Resveratrol attenuates arsenic-induced cognitive deficits via modulation of Estrogen-NMDAR-BDNF signalling pathway in female mouse hippocampus.

BACKGROUND: Chronic inorganic arsenic (iAs) exposure induces deleterious effects on CNS including oxidative stress, cognitive deficits and altered brain neurochemistry. Little is known about the association between iAs and estrogen receptor expression in brain regions. AIMS AND OBJECTIVES: Owing to the neuroprotective and estrogenic activities of resveratrol (RES), we examined the combined effects of arsenic trioxide (As2O3) and RES on neurobehavioural functions, estrogen signalling and associated neurochemical changes in mouse hippocampus. MATERIALS AND METHODS: As2O3 alone (2 and 4 mg/kg bw) or along with RES (40 mg/kg bw) was administered orally for 45 days to adult female mice. From days 33 to 45, open field, elevated plus maze and Morris water maze tests were conducted to evaluate locomotion, anxiety and learning and memory. On day 46, animals were euthanized and brain tissue and hippocampi obtained therefrom were processed for atomic absorption spectrophotometry and western blotting respectively. RESULTS: As2O3 alone exposure resulted in enhanced anxiety levels, reduced locomotion and impaired learning and memory. As2O3-induced behavioural deficits were accompanied by downregulation of estrogen receptor (ERα) expression with a concomitant reduction of BDNF and NMDAR 2B levels in the hippocampus. However, the behavioural alterations and expression of these markers were restored in RES-supplemented mice. Moreover, a dose-dependent iAs accumulation was observed in serum and brain tissues of mice receiving As2O3 alone whereas simultaneous administration of As2O3 with RES facilitated iAs efflux. CONCLUSIONS: These results suggest that reduced ERα expression with associated downregulation of BDNF and NMDAR 2B levels could be a mechanism by which iAs induces cognitive impairment; hence, the modulation of estrogen-NMDAR-BDNF pathway by RES represents a potential avenue to recover behavioural deficits induced by this neurotoxin.

Curcumin supplementation shows modulatory influence on functional and morphological features of hippocampus in mice subjected to arsenic trioxide exposure.

Since, oxidative stress has been suggested as one of the mechanisms underlying arsenic-induced toxicity, the present study focused on the role of antioxidant (curcumin) supplementation on behavioral, biochemical, and morphological alterations with context to mice hippocampus (CA1) following arsenic trioxide (As2O3) administration. Healthy male Swiss albino mice were divided into control and experimental groups. As2O3 (2 mg/kg bw) alone or along with curcumin (100 mg/kg bw) was administered to experimental groups by oral route for 45 days whereas the control groups received either no treatment or vehicle for curcumin. Animals were subjected to behavioral study towards the end of the experimental period (day 33-45). On day 46, the brain samples were obtained and subjected either to immersion fixation (for morphometric observations) or used afresh for biochemical test. Behavioral tests (open field, elevated plus maze, and Morris water maze) revealed enhanced anxiety levels and impairment of cognitive functions in As2O3 alone treated groups whereas a trend of recovery was evident in mice simultaneously treated with As2O3 and curcumin. Morphological observations showed noticeable reduction in stratum pyramidale thickness (CA1), along with decrease in density and size of pyramidal neurons in As2O3 alone exposed group as compared to As2O3+Cu co-treated group. Hippocampal glutathione levels were found to be downregulated in animals receiving As2O3 as against the levels of controls and curcumin supplemented animals, thereby, suggestive of beneficial role of curcumin on As2O3 induced adverse effects.

Effect of α-lipoic acid on spatial memory and structural integrity of developing hippocampal neurons in rats subjected to sodium arsenite exposure.

BACKGROUND: Exposure to arsenic has been reported to affect the nervous system in a number of ways. Various epidemiological studies suggest cognitive impairment in subjects following exposure to environmental arsenic. The goal of the present study was to determine if supplementation of exogenous α-lipoic acid (ALA) could ameliorate sodium arsenite (NaAsO2) induced adverse effects on learning and memory and synaptic connectivity in rat hippocampus. METHODS: Accordingly, NaAsO2 alone (1.5/2.0 mg/kg bw) or NaAsO2 along with ALA (70 mg/kg bw) was administered by intraperitoneal (i.p.) route from postnatal day (PND) 4-17 to Wistar rat pups (experimental groups) and the Control groups received either distilled water or no treatment at all. After carrying out Elevated Plus Maze (EPM) and Morris Water Maze (MWM) test, the fresh brain tissues were collected on PND 18 and processed for Golgi Cox staining. RESULTS: Observations of MWM test revealed impaired learning and memory in iAs alone treated animals as against those co-exposed to iAs and ALA. In Golgi stained hippocampal sections of iAs alone treated animals, decreased dendritic arborization and reduced number of spines in pyramidal neurons (CA1) and granule cells (DG) was observed whereas neuronal morphology was preserved in the controls and ALA supplemented groups CONCLUSIONS: These observations are suggestive of beneficial effects of ALA on iAs induced effects on learning and memory as well as on hippocampal neuronal morphology.

Ameliorative role of antioxidant supplementation on sodium-arsenite induced adverse effects on the developing rat cerebellum.

BACKGROUND: Arsenic is an environmental contaminant of global concern. Consumption of ground water contaminated with inorganic arsenic (iAs) continues to be the major source of its exposure. The developing nervous system is especially vulnerable to environmental insults due to its higher rate of oxygen consumption and provision of weaker antioxidant (AOX) machinery. OBJECTIVE: Since oxidative stress has been reported as one of the major factors underlying iAs induced toxicity, the aim of the present study is to study the effect of two AOXs i.e., Alpha Lipoic Acid (ALA) and Curcumin (Cur) in developing cerebellum of rats exposed to arsenic during postnatal period. MATERIALS AND METHODS: The study was carried out on mother reared neonatal rat pups grouped as normal (Ia) and sham (vehicle) controls (Ib,c,d), while the experimental groups IIa/ IIb received sodium arsenite (NaAsO2) [(1.5/2.5 mg/kg body weight (bw)] alone or along with ALA (70 mg/kg bw)- IIIa/ IIIb or along with Cur (150 mg/kg bw)- IVa/ IVb. Behavioural, biochemical and immunohistochemical procedures were carried out to understand the underlying mechanisms. RESULTS: The observations indicated deficits in locomotor function, accumulation of iAs, increased levels of oxidative stress markers along with downregulation of the expression of proteins closely associated with synaptic functioning (Synaptophysin and Postsynaptic density protein95) in the cerebellum of iAs treated animals. Substantial recovery in all these parameters was observed in AOX co-treated groups. CONCLUSION: Our results support the potential of ALA and Cur in amelioration of iAs induced developmental neurotoxicity. ALA and Cur can be proposed as dietary adjuvants amongst populations inhabiting areas with high iAs contamination as a safe and cost effective antidotes.

Antioxidant supplementation upregulates calbindin expression in cerebellar Purkinje cells of rat pups subjected to post natal exposure to sodium arsenite.

Optimal cytoplasmic calcium (Ca2+) levels have been associated with adequate cell functioning and neuronal survival. Altered intracellular Ca2+ levels following impaired Ca2+ homeostasis could induce neuronal degeneration or even cell death. There are reports of arsenite induced oxidative stress and the associated disturbances in intracellular calcium homeostasis. The present study focused on determining the strategies that would modulate tissue redox status and calcium binding protein (CaBP) (Calbindin D28k-CB) expression affected adversely by sodium arsenite (NaAsO2) exposure (postnatal) of rat pups. NaAsO2 alone or along with antioxidants (AOXs) (alpha lipoic acid or curcumin) was administered by intraperitoneal (i.p.) route from postnatal day (PND) 1-21 (covering rapid brain growth period - RBGP) to experimental groups and animals receiving sterile water by the same route served as the controls. At the end of the experimental period, the animals were subjected to euthanasia and the cerebellar tissue obtained therefrom was processed for immunohistochemical localization and western blot analysis of CB protein. CB was diffusely expressed in cell body as well as dendritic processes of Purkinje cells (PCs) along the PC Layer (PCL) in all cerebellar folia of the control and the experimental animals. The multilayered pattern of CB +ve cells along with their downregulated expression and low packing density was significantly evident in the arsenic (iAs) alone exposed group as against the controls and AOX supplemented groups. The observations are suggestive of AOX induced restoration of CaBP expression in rat cerebellum following early postnatal exposure to NaAsO2.

Alpha lipoic acid (ALA) modulates expression of apoptosis associated proteins in hippocampus of rats exposed during postnatal period to sodium arsenite (NaAsO2).

The present study focused on the role of exogenous alpha lipoic acid (ALA) in amelioration of inorganic arsenic (iAs) induced effects on apoptosis and apoptosis associated proteins in developing rat hippocampus. NaAsO2 (1.5/2.0 mg/kg bw) alone or along with ALA (70 mg/kg bw) was administered to rat pups (experimental groups) by intraperitoneal (i.p.) route from postnatal day (PND) 4-15. Controls received no treatment/distilled water/ALA. On PND 16, the animals were perfusion fixed and the brains were processed for paraffin embedding (CV and TUNEL staining) and cryopreservation (immunohistochemistry). The fresh brain tissue was used for Western blotting. Significant increase was observed in TUNEL positive cells and Bax (pro-apoptotic protein) expression in hippocampal sub-regions of iAs alone treated groups, whereas Bcl-2 expression was intensified in animals receiving ALA with iAs. Densitometric analysis (Western blots) revealed optimal restoration of Bax and Bcl-2 ratio in animals receiving ALA with iAs, thereby suggesting the protective role of ALA in iAs induced developmental neurotoxicity.

Hepatic flexure in right sub diaphragmatic space and its embryological basis and clinical importance.

The knowledge of variations in the large intestine and liver is of clinical importance from the anatomical and embryological points of view. Different positions of the hepatic flexure of large intestine, although generally asymptomatic, may have different impact on manifestations of disease. During routine cadaveric study of the abdominal region we observed a case where the hepatic flexure was interposed between the right dome of the diaphragm and the anterior surface of the liver. The liver appeared bilobulated and on the anterior surface the right and left hepatic lobes were separated by a deep furrow. The left wall of the furrow was attached to the falciform ligament. We have tried to explain such high position of hepatic flexure from an embryological point of view and to evaluate its possible clinical relevance. This abnormal site of hepatic flexure could cause chronic respiratory infections, twisting of the gut, volvulus and intestinal obstruction. Moreover it may alter the normal liver dullness on percussion. So clinicians and surgeons should be aware of this variant position of the hepatic flexure.

Surgical importance of arterial segments of human kidneys: an angiography and corrosion cast study.

OBJECTIVE: Variations in the arterial supply of human kidney have been observed frequently, either in routine dissections or surgical practice. The main objective of the present study was to describe the arterial segmental pattern of human kidneys and its variation by angiography and corrosion cast techniques. MATERIALS AND METHODS: Forty kidneys were washed and a plastic cannula was inserted into renal artery and the omnipaque dye was injected into it and X-ray was taken. The corrosion casts were prepared by injecting coloured acetate butyrate (CAB) granules solution. RESULTS: Five vascular segments of kidney were seen based on the branching pattern of the renal artery by angiography and corrosion cast techniques. The renal artery was divided into anterior and posterior branches. The anterior branch further divided into four branches viz. apical segmental artery (ASA), Upper segmental artery (USA), middle segmental artery (MSA), lower segmental artery (LSA) while the posterior branch continue as posterior segmental artery. The origins of segmental arteries were variable. In 60% cases apical segmental artery (ASA) had common origin with upper segmental artery (USA) while in 40% cases it took origin directly from the main renal artery. Similarly the variations in the origin of the other branches of anterior division of renal artery were observed. The posterior segmental artery (PSA) however was single and comparatively small and supplied the posterior surface of the kidney. CONCLUSION: The knowledge of the vascular pattern of the kidney is thus important for the purpose of angiography and surgical procedures especially for nephrectomy and kidney transplantation.

Preliminary morphological and immunohistochemical changes in rat hippocampus following postnatal exposure to sodium arsenite.

The effects of arsenic exposure during rapid brain growth period (RBGP) (postnatal period 4-11) on pyramidal neurons of cornu ammonis (specifically CA1 and CA3 regions) and granule cells of dentate gyrus (DG) of rat hippocampus were studied. Wistar rat pups, subdivided into the control (group I) and the experimental groups (group II, III, and IV), received distilled water and sodium arsenite (aqueous solution of 1.0, 1.5, and 2.0 mg/kg body weight, respectively) by intraperitoneal (i.p.) route. On postnatal day (PND) 12, the animals were sacrificed and brain tissue obtained. Paraffin sections (8 µm thick) stained with Cresyl Violet (CV) were observed for morphological and morphometric parameters. Arsenic induced programmed cell death (apoptosis) was studied using Terminal deoxyribonucleotidyl transferase mediated dUTP biotin Nick End Labeling (TUNEL) technique on the paraffin sections. Microscopy revealed decreased number and isolation of pyramidal neurons in superficial layers, misalignments of pyramidal cells in stratum pyramidale (SP) of CA1 and CA3 in experimental group III and IV, and presence of polymorphic cells in subgranular zone of ectal limb of dentate gyrus (suggestive of arsenic induced proliferation and migration of granule cells in the dentate gyrus). Morphometric assessments quantified and confirmed the microscopic findings. The mean nuclear area of pyramidal cells was increased and cell density was decreased in the CA1, CA3, and DG of experimental groups in comparison to the control group. Increase in the TUNEL positive cells in DG was observed in the experimental group IV, suggestive of increased apoptosis. These observations confirm vulnerability of pyramidal (CA1, CA3) and granule cells (DG) of hippocampus during RBGP.

Postnatal Exposure to Sodium Arsenite (NaAsO(2)) Induces Long Lasting Effects in Rat Testes.

OBJECTIVE: The present study was undertaken to investigate the effects of early postnatal exposure to sodium arsenite (NaAsO(2)) on rat testis. MATERIALS AND METHODS: Wistar rat pups were administered aqueous solution of NaAsO(2,) 1.5 mg/kg body weight (bw) (experimental) and distilled water (control), respectively, by intraperitoneal route (i.p.) from postnatal day (PND) 1 to 14. Testes were collected after 1, 7 and 36 days (at PND 15, 21 and 50) after the treatment period (PND1-14) from the animals and immersion fixed in Bouin's fluid followed by paraffin embedding. Seven micrometer thick serial sections were cut and stained with hematoxylin and eosin for light microscopic observations. At PND 50, morphological features of sperms and their counting was carried out besides processing the perfusion-fixed testes for electron microscopy (EM). RESULTS AND CONCLUSIONS: The observations revealed an altered morphology of the seminiferous tubules (ST) along with degeneration and dissociation of spermatogenic cells in the experimental animals at PND 15, 21 and 50. Also, increased number of sperms with abnormal morphology and decreased sperm count was noted in the experimental animals. These features together with electron microscopic observations of abnormal mitochondria and apoptotic nuclei of spermatogonia and spermatocytes could be indicative of long-lasting adverse effects on the rat testis induced by exposure to As during early postnatal period.

Preliminary morphological and biochemical changes in rat liver following postnatal exposure to sodium arsenite.

The effects of sodium arsenite exposure on the hepatic maturation period of cellular and functional reorganization in developing rat livers were evaluated. Animals received intraperitoneal injections of sodium arsenite (1.5 mg/kg body weight) or distilled water on days 9 to 28 after birth. On day 29, the animals were sacrificed either by cervical dislocation or by perfusion fixation. The perfusion fixed liver tissue was processed for paraffin embedding, sectioning and hematoxylin and eosin staining. The fresh liver tissue was processed for cryo-sectioning followed by Sudan Black B staining and for biochemical estimation of reduced glutathione. Microscopic observation revealed comparable preserved hepatic lobular patterns and distributions of uninucleate and binucleate hepatocytes in the control and the experimental groups. The mean nuclear area and diameter of the hepatocytes was increased in the experimental group. Lipid droplet distribution pattern in Sudan Black B stained sections revealed higher staining intensity towards the centrilobular area in both groups. Semiquantitative estimation of staining intensity showed lower mean gray values in zone 3 than in zones 2 and 1 (suggestive of the setting in of the adult pattern) in both groups. The reduced glutathione levels in the liver tissue and the altered nuclear size of the hepatocytes in the experimental group suggested the impairment of morphological and biochemical processes induced by arsenic exposure during the postnatal period.

Protective role of exogenous α-lipoic acid (ALA) on hippocampal antioxidant status and memory function in rat pups exposed to sodium arsenite during the early post-natal period.

The present work focussed on the effect of exogenous α-lipoic acid (ALA) administration on retention memory and oxidative stress markers in the hippocampus subsequent to early post-natal exposure of rat pups to sodium arsenite (NaAsO(2)). Wistar rat pups were divided into the control groups receiving either no treatment (Ia) or distilled water by intraperitoneal route (i.p.) (Ib) and the experimental groups receiving either NaAsO(2) alone (1.5 and 2.0 mg/kg body wt.) (IIa, IIb) or NaAsO(2) (1.5 and 2.0 mg/kg body wt.) followed by ALA (70 mg/kg body wt.) (IIIa, IIIb) (i.p.) from post-natal day (PND) 4-15. The initial and retention transfer latency (ITL and RTL) was determined on PND 14 and 15 using elevated plus maze. The animals were sacrificed by cervical decapitation (PND 16) and the brains were obtained. The dissected out hippocampus was processed for estimation of oxidative stress markers, glutathione (GSH), and superoxide dismutase (SOD). NaAsO(2) exposure resulted in longer RTL in animal groups IIa and IIb, thereby suggestive of arsenic-induced impairment in retention memory. RTL was significantly shorter in animal groups (IIIa, IIIb) receiving ALA following NaAsO(2), thereby suggestive of improvement in retention memory. GSH and SOD levels were significantly decreased in animals receiving NaAsO(2) alone as against group Ib and administration of ALA following NaAsO(2) increased the levels of hippocampal GSH and SOD. These observations are suggestive of the role of exogenous ALA in ameliorating the adverse effects induced by NaAsO(2) exposure of rat pups on retention memory and oxidative stress markers.

Preliminary morphological and morphometric study of rat cerebellum following sodium arsenite exposure during rapid brain growth (RBG) period.

The effects of arsenic exposure during rapid brain growth (RBG) period were studied in rat brains with emphasis on the Purkinje cells of the cerebellum. The RBG period in rats extends from postnatal day 4 (PND 4) to postnatal day 10 (PND 10) and is reported to be highly vulnerable to environmental insults. Mother reared Wistar rat pups were administered intraperitoneal injections (i.p.) of sodium arsenite (aqueous solution) in doses of 1.0, 1.5 and 2.0mg/kg body weight (bw) to groups II, III and IV (n=6 animals/group) from PND 4 to 10 (sub acute). Control animals (group I) received distilled water by the same route. On PND 11, the animals were perfusion fixed with 4% paraformaldehyde in 0.1M phosphate buffer (PB) with pH 7.4. The cerebellum obtained from these animals was post-fixed and processed for paraffin embedding. Besides studying the morphological characteristics of Purkinje cells in cresyl violet (CV) stained paraffin sections (10 microm), morphometric analysis of Purkinje cells was carried out using Image Analysis System (Image Proplus software version 4.5) attached to Nikon Microphot-FX microscope. The results showed that on PND 11, the Purkinje cells were arranged in multiple layers extending from Purkinje cell layer (PL) to outer part of granule cell layer (GL) in experimental animals (contrary to monolayer arrangement within PL in control animals). Also, delayed maturation (well defined apical cytoplasmic cones and intense basal basophilia) was evident in Purkinje cells of experimental animals on PND 11. The mean Purkinje cell nuclear area was significantly increased in the arsenic treated animals compared to the control animals. The observations of the present study (faulty migration, delayed maturation and alteration in nuclear area measurements of Purkinje cells subsequent to arsenic exposure) thus provided the morphological evidence of structural alterations subsequent to arsenite induced developmental neurotoxicity which could be presumed to be the underlying basis for some of the functional deficits encountered in the later period of life.

Main and accessory renal arteries--a morphological study.

The present work was undertaken to document the incidence of accessory renal arteries in kidney specimens obtained from subjects of Indian origin. Comprehensive dissection carried out in the dissection hall of Anatomy Department of All India Institute of Medical Sciences (India) on forty cadavers over a period of five years revealed a single main renal artery on either side in 80% of the specimens. The mean length of the main renal artery was 31.05 +/- 12 and 25.0 +/- 9.5 mm on the right and the left side respectively. Multiple (accessory) renal arteries were observed in 20% of the specimens with unilateral anomaly (15%) being more commonly encountered than bilateral anomaly (5%). The mean length of the accessory renal artery was 36.4 +/- 10 and 36.6 +/- 11 mm on the right and the left side respectively. The accessory artery when present, invariably crossed the anterior aspect of the ureter. Familiarity about the possible variations in the renal arterial pattern are especially important for the personnel dealing with kidney retrieval and transplantation, various endourologic procedures and innumerable interventional techniques. In the majority of such situations, it is the comprehensive knowledge of the renal arterial pattern which remains the key issue in determining the technical feasibility of surgical interventions as well as the post operative management.

An additional renal vein.

A routine cadaveric dissection in an adult male revealed the emergence of two renal veins at the hilum of the right kidney that presented two separate openings in the inferior vena cava (IVC), one above the other. At the hilum the segmental branches of the right renal artery were sandwiched between the two veins. An additional venous tributary was present posterior to the right renal pelvis. A single renal vein emerged at the hilum of the left kidney. The various tributaries observed on the right and left sides were measured. The possible developmental basis for this variation suggests the persistence of the embryonic pattern observed in the 22 mm embryo.