Pushpa Dhar

Chronic exposure to estrogen and tamoxifen regulates synaptophysin and phosphorylated cAMP response element-binding (CREB) protein expression in CA1 of ovariectomized rat hippocampus

We report here the in vivo effects of estrogen (E2) on modulation of synaptic plasticity and the agonistic (estrogen-like) role of selective estrogen receptor modulator (SERM), tamoxifen (TAM) in the CA1 of the rat hippocampus. Effects on synaptophysin (SYP), a presynaptic vesicular protein, and phosphorylated cyclic AMP responsive element-binding (p-CREB) protein, a signal transduction pathway molecule, were studied using the ovariectomized (OVX) experimental rat model. Bilateral ovariectomy was performed on 40 rats and these were divided into 4 groups based on the treatment they received (at 2 weeks post-ovariectomy, a subcutaneous injection daily for 4 weeks) viz., OVX+E2 (0.1 mg/kg body weight), OVX+TAM (0.05 mg/kg body weight), OVX+vehicle and one group served as OVX control. An additional 10 animals served as the ovary intact control group. At the end of the treatment schedule, five animals/group were used for immunohistochemical staining of SYP and p-CREB using specific antibodies with peroxidase anti-peroxidase technique on paraformaldehyde-fixed cryostat sections. Protein estimation and Western blot analysis coupled with densitometric analysis (using gel-documentation system and image analysis software) were performed on unfixed hippocampus collected from rest of the five animals/group. Serum estradiol levels were estimated with radioimmunoassay prior to sacrifice. The results revealed that ovariectomy reduced SYP and p-CREB expression whereas E2 or TAM administration resulted in their upregulation. Serum estradiol levels of E2 administered animals were comparable with the ovary intact group whereas those of TAM administered group persisted in the range of OVX controls. To conclude, long-term estrogen therapy modulates the synaptic plasticity of hippocampal neurons and presumably, the agonist biocharacter of TAM as observed in the present investigations, may in the long run have a potential in the treatment and prevention of various estrogen-related disorders.

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.

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.

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 (NaAsO2). 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 NaAsO2 alone (1.5 and 2.0 mg/kg body wt.) (IIa, IIb) or NaAsO2 (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). NaAsO2 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 NaAsO2, thereby suggestive of improvement in retention memory. GSH and SOD levels were significantly decreased in animals receiving NaAsO2 alone as against group Ib and administration of ALA following NaAsO2 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 NaAsO2 exposure of rat pups on retention memory and oxidative stress markers.

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.

Effects of arsenic exposure during early postnatal period on Leydig cells of rat testis

The effects of sodium arsenite exposure from postnatal day (PND) 1 to PND 14 on Leydig cells of Wistar rat testes were investigated at PND 15 and 21. Gross morphometric observations of testes did not reveal a significant change in the numerical density and volume of the testes in exposed animals compared to controls either at PND 15 or 21. However, there was significant decrease in testicular weight at PND 21 in treated animals. Measurement of nuclear area of Leydig cells revealed a decrease in nuclear area of these cells in exposed groups at PND 15 and 21. A significant decrease in the total number of Leydig cells was apparent at PND 21 in the treated group. The observations of the present study are indicative of adverse effects on rat testes Leydig cells following exposure to low doses of sodium arsenite during critical window periods. The persistence of these observations at PND 21 is suggestive of irreversible damage to testicular tissue.

Role of Estrogen in Regulation of Morphology and Synaptic Connectivity in Female Rat Subiculum

Abstract Estrogen is reported to exert neuroprotective influences on the cornu ammonis as well as the dentate gyrus, two of the three components of the hippocampal memory system. Subiculum, the third and the output component of this system, plays a vital role in retrieval of previously learnt information (recall tasks). We undertook these studies to understand estrogens role in regulating the synaptic connectivity, neurotransmission and neural degeneration in subiculum. For this, we used brains of aged (16–18 months) post-estropausal female Wistar rats, and compared them with those of adult (4–5 months) ovary-intact, ovariectomized, ova riectomized-vehicle treated and ova riectomized-estrogen treated rats. The vehicle (sesame oil) and estrogen therapy. (17β estradiol, E2) were administered as daily subcutaneous injections (0.1 mg/kg of E2 in 0.1 ml vehicle) for 30 days. Serum estradiol assay was done to determine the levels of circulating serum estradiol levels. Brains were processed for Cresyl Violet, Golgi staining and Immunohistochemical studies [using anti-synaptophysin antibody]. Our results indicate that estrogen deficiency (induced by normal ageing or by ovariectomy) results in subicular neuronal degenerative changes, decreased synaptic connectivity and reduced dendritic arborization, most of which are reasonably reversed with estrogen replenishment. These studies suggest a unique role of estrogen in preventing and even reversing the senile neurodegenerative changes in female subiculum.

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.

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.