iba-1) and protein expression of nuclear factor- kappa B (NF-КB) in the brain. Obesity also caused significant (p < 0.05) increase in lipid peroxidation accompanied by reduced activities of antioxidant enzymes (glutathione S-transferase, catalase and glutathione peroxidase) and decrease in reduced-glutathione (p < 0.05) when compared to non-obese rats with BPA treatment. Overall, study revealed that obesity serves as a risk factor in the toxicity of BPA which may exacerbate the progression of neurological diseases.

Different Biofluids, Small Extracellular Vesicles or Exosomes: Structural Analysis in Atherosclerotic Cardiovascular Disease Using Electron Microscopy Techniques.

Small extracellular vesicles (sEVs) or exosomes are secretory vesicles largely involved in cell-cell communications and found to play a role in development as well as diseases including atherosclerosis. They hold a huge potential for translational research by devising better clinical diagnostics, biomarker discovery, drug delivery, and therapeutic strategies. Variations terms of morphology and distribution are crucial to biological function integrity. Moreover, it is dependent on susceptibility to influential factors of the environment like cell stress, inflammation, and secretion by different cells in subsequent biofluids. We have observed the morphological variations in sEVs or exosomes freshly isolated from patients with atherosclerotic cardiovascular disease (AsCVD), in blood plasma, saliva, and urine biofluids compared to healthy controls. High-resolution images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) for the characterization of sEVs morphology. Western blotting and immuno-TEM gold labeling confirmed the presence of exosome markers. For the first time, we report size and shape variations, which suggest the existence of different functions of sEVs in the disease state. Morphological variations in sEVs were observed significantly in noninvasive AsCVD saliva and urine samples, important to understand the cell behavior and physiological state. These variations will be useful to investigate their possible role in the disease process.

Evaluation of two rehydration protocols for fractured tooth fragments for characteristics of penetration of resin tags using confocal laser scanning microscopy.

BACKGROUND: Fragment reattachment is the recommended treatment modality in uncomplicated crown fractures. There is a paucity of literature regarding the mechanisms responsible for increased resistance to fracture after fragment rehydration in such cases. Hence, the aim of this proof-of-concept study was to decipher the microscopic changes in the penetration characteristics of resin in tooth fragments after different rehydration protocols. MATERIAL AND METHODS: Sixty bovine incisors free of structural deformities were fractured as per a standard protocol and the fragments were stored in saline at 4°C. They were randomly allocated into three groups (n = 20)-Group 1: negative-control, no-rehydration, Group 2: rehydration by immersion in distilled water for 15 min, Group 3: rehydration by humidification for 15 min. They were subjected to the "experimental bonding protocol" using an eighth-generation bonding agent mixed with rhodamine-B dye. The samples were subjected to decalcification and sectioned into cubical blocks (2 × 2 × 2 mm3 ). They were embedded in paraffin wax, sectioned by an ultramicrotome and evaluated by using a confocal laser scanning microscope. The depths and width of the resin tags were assessed by image analysis software, and the number of tags was counted manually by blinded evaluators. Statistical analysis was done with Stata-14. RESULTS: The depth of penetration of the resin tags was greatest in Group 2 (927.81 ± 280.38 µm) followed by Group 3 (902.03 ± 371.85 µm) and Group 1 (287.74 ± 124.80 µm). Similarly, the width of the penetrated resin tags was greatest in Group 2 (58.29 ± 21.15), followed by Group 3 (35.53 ± 22.15) and Group 1 (23.89 ± 6.88). The number of resin tags in the majority of the samples in Group 1 had less than 25 tags (65%), whereas there were more than 25 tags in Group 2 (70%) and Group 3 (75%). These differences were statistically significant (p < .05). CONCLUSION: The resin penetration, as observed by the number of tags and their depth and width, was significantly affected by the rehydration of the fragments. The fragments rehydrated in the distilled water had greater penetration of resin tags than those rehydrated in a humidification chamber.

Ultracellular Imaging of Bronchoalveolar Lavage from Young COVID-19 Patients with Comorbidities Showed Greater SARS-COV-2 Infection but Lesser Ultrastructural Damage Than the Older Patients.

In this study, we examined the cellular infectivity and ultrastructural changes due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the various cells of bronchoalveolar fluid (BALF) from intubated patients of different age groups (≥60 years and <60 years) and with common comorbidities such as diabetes, liver and kidney diseases, and malignancies. BALF of 79 patients (38 cases >60 and 41 cases <60 years) were studied by light microscopy, immunofluorescence, scanning, and transmission electron microscopy to evaluate the ultrastructural changes in the ciliated epithelium, type II pneumocytes, macrophages, neutrophils, eosinophils, lymphocytes, and anucleated granulocytes. This study demonstrated relatively a greater infection and better preservation of subcellular structures in these cells from BALF of younger patients (<60 years compared with the older patients (≥60 years). The different cells of BALF from the patients without comorbidities showed higher viral load compared with the patients with comorbidities. Diabetic patients showed maximum ultrastructural damage in BALF cells in the comorbid group. This study highlights the comparative effect of SARS-CoV-2 infection on the different airway and inflammatory cells of BALF at the subcellular levels among older and younger patients and in patients with comorbid conditions.

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.

Cardioprotective effects of azilsartan compared with that of telmisartan on an in vivo model of myocardial ischemia-reperfusion injury.

Azilsartan is found to be more potent than other angiotensin receptor blockers in reducing blood pressure. However, its effect on the heart following myocardial infarction remains to be established. For the first time, we investigated the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonistic and cardioprotective properties of azilsartan. Computational modeling studies of interactions between azilsartan and PPAR-γ revealed azilsartan as an agonist of PPAR-γ and showed the mechanism of azilsartan in cardioprotection. Our study compared the cardioprotective potential of telmisartan to that of azilsartan in a murine model of myocardial ischemia-reperfusion injury by comparing their antioxidant, ant apoptotic, anti-inflammatory, mitogen-activated protein kinase (MAPK)-modulating ability, and PPAR-γ agonistic activity. Male Wistar rats were grouped into four to receive vehicle (dimethyl sulfoxide [0.05%] 2 ml/kg) telmisartan (10 mg/kg p.o.), azilsartan (10 mg/kg p.o.) or azilsartan with specific PPAR-γ blocker, GW 9662 for 28 days. Ischemia was induced for 45 min on the 29th day followed by 60 min of reperfusion. Telmisartan and azilsartan pretreatment significantly nearly normalized cardiac parameters and preserved structural changes. Both drugs inhibited oxidative burst, inflammation, as well as cell death by modulating apoptotic protein expression along with reduction in 4',6-diamidino-2-phenylindole/terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. An increment in pro-survival kinase ERK paralleled with a reduction in p38 and JNK was also revealed by MAPK pathway studies, after administration of these drugs. Interestingly, the aforementioned changes induced by both drugs were reversed by administration of the specific PPAR-γ antagonist, GW9662. However, we found that azilsartan upregulated PPAR-γ to a lesser extent as compared to telmisartan and the latter may be preferred in hypertensive patients at risk of myocardial infarction.

Plasminogen activator inhibitor-1 promoter sequence variations in idiopathic osteonecrosis of head of femur.

Background & objectives: Primary or idiopathic osteonecrosis of femur head (ONFH) is the second most commonly observed cause among Indian patients suffering from ischemic ONFH. Although a number of genetic polymorphisms have been associated with idiopathic ONFH pathogenesis in Korean and Chinese populations, there are no studies in the Indian population. This is an exploratory study designed to implicate in promoter sequence polymorphisms of a critical fibrinolytic system regulator, plasminogen activator inhibitor-1 (PAI-1) gene, in cases of idiopathic osteonecrosis. Promoter sequence variations can affect expression levels of PAI-1 gene and may disrupt the coagulation/fibrinolytic equilibrium, which may finally culminate into osteonecrosis. Hence, the aim of the study was to investigate the role of single-nucleotide polymorphisms (SNPs) in the promoter region of PAI-1 gene and osteonecrosis development. Methods: Two SNPs of the PAI-1 gene (rs2227631, -844 G/A; rs1799889, -675 4G/5G) were genotyped in 25 patients diagnosed with idiopathic ONFH and 25 control subjects, using direct sequencing. Subsequently, association analyses were performed for the genotyped SNPs. Results: Both the rs2227631 and rs1799889 genotype and allele frequencies of PAI-1 gene showed an insignificant association with osteonecrosis risk (P=0.717, 0.149). Haplotype frequencies of rs2227631 and rs1799889 were also calculated in patients having idiopathic ONFH and controls. Although the distribution of haplotype GA-4G 4G was found to be the highest among the cases, it was not significantly different when compared with the controls. Interpretation & conclusions: Our findings demonstrate that the minor alleles of promoter region sequences of the PAI-1 gene do not contribute to an increase in ONFH predisposition. However, this is a preliminary study and its findings should be considered as suggestive for studies to be done in a larger sample size.

Novel role of mortalin in attenuating HIV-1 Tat-mediated astrogliosis.

BACKGROUND: In human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation. METHODS: Expression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed. RESULTS: HIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat. CONCLUSION: Overall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.

The molecular mechanism involved in cardioprotection by the dietary flavonoid fisetin as an agonist of PPAR-γ in a murine model of myocardial infarction.

The methodology exploring the cardioprotective potential of the flavonoid Fisetin through its ability to modulate PPAR-γ was unraveled in the present study. Computational modelling through molecular docking based binding study of interactions between Fiestin and PPAR-γ revealed the potential role of Fisetin as an agonist of PPAR-γ. A murine model of cardiac ischemia-reperfusion injury was used to explore this further. Male Wistar Rats were randomly assigned to five groups. Fisetin (20 mg/kg; p. o) was administered for 28 days. Ischemia was induced for 45 min on the 29th day followed by 60 min of reperfusion. Fisetin pretreatment upregulated the expression of PPAR-γ in heart tissue significantly Cardioprotection was assessed by measurement of hemodynamic parameters, infarct size, ELISA for oxidative stress, immunohistochemistry and TUNEL assay for apoptosis, and western blot analysis for MAPK proteins and inflammation. PPAR-γ activation by fisetin led to significantly reduced infarct size, suppression of oxidative stress, reduction of cardiac injury markers, alleviation of inflammation, and inhibition of apoptosis The MAPK-based molecular mechanism showed a rise in a key prosurvival kinase, ERK1/ERK2 and suppression of JNK and p38 proteins. The aforementioned beneficial findings of fisetin were reversed on the administration of a specific antagonist of PPAR-γ. In conclusion, through our experiments, we have proved that fisetin protects the heart against ischemia-reperfusion injury and the evident cardioprotection is PPAR-γ dependant. In conclusion, our study has revealed a prime mechanism involved in the cardioprotective effects of fisetin. Hence, Fisetin may be evaluated in further clinical studies as a cardioprotective agent in patients undergoing reperfusion interventions.

The dietary isoflavone daidzein mitigates oxidative stress, apoptosis, and inflammation in CDDP-induced kidney injury in rats: Impact of the MAPK signaling pathway.

Cisplatin-induced nephrotoxicity persists as a clinical problem despite several supportive measures to alleviate renal damage. Daidzein (DZ), a dietary isoflavone having antioxidant and anti-inflammatory activity, is investigated in this study for protective effects against cisplatin-induced renal injury in rats. DZ (25, 50, or 100 mg/kg; intraperitoneally; 10 days) was administered along with Cisplatin, single dose, on the 7th day of the experiment. On the 11th day, the rats were euthanized, and different samples were collected for analysis. Biochemical, histopathological, and molecular parameters were assessed to evaluate the effect of daidzein. Cisplatin injection resulted in renal dysfunction, lipid peroxidation that led to consumption of antioxidants, exaggerated apoptosis, and inflammation. These changes were associated with increase in the signaling proteins. DZ attenuated the toxic effects of cisplatin on the kidney at 100 mg/kg dose. The study concludes with the finding that daidzein imparts protection against the nephrotoxic effect of Cisplatin and can be considered as a novel, potential therapy.

Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their changes and significance with duration of injury.

Fibroblasts and myofibroblasts have been known to be present in both ruptured and intact human anterior cruciate ligament (ACL), and although their relevant histology and immunochemistry have been studied in the past, ultrastructural features of these cells are largely lacking. Therefore, we aim to characterise the ultrastructural details of these cells with the help of transmission electron microscopy (TEM) and to study the changes and their significance with duration of injury. Samples from 60 ruptured human ACL undergoing surgery were obtained and categorised according to duration of injury and observed under TEM with main focus on the following ultrastructural features: cellular morphology, presence of rough endoplasmic reticulum, Golgi apparatus, lamina, myofilaments, and presence of myofibroblasts. These features were further correlated with the duration of injury and association, if any, determined using appropriate statistical analysis. A total of 54 male and 6 female patients with mean duration of the injury of 23.01 ± 26.09 weeks (2-108 weeks) were included in the study and categorised into five groups based on duration of injury as follows: I (< 6 weeks), II (7-12 weeks), III (13-20 weeks), IV (21-50 weeks) and V (> 50 weeks). There was a significant association between the above-mentioned ultrastructural features and the duration of injury (p < 0.05) except for the presence of ovoid fibroblast cells (p = 0.53). Furthermore, number of myofibroblasts and cells with Golgi apparatus and rough endoplasmic reticulum was seen to peak at 13-20 weeks following injury. We describe ultrastructural features of fibroblast of different morphology along with myofibroblasts in the ligaments following injury, the changes in which might have a potential bearing on ligament healing.

Mangiferin attenuates cisplatin-induced acute kidney injury in rats mediating modulation of MAPK pathway.

Cisplatin has been confined due to the reported cases of nephrotoxicity. In the present study, an active xanthone, Mangiferin (from Mangifera indica) was investigated for its defensive role in cisplatin-induced nephrotoxicity. Male wistar albino rats were divided into six groups i.e., group 1 (normal); group 2 (cisplatin control); group 3, 4, and 5 (mangiferin 10, 20, and 40 mg/kg, i.p.); and per se (40 mg/kg; i.p.). The treatment was given for 10 days. On day 7, single dose of cisplatin 8 mg/kg i.p. was administered to induce nephrotoxicity in all groups except normal and per se. On day 11, animals were anesthetized, blood was taken from heart and serum was separated. Thereafter, rats were sacrificed and kidneys were isolated and preserved for histopathological, ultrastructural, immunohistochemical, and western blot analysis. Cisplatin control group showed significant impairment in renal function due to increased inflammation and oxidative stress which was also confirmed by histopathology and MAPK pathway proteins expression. However, pretreatment with mangiferin 20 and 40 mg/kg significantly reversed the renal function along with the structural changes and the levels of antioxidants. Mangiferin treatment attenuated DNA damage and apoptotic pathway.

Ultrastructural analysis of collagen fibril diameter distribution in cleft lip.

OBJECTIVE: A preliminary study to determine collagen fibril diameter (CF-ED) distribution on medial and lateral sides of cleft lip (CL). MATERIAL AND METHODS: Tissue samples from medial and lateral sides of CL were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in Araldite CY212 resin for transmission electron microscopy. The analysis of CF-ED was performed using the ImageJ program. To characterize the packaging of collagen fibrils (CFs) in the two tissues, we estimated the collagen number density (CF-ND) and fibril-area-fraction (FAF). Differences in measurements across the two sides were calculated using Wilcoxon signed-rank test. RESULTS: The CF-ED was statistically significantly (p < 0.001) smaller on the medial side (45.69 ± 7.89 nm) than on the lateral side (54.18 ± 7.62 nm). The medial side had a higher CF-ND and a higher percentage of FAF than the lateral side. CONCLUSION: Our finding of a smaller CF-ED and higher CF-ND and FAF for the medial side suggests possible differences in size and distribution of CFs between medial and lateral sides of CL. This finding provides knowledge toward underlying tissue biomechanics that may help reconstruction of perioral tissue scaffolds, ultimately resulting in better treatment of patients with oral clefts.

Ameliorative potential of rutin in combination with nimesulide in STZ model of diabetic neuropathy: targeting Nrf2/HO-1/NF-kB and COX signalling pathway.

Emerging role of Nrf-2/HO-1 in pathogenesis of diabetic neuropathy has been suggested. Diabetic neuropathy is one of the most common complications of diabetes and more than 50% patients of diabetes develop diabetic neuropathy. Rutin has been well documented to show protective effect in various complications, e.g., diabetic neuropathy. However, its mechanistic insight is still not completely understood. The present study has been designed to explore the protective effect of rutin and its interaction with COX-2 inhibitor, nimesulide in diabetic neuropathy. DN (diabetic neuropathy) rats were maintained with or without rutin (100 and 200 mg/kg), nimesulide (5 and 10 mg/kg), and their combinations for 8 weeks. Body weight, serum glucose, pain assessment (mechanical allodynia, cold allodynia, mechanical hyperalgesia, and thermal hyperalgesia), and motor nerve conduction velocity (MNCV) were measured in all groups. Oxidative damage was assessed through biochemical estimation and mitochondrial ROS production, followed by inflammatory and apoptotic markers (TNF-α, caspase-3, Nrf-2, HO-1, and NF-kBp65) for their activity, protein, and gene expression. The structural changes were also reported through transmission electron microscope. Streptozotocin injection (55 mg/kg) induced diabetes reduced body weight, reduced the threshold for pain in various pain assessment parameters. Oxidative damage (increased MDA, decreased SOD, catalase, and GSH levels) increased mitochondrial ROS production followed by increased expression of inflammatory markers and decreased expression of Nrf-2/HO-1 in sciatic nerve. Treatment with rutin (100 and 200 mg/kg) and nimesulide (5 and 10 mg/kg) significantly attenuates these alterations as compared to DN control rats. Furthermore, combination of rutin (200 mg/kg) and nimesulide (10 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect alone in streptozotocin-treated rats. The present study suggests the involvement of Nrf-2/HO-1 pathway in the protective effect of rutin against streptozotocin-induced diabetic neuropathy.

Global hypoxia induced impairment in learning and spatial memory is associated with precocious hippocampal aging.

Both chronological aging and chronic hypoxia stress have been reported to cause degeneration of hippocampal CA3 neurons and spatial memory impairment through independent pathways. However, the possible occurrence of precocious biological aging on exposure to single episode of global hypoxia resulting in impairment of learning and memory remains to be established. The present study thus aimed at bridging this gap in existing literature on hypoxia induced biological aging. Male Sprague Dawley rats were exposed to simulated hypobaric hypoxia (25,000ft) for different durations and were compared with aged rats. Behavioral studies in Morris Water Maze showed decline in learning abilities of both chronologically aged as well as hypoxic rats as evident from increased latency and pathlength to reach target platform. These behavioral changes in rats exposed to global hypoxia were associated with deposition of lipofuscin and ultrastructural changes in the mitochondria of hippocampal neurons that serve as hallmarks of aging. A single episode of chronic hypobaric hypoxia exposure also resulted in the up-regulation of pro-aging protein, S100A9 and down regulation of Tau, SNAP25, APOE and Sod2 in the hippocampus similar to that in aged rats indicating hypoxia induced accelerated aging. The present study therefore provides evidence for role of biological aging of hippocampal neurons in hypoxia induced impairment of learning and memory.

Association of ZEB1 and TCF4 rs613872 changes with late onset Fuchs endothelial corneal dystrophy in patients from northern India.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) results in loss of vision associated with progressive corneal edema and loss of corneal transparency. The aim of the study was to evaluate changes in ZEB1, COL8A2, SLC4A11, and TCF4 rs613872 and correlate them with clinical findings. METHODS: Eighty-two patients with clinically diagnosed FECD and 143 controls were recruited during the period 2007-2012. Clinical details, pedigree information up to three generations, and 5 ml of blood samples were collected. Histopathological and transmission electron microscopy studies were performed on host corneal buttons from patients who underwent keratoplasty. Genomic DNA from blood was processed for PCR amplification followed by direct sequencing to screen genetic changes in the candidate genes. The pathogenic nature of the genetic variants was assessed using Sorting Intolerant From Tolerant (SIFT) and MutationTaster. RESULTS: The mean age at the onset of symptoms was 59.14±1.41years, the male to female ratio was 1:1.5, and the mean specular count (endothelial cell density) was 1629±93.62 cells/mm(2) with a mean central corneal thickness (CCT) of 617.30±15.73 µm. ZEB1 showed a novel variant IVS2+276 C/T in 14% of the cases, a novel nonsense p.Leu947stop mutation in one patient, two novel missense mutations (p.Glu733Lys, p.Ala818Val) in one patient each, and one novel synonymous variation (p.Ser234Ser) in two patients. Reported mutation p.Gln840Pro and five polymorphisms were also identified. The TCF4 single nucleotide polymorphism (SNP) rs613872 was significantly higher in patients with FECD. CONCLUSIONS: This is the first report of genetic variations in ZEB1 and TCF4 SNP rs613872 in patients with FECD from northern India that suggests a possible role in disease pathogenesis and the regulation of endothelial cell density.

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.

Retinal neuroprotective effects of quercetin in streptozotocin-induced diabetic rats.

The aim of the present study was to evaluate the effects of Quercetin (Qctn), a plant based flavonol, on retinal oxidative stress, neuroinflammation and apoptosis in streptozotocin-induced diabetic rats. Qctn treatment (25- and 50 mg/kg body weight) was given orally for six months in diabetic rats. Retinal glutathione (GSH) and antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] were estimated using commercially available assays, and inflammatory cytokines levels [tumor necrosis factor-α (TNF-α), Interleukin-1ß (IL-1ß)] were estimated by ELISA method. Immunofluorescence and western blot studies were performed for nuclear factor kappa B (NF-kB), caspase-3, glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) expressions. Structural changes were evaluated by light microscopy. In the present study, retinal GSH levels and antioxidant enzyme (SOD and CAT) activities were significantly decreased in diabetic group as compared to normal group. However, in Qctn-treated rats, retinal GSH levels were restored close to normal levels and positive modulation of antioxidant enzyme activities was observed. Diabetic retinas showed significantly increased expression of pro-inflammatory cytokines (TNF-α and IL-1ß) as compared to that in normal retinas, while Qctn-treated retinas showed significantly lower levels of cytokines as compared to diabetic retinas. Light microscopy showed significantly increased number of ganglion cell death and decreased retinal thickness in diabetic group compared to those in normal retina; however, protective effect of Qctn was seen. Increased apoptosis in diabetic retina is proposed to be mediated by overexpression of NF-kB and caspase-3. However, Qctn showed inhibitory effects on NF-kB and caspase-3 expression. Microglia showed upregulated GFAP expression, and inflammation of Müller cells resulted in edema in their endfeet and around perivascular space in nerve fiber layer in diabetic retina, as observed through AQP4 expression. However, Qctn treatments inhibited diabetes-induced increases in GFAP and AQP4 expression. Based on these findings, it can be concluded that bioflavonoids, such as Qctn can be effective for protection of diabetes induced retinal neurodegeneration and oxidative stress.