Muscle fiber diameter assessment in cleft lip using image processing.

OBJECTIVE: To pilot investigation of muscle fiber diameter (MFD) on medial and lateral sides of the cleft in 18 infants with cleft lip with or without cleft palate (CL/P) using image processing. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue samples from the medial and lateral sides of the cleft were analyzed for MFD using an image-processing program (ImageJ). For within-case comparison, a paired Student's t test was performed. For comparisons between classes, an unpaired t test was used. RESULTS: Image processing enabled rapid measurement of MFD with majority of fibers showing diameter between 6 and 11 µm. There was no significant difference in mean MFD between the medial and lateral sides, or between CL and CLP. However, we found a significant difference on the medial side (p = .032) between males and females. CONCLUSION: The image processing on FFPE tissues resulted in easy quantification of MFD with finding of a smaller MFD on the medial side in males suggesting possible differences in orbicularis oris (OO) muscle between the two sexes in CL that warrants replication using larger number of cases. Moreover, this finding can aid subclinical phenotyping and potentially in the restoration of the anatomy and function of the upper lip.

Quantitative correlation of mechanoreceptors in tibial remnant of ruptured human anterior cruciate ligament with duration of injury and its significance: an immunohistochemistry-based observational study.

BACKGROUND: Proprioception is a specialized sensory modality encompassing the movement of the joint and its position in space. Reconstruction of the anterior cruciate ligament (ACL) does not always yield expected outcome, suggesting that successful reconstruction depends on not only the ultimate strength of the graft but also recovery of proprioception. Treatment delay is a significant concern in developing countries, e.g., in Asia. Thus, presence of mechanoreceptors is one of the factors having paramount importance for successful outcome. We conducted this study to identify mechanoreceptors via immunohistochemical staining and correlate their presence with duration of injury. MATERIALS AND METHODS: A total of 38 injured native ACL stumps were harvested from patients undergoing ACL reconstruction and stained with neurofilament protein stain to detect functional mechanoreceptors. RESULTS: Of the specimens, 44.7% stained positive for monoclonal antibody. No association was found between duration of injury and presence of mechanoreceptors (p = 0.897). No correlation was seen between age and side. CONCLUSIONS: No correlation was found between duration of injury and presence of viable mechanoreceptors, hence it is beneficial to preserve the native ACL stump irrespective of the time interval between injury and surgery. LEVEL OF EVIDENCE: III.

Clinical implications of microbial biofilms in chronic rhinosinusitis and orbital cellulitis.

BACKGROUND: Discovery of sessile mode of microbial existence (Biofilm state) focussed much interest, during the recent years, on the study of biofilms in many recurring and chronic infections. However, the exact role of microbial biofilms in chronic rhinosinusitis and orbital cellulitis were not elucidated earlier. The purpose of the present study was to look for the adherent property and biofilm producing ability of the clinical isolates in chronic rhinosinusitis and orbital cellulitis, and to look for the effects of antimicrobial agents on these biofilms by colorimetric assay and ultrastructural analysis. METHODS: Organisms were isolated and identified from various clinical samples in patients with chronic sinusitis and orbital cellulitis. Antimicrobial sensitivity testing was carried out by the standard protocol. Biofilms were developed; quantified and antimicrobial drug perfusion through the biofilm model was evaluated by the earlier devised procedure. Electronmicroscopic study of the biofilm was performed by the recommended technique. RESULTS: Of the total of 70 clinical samples processed, 48 i.e. 68.5 % grew bacteria and 13 i.e.(18.6 %) fungi. Staphylococcus aureus (20), S epidermidis (16) and Pseudomonas aeruginosa (6) accounted for the majority of the bacterial isolates. Aspergillus flavus (8), however was the commonest amongst the fungi. A total of 40 bacteria and 8 fungi could be tested for biofilm production. Eighteen (45 %) of the 40 bacterial isolates and 4(50 %) out of the 8 A flavus isolates were found to be biofilm producers. In vitro adherence testing revealed that majority i.e. 16 (88.8 %) of the 18 biofilm positive bacteria were adherent to artificial surfaces. Antimicrobial drug perfusion through the biofilm model was poor. Antimicrobial treatment was totally ineffective against strong biofilm producers, whose electron microscopic picture was quite similar to that observed for biofilm producers without any antimicrobial pre-treatment. CONCLUSIONS: Filamentous fungi, like bacteria were capable of forming biofilms, which could be one of the important virulence factors in determining the pathogenic potential of these organisms in causing chronic rhinosinusitis and orbital cellulitis.

Safety assessment and attenuation of cisplatin induced nephrotoxicity by tuberous roots of Boerhaavia diffusa.

Cisplatin (Cis-diaminedichloroplatinum II) is a chemotherapeutic agent having well documented adverse effect as nephrotoxicity. This study was designed to evaluate the nephroprotective role of Boerhaavia diffusa in cisplatin-induced acute kidney injury. Wistar rats (n = 6) were allocated into six groups constituting normal control, cisplatin-induced, Boerhaavia diffusa root extract in doses 50, 100 and 200 mg/kg and Boerhaavia diffusa per se group, administered orally for a period of ten days. Intraperitoneal injection of cisplatin was administered on day 7, to all groups except normal control and Boerhaavia diffusa per se group. On day 10, cisplatin resulted in substantial nephrotoxicity in Wistar rats with significant (p < 0.001) elevation in serum creatinine and blood urea nitrogen, decline in the concentrations of reduced glutathione and superoxide dismutase, elevation in TNF-α level in renal tissues. Boerhaavia diffusa at a dose of 200 mg/kg body weight significantly (p < 0.001) ameliorates increased in serum creatinine, blood urea nitrogen, oxidative stress and inflammatory markers. In parallel to this, it also exhibits antiapoptotic activity through the reduction of active caspase-3 expression in kidneys. Findings indicate that Boerhaavia diffusa is effective in mitigating cisplatin-induced nephrotoxicity and thus, for this the acute and sub-acute toxicity studies conducted to evaluate the safety profile of Boerhaavia diffusa. The no-observed adverse effect level (NOAEL) of tuberous roots of Boerhaavia diffusa root extract was 1000 mg/kg.

Molecular & phenotypic characterization of Staphylococcus epidermidis in implant related infections.

BACKGROUND & OBJECTIVES: The discrimination between the Staphylococcus epidermidis colonizing the deep seated indwelling devices and those which are mere commensals has always been a challenge for the clinical microbiologist. This study was aimed to characterize the S. epidermidis isolates obtained from device related infection for their phenotypic and molecular markers of virulence and to see whether these markers can be used to differentiate the pathogenic S. epidermidis from the commensals. METHODS: Fifty five S. epidermidis isolates from various device related infections such as endophthalmitis following intra-ocular lens (IOL) implantation, intravascular (IV) catheter related sepsis and orthopaedic implant infections, were studied for slime production, biotyping, antibiotic sensitivity; and mec A and ica positivity by the recommended procedures. RESULTS: Twenty three (41.8%) isolates were multi-drug resistant, 26 (65.2%) were slime producers, 30 (54.5%) were adherent, 23 (41.8%) possessed the intercellular adhesin (ica) gene, and 28 (50.9%) harboured the mec A gene. Biotypes I and III were the commonest, most members of which were multi- drug resistant. Twenty two (73.3%) of the 30 adherent bacteria were slime producers as opposed to only 4 (16%) of the 25 non-adherent bacteria (P<0.001). A vast majority i.e. 21 (91.3%) of the 23 ica positive organisms were adherent to artificial surfaces in contrast to only 9 (28.1%) of the 32 non-ica positive organisms (P<0.001). Twenty (86.9%) of the 23 ica positive bacteria were slime producers, as opposed to only 6 (18.7%) of the 32 ica negative bacteria (P<0.001). Of the 23 multi-drug resistant isolates, 19 (82.6%) carried the mec A gene. INTERPRETATION & CONCLUSIONS: The present findings showed that ica AB and mec A were the two important virulence markers of S. epidermidis in implant infections and slime was responsible for the sessile mode of attachment on the devices.

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

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

The eye of the tongue sole Cynoglossus bilineatus (Lacepède, 1802) (Teleostei: Pleuronectiformes).

We report the ocular features of the tongue sole, Cynoglossus bilineatus (Lacepède, 1802), a marine, bottom-dwelling flatfish. In this species, both eyes are located juxtaposed on the same side of the flat head. Histology revealed the sclera to be fibrous (collagenous) in nature. The choroid possesses the choriocapillaris, and adjacent to it, 3-4 rows of iridophores with stacks of cytoplasmic platelets. No choroidal gland is present. The retinal pigment epithelium (RPE) contains scanty melanin granules. Its vitread half is modified into a dense tapetum with lipid spheres (about 0.34 µm in diameter). In juveniles, the tapetal spheres arise by budding from the smooth endoplasmic reticulum of the RPE. There are blood vessels within the retina; the vitreal vessels penetrate the retina and ramify close to the level of the outer limiting membrane. The vessels are capillaries in nature. The photoreceptor layer contains abundant rods, and twin cones and single cones, being arranged into square mosaics. The optic disc is non-pleated and shows pan- cytokeratin immunopositivity, which is related to the bundled cytokeratin filaments detected in astrocytes by electron microscopy. The retinal tapetum and choroidal iridophores help the species to live in a muddy bottom having dim-light environment. The lack of a choroidal gland, hypoxic aquatic condition and presence of a dense retinal tapetum (that limits O2 transport to the photoreceptors) appear to have favored the proliferation of vitreal vessels within the retina in this species. The fibrous sclera has probably arisen to provide structural support to the eye in migration from the lateral to the dorsal aspect of the head during larval metamorphosis.

Telmisartan, a dual ARB/partial PPAR-γ agonist, protects myocardium from ischaemic reperfusion injury in experimental diabetes.

AIM: Apart from its angiotensin receptor blocker (ARB) activity, telmisartan is also a partial agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ). Therefore, we assessed whether telmisartan treatment attenuates myocardial ischaemia/reperfusion (I/R) injury in diabetic rats through PPAR-γ pathway. METHODS: Diabetic rats were randomized to receive vehicle (sham and I/R), telmisartan (10 mg/kg/day, orally), PPAR-γ antagonist GW9662 (1 mg/kg/day, intraperitoneally) or both for 14 days. On 15th day, excluding sham group, left anterior descending coronary artery occlusion was performed for 45 min followed by 1 h of reperfusion. Haemodynamic, biochemical, histopathological, ultrastructural, immunohistochemical (Bax and Bcl-2 protein), TUNEL positivity, infarct size and western blot studies were performed. RESULTS: Telmisartan treatment significantly improved cardiac function by normalizing mean arterial pressure, left ventricular pressure (±LVdP/dt(max) , a marker of myocardial contraction and relaxation), by decreasing left ventricular end-diastolic pressure (a marker of preload, 3.7 ± 0.41 vs. 7.3 ± 0.89, p < 0.001) and percent infarct area (37.52 ± 5.83 vs. 46.27 ± 3.20, p < 0.01) as compared to diabetic I/R group. Interestingly, GW9662 worsens the I/R injury (percent infarct area, 54.38 ± 6.48 vs. 46.27 ± 3.20, p < 0.01), whereas telmisartan with GW9662 (percent infarct area, 41.16 ± 8.23 vs. 46.27 ± 3.20, p < 0.05) showed lesser significant results as compared to telmisartan alone. Additionally, telmisartan significantly ameliorates activities of endogenous antioxidants, creatine kinase-MB isoenzyme, lactate dehydrogenase and prevented the increase of tumour necrosis factor-alpha and malondialdehyde in myocardium. Furthermore, telmisartan also decreased Bax expression (4.45 ± 1.24% vs. 10.25 ± 0.96%, p < 0.01), number of TUNEL-positive cells (6.2 ± 0.98% vs. 13.0 ± 1.6, p < 0.01), inflammation, myonecrosis and increased Bcl-2 expression (5.45 ± 0.15% vs. 1.24 ± 0.3%, p < 0.01). On the other hand, GW9662 treatment alone increased the Bax expression, TUNEL positivity and decreased Bcl-2 expression. Telmisartan protective effects were partially attenuated by a co-administration with GW9662. Western blot analysis showed that telmisartan treatment enhanced PPAR-γ expression, whereas GW9662 decreased it in myocardium. CONCLUSIONS: In addition to the class effect of ARBs, telmisartan has a beneficial effect in I/R injury in diabetic rats in part because of activation of PPAR-γ.

Ultrastructural changes in cristae of lymphoblasts in acute lymphoblastic leukemia parallel alterations in biogenesis markers.

We explored the link between mitochondrial biogenesis and mitochondrial morphology using transmission electron microscopy (TEM) in lymphoblasts of pediatric acute lymphoblastic leukemia (ALL) patients and compared these characteristics between tumors and control samples. Gene expression of mitochondrial biogenesis markers was analysed in 23 ALL patients and 18 controls and TEM for morphology analysis was done in 15 ALL patients and 9 healthy controls. The area occupied by mitochondria per cell and the cristae cross-sectional area was observed to be significantly higher in patients than in controls (p-value = 0.0468 and p-value< 0.0001, respectively). The mtDNA copy numbers, TFAM, POLG, and c-myc gene expression were significantly higher in ALL patients than controls (all p-values< 0.01). Gene Expression of PGC-1α was higher in tumor samples. The analysis of the correlation between PGC-1α expression and morphology parameters i.e., both M/C ratio and cristae cross-sectional area revealed a positive trend (r = 0.3, p = 0.1). The increased area occupied by mitochondria and increased cristae area support the occurrence of cristae remodelling in ALL. These changes might reflect alterations in cristae dynamics to support the metabolic state of the cells by forming a more condensed network. Ultrastructural imaging can be useful for affirming changes occurring at a subcellular organellar level.

Lipo-PEG nano-ocular formulation successfully encapsulates hydrophilic fluconazole and traverses corneal and non-corneal path to reach posterior eye segment.

The present study describes a special lipid-polyethylene glycol matrix solid lipid nanoparticles (SLNs; 138 nm; -2.07 mV) for ocular delivery. Success of this matrix to encapsulate (entrapment efficiency - 62.09%) a hydrophilic drug, fluconazole (FCZ-SLNs), with no burst release (67% release in 24 h) usually observed with most water-soluble drugs, is described presently. The system showed 164.64% higher flux than the marketed drops (Zocon®) through porcine cornea. Encapsulation within SLNs and slow release did not compromise efficacy of FCZ-SLNs. Latter showed in vitro and in vivo antifungal effects, including antibiofilm effects comparable to free FCZ solution. Developed system was safe and stable (even to sterilisation by autoclaving); and showed optimal viscosity, refractive index and osmotic pressure. These SLNs could reach up to retina following application as drops. The mechanism of transport via corneal and non-corneal transcellular pathways is described by fluorescent and TEM images of mice eye cross sections. Particles streamed through the vitreous, crossed inner limiting membrane and reached the outer retinal layers.

Ultrastructural and histological changes in tibial remnant of ruptured anterior cruciate ligament stumps: a transmission electron microscopy and immunochemistry-based observational study.

OBJECTIVES: Anterior cruciate ligament (ACL) rupture is a common injury and has a non-union rate of 40-100%. Important cellular events, such as fibroblast proliferation, angiogenesis and change in collagen fibril thickness in the ACL remnant, as described in other dense connective tissue, might have an implication in graft recovery following ACL reconstruction. Thus we conducted a study with an aim to characterize the ultrastructural and histological features of ruptured ACL tibial stump and correlate the same with the duration of injury. MATERIALS AND METHODS: This was a prospective observational study in which 60 ruptured human ACLs were evaluated for collagen fibril thickness, blood vessel density (per mm2) and fibroblast density (per mm2) with the help of transmission electron microscopy, immunohistochemistry via CD34 antibody staining and light microscopy (H&E staining). The findings were correlated with duration of injury. RESULTS: Fifty-four male and six female patients with a mean duration of the injury of 23.01 weeks (SD = 26.09; range 2-108 weeks) were included for the study and were divided on the basis of duration of injury as follows: Group I (≤ 6 weeks; N = 16), Group II (7-12 weeks; N = 18), Group III (13-20 weeks; N = 7), Group IV (21-50 weeks; N = 12), Group V (> 50 weeks; N = 7). A significant correlation was seen with blood vessel density (r = 0.303, p = 0.01) and fibroblast density (r = - 0.503, p = 0.001). Thickness of collagen fibril did not correlate with the duration of injury (r = 0.15, p = 0.23). The thickness of the collagen reached its peak after 50 weeks following injury, whereas highest density of blood vessel and fibroblast was seen at 12-20 weeks. Matched pair analysis revealed a significant decrease in collagen fibril thickness and an increase in fibroblast density at 7-12 weeks. CONCLUSION: Following injury to ACL, the ruptured tibial stump undergoes a series of changes at the cellular level vis-à-vis changes in collagen fibril thickness, vascular density and fibroblast density that possibly suggest an intrinsic healing response. This further may have implications on the functional outcome following ACL reconstruction with remnant preservation. LEVEL OF EVIDENCE: III.

Immunomodulatory effect of DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) on complement system and macrophages.

DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) is responsible for many immuno-dysregulatory functions in exposed animals, but data particularly on complement system and macrophages are limited. In this study we have shown that DDT activates the complement system through the alternative pathway in the absence of any pathogen. A significant (p<0.05) increase in C3b, C3d and C3a generation, and decline in complement hemolytic activity was observed in insecticide exposed sera. The uncontrolled complement consumption reduces the lytic activity of the complement, which enhances the susceptibility to pyogenic infection if the exposure to DDT remains unabated. Further, DDT induced the significant (p<0.05) production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages and thus contributes inflammatory reactions, cytokine imbalance and immune-dysregulation. These molecular changes in macrophages lead to structural aberrations like heterochromatin condensation, loss of pseudopodia, cytoplasmic vacuolization, DNA fragmentation and hypodiploid nuclei as seen in our study, suggesting apoptosis. However, in presence lipopolysaccharide, DDT induced significant (p<0.05) suppression of TNF-alpha and NO generation, suggestive of impairment of macrophage microbiocidal effects. This study concludes that the functional and structural derangements of macrophages in association with uncontrolled and excessive complement consumption by DDT are perhaps one of the major mechanisms contributing to the immunosuppressive effects of insecticide.

Comparison of unbiased stereological estimation of total number of cresyl violet stained neurons and parvalbumin positive neurons in the adult human spiral ganglion.

Estimation of total number of neurons in the spiral ganglion (SG) at various ages and their functional status is important as these neurons are constantly exposed to noise and other environmental factors that may lead to neuronal loss with aging due to excitotoxic damage. Parvalbumin (PV) is a calcium-binding protein (CBP), found in highly metabolically active neurons. It helps in buffering cytosolic calcium, which is essential for neurotransmitter release. The neurons in the adult human SG express PV more strongly than other CBPs like calbindin and calretinin. These CBPs can be used as signatures to recognise neurons. In the present study, we quantified the number of neurons expressing PV by unbiased stereology and compared it to the number of neurons stained by cresyl violet (CV), which is a Nissl stain, in the adult human SG. Five adult human cadaveric temporal bones were obtained from the forensic science mortuary, after due clearance from the institute ethics committee. Independent CV stained and PV immunostained sections were used to estimate the total number of neurons (optical fractionator), with StereoInvestigator (SI) software. The estimated total number of SG neurons was 27,485±3251 and 26,705±1823 in the PV and CV stained sections, respectively. There was no significant difference between the estimates (p=0.552). Therefore, CV staining is simpler and more cost effective when estimating neuronal number. Although PV stains spiral ganglion neurons (SGNs) with a greater intensity and provides a functional status, its tedious protocol limits its use for quantification.

Age-related decrease in rod bipolar cell density of the human retina: an immunohistochemical study.

During normal ageing, the rods (and other neurones) undergo a significant decrease in density in the human retina from the fourth decade of life onward.Since the rods synapse with the rod bipolar cells in the outer plexiform layer, a decline in rod density (mainly due to death)may ultimately cause an associated decline of the neurones which,like the rod bipolar cells,are connected to them.The rod bipolar cells are selectively stained with antibodies to protein kinase C-alpha.This study examined if rod bipolar cell density changes with ageing of the retina, utilizing donor human eyes (age: 6-91 years).The retinas were fixed and their temporal parts from the macula to the mid-periphery sectioned and processed for protein kinase C-alpha immunohistochemistry.The density of the immunopositive rod bipolar cells was estimated in the mid-peripheral retina (eccentricity: 3-5 mm)along the horizontal temporal axis.The results show that while there is little change in the density of the rod bipolar cells from 6 to 35 years (2.2%), the decline during the period from 35 to 62 years is about 21% and between seventh and tenth decades,it is approximately 27%.

Ultrastructural analysis of slime positive & slime negative Staphylococcus epidermidis isolates in infectious keratitis.

BACKGROUND & OBJECTIVE: Slime is a major determinant of Staphylococcus epidermidis adherence. The established methods of laboratory detection of slime production by this organism i.e., Christensen's tube method and congo red agar plate method, can both yield inconclusive and/or intermediate results. We, therefore tried to find out electronmicroscopically the localization of slime in relation to the bacterial cell wall and look for the effect, if any of the slime location on the staphylococcal adherence as well as on the quantum of slime production. METHODS: A total of 132 coagulase negative staphylococci from cases of infectious keratitis were identified as S. epidermidis following the recommended protocol. Slime was detected both by Christensen's tube method and congo red agar plate method. Antibiotic sensitivity testing was performed by standardized disc diffusion method. Adherence of the organisms to artificial surfaces was determined by a quantitative method and transmission electron microscopy was carried out by the conventional techniques. RESULTS: Of the total 132 isolates, 57 (43.2%) were slime positive and 75 (56.8%) were slime negative. Twenty seven (47.4%) of the 57 slime producing organisms were multi drug resistant as compared to only 12 (16%) of 75 nonslime-producing organisms (P<0.001). A majority i.e., 45 (78.9%) of 57 adherent organisms were slime producers as against 12 (16%) of 75 nonadherent organisms. Electron microscopic study revealed a thick viscid layer of slime anchoring to the bacterial cell wall, especially in adherent organisms and those yielding positive slime test. Some of the organisms showed loose nonadherent slime and those were mostly nonadherent to artificial surfaces. INTERPRETATION & CONCLUSION: Slime and multi drug resistance were the important virulence factors of S. epidermidis in bacterial keratitis. It was the adherent slime (i.e., slime in intimate association with the bacterial cell wall as shown by electron microscopy) only, which was responsible for resistance to multiple antibiotics and for the adhesion phenomenon observed in the quantitative slime test.

Cardioprotective response to chronic administration of vitamin E in isoproterenol induced myocardial necrosis: Hemodynamic, biochemical and ultrastructural studies.

The present study evaluated the cardioprotective potential of vitamin-E by studying its effect on hemodynamic parameters, lipid peroxidation, myocyte injury marker and ultrastructural changes in model of isoproterenol-induced myocardial necrosis in rats. Wistar albino male rats (150-200 g) were randomly divided into saline, ISP control, and vit E groups. Vitamin E group was administered vitamin E at a dose of 100mg/kg/day while saline and ISP control groups received saline orally for one month. On 29(th) and 30(th) day, ISP (85 mg/kg, sc) was administered at an interval of 24 h to vit E and ISP control rats. On 31(st) day, rats of all groups were anesthetized and hemodynamic parameters were recorded. At the end of experimentation, animals were sacrificed; hearts were excised and processed for biochemical and ultrastructural studies. ISP administration produced marked cardiac necrosis as evidenced by significant decrease in my ocardial creatine kinase-MB as well as increase in malonaldialdehyde levels. ISP-induced myocardial necrosis resulted in myocardial dysfunction as evidenced by significant depression in heart rate and mean arterial pressure in the ISP control group as compared to saline control. Salient ultrastructural changes including extensive loss of myofibrils, muscle necrosis, loss of mitochondria, and formation of several intracytoplasmic vacuoles and lipid droplets further confirmed the ISP-induced myocardial damage. However, subsequent to ISP challenge, vit E treatment significantly preserved the myocardium by restoring myocardial CK-MB activity, inhibiting the ISP-induced lipid peroxidation and ultrastructural changes. Additionally, pre-and co-treatment of vit E prevented the deleterious ultrastructural changes caused by ISP. These beneficial effects of chronic vit E treatment also translated into significant restoration of the altered hemodynamic parameters. The present study clearly demonstrated the cardioprotective potential of vit E at dose of 100 mg/kg in ISP-induced model of myocardial necrosis in rats. The significant restoration of altered hemodynamic parameters, myocardial CK-MB activity, prevention of ISP-induced rise in lipid peroxidation and ultrastructural changes may confirm its cardioprotective effect.

Arsenic induced apoptosis in rat liver following repeated 60 days exposure.

BACKGROUND: Accumulation of the wide spread environmental toxin arsenic in liver results in hepatotoxcity. Exposure to arsenite and other arsenicals has been previously shown to induce apoptosis in certain tumor cell lines at low (1-3 microM) concentration. AIM: The present study was focused to elucidate the role of free radicals in arsenic toxicity and to investigate the nature of in vivo sodium arsenite induced cell death in liver. METHODS: Male wistar rats were exposed to arsenite at three different doses of 0.05, 2.5 and 5mg/l for 60 days. Oxidative stress in liver was measured by estimating pro-oxidant and antioxidant activity in liver. Histopathological examination of liver was carried out by light and transmission electron microscopy. Analysis of DNA fragmentation by gel electrophoresis was used to identify apoptosis after the exposure. Terminal deoxy-nucleotidyl transferase mediated dUTP Nick end-labeling (TUNEL) assay was used to qualify and quantify apoptosis. RESULTS: A significant increase in cytochrome-P450 and lipid peroxidation accompanied with a significant alteration in the activity of many of the antioxidants was observed, all suggestive of arsenic induced oxidative stress. Histopathological examination under light and transmission electron microscope suggested a combination of ongoing necrosis and apoptosis. DNA-TUNEL showed an increase in apoptotic cells in liver. Agarose gel electrophoresis of DNA of hepatocytes resulted in a characteristic ladder pattern. CONCLUSION: Chronic arsenic administration induces a specific pattern of apoptosis called post-mitotic apoptosis.

The effects of an electromagnetic field on the boundary tissue of the seminiferous tubules of the rat: A light and transmission electron microscope study.

Human beings are unavoidably exposed to ambient electromagnetic fields (EMF) generated from various electrical devices and from power transmission lines. Controversy exists about the effects of EMF on various organs. One of the critical issues is that EMF may adversely affect the reproductive system. In order to examine this 30 rat pups were exposed to 50 Hz EMF (non-ionising radiation) during in utero development (approximately 3 weeks) and postnatal life (5 weeks). Groups of exposed rats were subsequently left in an environment free of EMF in order to observe recovery, if any, from the changes induced by EMF on the boundary tissue of the seminiferous tubules. The materials were processed and observed under a light and a transmission electron microscope. In the experimental rats boundary tissue was found disrupted at various layers. This tissue showed infoldings, which were perhaps due to the loss of collagen and reticular fibrils from the inner and outer non-cellular layers. The outer non-cellular layer, which was thinner than that of the control, was stripped away from the myoid cell layer in multiple regions, giving a "blister-like" appearance. The myoid cells showed fewer polyribosomes, pinocytotic vesicles and glycogen granules. Most mitochondria were found to lack cristae. The connections between individual myoid cells were apparently lost. There were signs of recovery in the boundary tissue following withdrawal from EMF exposure. These results suggest that EMF exposure may cause profound changes in the boundary tissue of the seminiferous tubules. Therefore exposure to EMF may result in pathological changes that lead to subfertility and infertility.

Chrysin, a PPAR-γ agonist improves myocardial injury in diabetic rats through inhibiting AGE-RAGE mediated oxidative stress and inflammation.

AGE-RAGE interaction mediated oxidative stress and inflammation is the key mechanism involved in the pathogenesis of cardiovascular disease in diabetes. Inhibition of AGE-RAGE axis by several PPAR-γ agonists has shown positive results in ameliorating cardio-metabolic disease conditions. Chrysin, a natural flavonoid has shown to possess PPAR-γ agonist activity along with antioxidant and anti-inflammatory effect. Therefore, the present study was designed to evaluate the effect of chrysin in isoproterenol-induced myocardial injury in diabetic rats. In male albino Wistar rats, diabetes was induced by single injection of streptozotocin (70 mg/kg, i.p.). After confirmation of the diabetes, rats were treated with vehicle (1.5 mL/kg, p.o.), chrysin (60 mg/kg, p.o.) or PPAR-γ antagonist GW9662 (1 mg/kg, i.p.) for 28 days. Simultaneously, on 27th and 28th day myocardial injury was induced by isoproterenol (85 mg/kg, s.c.). Chrysin significantly ameliorated cardiac dysfunction as reflected by improved MAP, ±LVdP/dtmax and LVEDP in diabetic rats. This improvement was associated with increased PPAR-γ expression and reduced RAGE expression in diabetic rats. Chrysin significantly decreased inflammation through inhibiting NF-κBp65/IKK-ß expression and TNF-α level. Additionally, chrysin significantly reduced apoptosis as indicated by augmented Bcl-2 expression and decreased Bax and caspase-3 expressions. Furthermore, chrysin inhibited nitro-oxidative stress by normalizing the alteration in 8-OHdG, GSH, TBARS, NO and CAT levels and Nox4, MnSOD, eNOS and NT expressions. Co-administration of GW9662 significantly blunted the chrysin mediated cardioprotective effect as there was increase in oxidative stress, inflammation and apoptosis markers. Chrysin significantly ameliorated isoproterenol-induced myocardial injury in diabetic rats via PPAR-γ activation and inhibition of AGE-RAGE mediated oxidative stress and inflammation.