Raj D. Mehra

Long-term administration of estrogen or tamoxifen to ovariectomized rats affords neuroprotection to hippocampal neurons by modulating the expression of Bcl-2 and Bax

Recently we reported that chronic treatment with 17beta-estradiol (E2) or tamoxifen (TAM) regulates the ovariectomy-induced downregulation of the key molecules linked to hippocampal synaptic plasticity and signal transduction pathway. We now report modulation of the antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in the hippocampus of both the ovariectomized (OVX) rats as well as those given E2 or TAM subcutaneously as a daily dose for four weeks post-ovariectomy. Forty bilaterally OVX animals were divided into four groups of 10 each, namely i) OVX+E2 (0.1 mg/kg body weight), ii) OVX+TAM (0.05 mg/kg body weight), iii) OVX+vehicle (0.1 ml of sesame oil) and iv) OVX controls. An additional group of 10 animals constituted the ovary intact controls. Following culmination of treatment regimen, brain tissues of five animals from each group were processed for immunohistochemical staining of Bcl-2 and Bax on perfusion fixed cryo-sections. The remaining animals in each group were utilized for protein and Western blot analyses using unfixed hippocampal tissue. The results revealed that chronic administration of both E2 and TAM prevented the ovariectomy-induced downregulation of Bcl-2 and upregulation of Bax expression while restoring the Bcl-2/Bax ratio as observed in the ovary intact rats. Furthermore, TUNEL assay demonstrated a decline in the percentage of TUNEL positive cells in E2 or TAM treated groups. Confocal microscope studies of ERalpha and the apoptotic markers revealed that these two proteins co-reside in the same ERalpha positive hippocampal neurons. Thus, long-term E2 or TAM therapy modulates the apoptotic proteins and affords neuroprotection to the hippocampal neurons. Furthermore the estrogen-like effects of TAM point towards its potential as a beneficial therapeutic agent for neurodegenerative disorders, particularly in the postmenopausal women.

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.

Neuroprotective effects of estrogen treatment on ischemia-induced behavioural deficits in ovariectomized gerbils at different ages

Although much is known about the protective effect of acute estrogen therapy in cerebral ischemia, relatively little is known about its effect on functional outcome at different ages. The impact of age is, however, important on the efficacy of steroids in the central nervous system. We investigated whether a single dose of estradiol pre-treatment would be neuroprotective in young (4 months), middle-aged (9 months) and old (18 months) female gerbils following 10min global brain ischemia. Apoptotic and necrotic cells were labelled and quantified in the affected hippocampus; exploratory activity, attention and memory functions were tested using open field, spontaneous alternation, novel object recognition and hole-board test. Age effect and treatment effect were analysed. High single dose (4mg/kg b.w.) of estradiol pre-treatment exposed a marked neuroprotective effect against hippocampal cell loss in all age groups. In behavioural tests, however, age-related differences could be observed. In middle-aged and old animals the worsening in memory function following ischemia was more prominent compared to that in the young ones. In the Y-maze and the novel object recognition tests the middle-aged, in the hole-board test (investigating working memory and total time) the old gerbils had the worst functional outcome. Only reference memory in hole-board test did not change by age. Estrogen improved memory performances in all the tests at every age. We can conclude that age of experimental animals is a factor worsening the outcome following brain ischemia. A single-dose estrogen therapy prevents the lesion-induced behavioural dysfunctions and the hippocampal cell loss.

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.

L-type calcium channel blockers, morphine and pain: Newer insights

Earlier, we had reported that co-administration of opioids and L-type calcium channel blockers (L-CCBs) like diltiazem could prove useful in the treatment of cancer pain. Much of this report was based upon earlier published work involving animal models of pain exposed to brief periods of noxious radiant heat without any tissue injury. However, pain in clinical situations usually result from tissue injury. Thus, the aim of the current investigation was to study the analgesic effect of this combination of drugs in the rat formalin test which is associated with actual tissue injury. Wistar rats (n=60) received either L-CCB (nifedipine/nimodipine/verapamil/diltiazem i.p.) or morphine (s.c.) or both drugs. The formalin test was done 30 min after morphine or placebo injection. The naloxone reversal test was also done. Administration of L-CCBs alone, particularly diltiazem, increased pain in the formalin test. In contrast, co-administration of these L-CCBs with morphine led to decreased pain response, though statistically significant decrease was noted only with nimodipine + morphine. Naloxone reversed this analgesic effect, indicating that it was primarily an opioid-mediated effect. The results show that administration of L-CCBs alone may prove counterproductive in the therapeutic management of pain (anti-analgesic effect). However, co-administration of both drugs (morphine and nimodipine) in quick succession could lead to adequate pain relief.

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.

Ultrastructure of marginal zone during prenatal development of human spinal cord

SummaryElectron microscopic studies were conducted in the marginal zone (lamina I) in human fetuses ranging from 8–25 weeks of gestational age. At 8 weeks the neurons have indented nuclei and sparse organelles in the cytoplasm. The neuropil shows contacts between the axons and dendritic profiles. Some of them are well defined synapses with post synaptic thickening and agranular spherical vesicles in the presynaptic terminal. At 18 weeks compactly packed organelles with long cisternae of rough endoplasmic reticulum could be visualized in the neuronal cytoplasm. At 25 weeks the neurons have heterochromatin patches in the nuclei. Axosomatic, dendrodendritic, axoaxonic, symmetrical, asymmetrical and multisynaptic contacts with agranular and dense core vesicles are seen at different sequential age periods.

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.

Brain Plasticity Following Ischemia: Effect of Estrogen and Other Cerebroprotective Drugs

Edina A. Wappler1,2, Klara Felszeghy3, Mukesh Varshney4, Raj D. Mehra4, Csaba Nyakas3 and Zoltan Nagy5 1Department of Pharmacology, Tulane University, New Orleans 2Department of Anaesthesiology and Intensive Therapy, Semmelweis University, Budapest 3Neuropsychopharmacology Research Unit of Semmelweis University and Hungarian Academy of Sciences, Budapest 4Department of Anatomy, All India Institute of Medical Sciences, New Delhi 5Cardiovascular Center, Department Section of Vascular Neurology, Semmelweis University, Budapest 1USA 4India 2,3,5Hungary