Faculty of Basic Medical Sciences

THE RELATIONSHIP OF ENDOCRINE DISRUPTORS WITH PITUIARY GONADAL, THYROID HORMONES AND SOME RECEPTORS IN NIGERIAN WOMEN WITH BREAST CANCER

Tue, 01 Mar 2016 00:00:00 GMT

THE RELATIONSHIP OF ENDOCRINE DISRUPTORS WITH PITUIARY GONADAL, THYROID HORMONES AND SOME RECEPTORS IN NIGERIAN WOMEN WITH BREAST CANCER AJAYI, O. O. Endocrine Disruptors (EDs) such as lead, cadmium, arsenic, polychlorinated biphenyls (PCBs) and bisphenol-A (BPA) are associated with increased risk of Breast Cancer (BCa). Human epithelial receptor, oestrogen, progesterone and their receptors, gonadotrophins and thyroid hormones are also implicated in the aetiology of BCa but remains controversial. Although, several studies have been conducted on BCa, the relationship of EDs with the hormones and receptors has not been fully explored. This study was designed to examine the relationship of these endocrine disruptors with pituitary, gonadal, thyroid hormones and selected receptors in Nigerian women with breast cancer. One hundred and seventy non-pregnant women aged 48.3±1.3 years were purposively selected from the Surgical Oncology Clinic, University College Hospital, Ibadan. The study comprised of 85 Histologically-Confirmed Breast Cancer pre-therapy (HCBCa) which were sub-divided into premenopausal-HCBCa and postmenopausal-HCBCa. These were matched with 85 Apparently Healthy Women without BCa (AHWB) comprising of premenopausal-AHWB and postrnenopausal-AHWB according to age and menstrual phase. Anthropometry and Blood Pressure (BP) were determined with standard methods. Blood was obtained from participants and centrifuged to obtain serum. Serum lead, cadmium and arsenic were determined using atomic absorption spectrophotometry. Bisphenol-A and PCBs were determined using HPLC and gas chromatography, respectively. Oestradiol, progesterone, Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone {TSH), Free Thyroxine (FT4) and Free Triiodothyronine (FT3) levels were determined using ELISA. Expression of Oestrogen Receptor (ER), Progesterone Receptor (PR) and Human Epithelial Receptor 2 (HER2) were determined using immunohistochernistry. Data were analysed using descriptive statistics, Student's t-test and multiple regression at a0.05 Waist Circumference (WC), Hip Circumference (HC), weight, height, Waist-Hip-Ratio (WHR), WaistHeight- Ratio (WHtR) were significantly higher in HCBCa compared with AHWB (89.9± l. l vs 82.6±1.2 cm; 101.8±1.1 vs 98.5±1.0 cm; 69.2±1.4 vs 62.1±1.1 kg; 1.63±0.0 vs 1.58±0.0 m; 0.9±0.0 vs 0.8±0.0; 55.3±0.7 vs 52.4±0.7, respectively). Toe HCBCa had significantly higher levels of lead, cadmium, arsenic, PCBs and bisphenol-A compared with AHWB (5.5±0.2 vs 1.8±0.0 μgldL; 0.04±0.0 vs 0.01±0.0 μgldL; 0.30±0.0 vs 0.04±0.0 μg/dL; 0.8±0.5 vs 0.3±0.0 μg/dL; 0.8±0.7 vs 0.4±0.0 mg/dL, respectively). The FT4 was significantly higher in HCBCa than AHWB (17.8±0.4 vs 14. 7±0.3 pmol/L). There was no difference in LH, FSH and TSH in HCBCa compared with AHWB. Progesterone and oestradiol were higher in postmenopausal-HCBCa compared with postmenopausal-AHWB. (2.09±0.35 vs 1.04±0.10 nmol/L;l56.48±12.42 vs 90.42±3.59 pmol/L, respectively) Regression analysis showed that in HCBCa, oestradiol and WC jointly predicted lead and cadmium (P>0.348, P>S.830, respectively). Diastolic-BP predicted cadmium (P=0.299), WHR and HC predicted arsenic (P>2.732) while FT3, WHtR and height positively predicted BPA (P>0.404). Contrarily, these relationships were absent in AHWB. Fifty-five tumour samples (69.6%) exhibited ER-, PR- and HER2- expressions (triple negative) while 2 (2.5%) demonstrated ER+, PR+ and HER2+ expressions. Fifty-two tumour samples (100%) in premenopausal-HCBCa exhibited ER-IPR- co-expressions while 46 (88.5%) exhibited HER2- expression. However, 18 (66.7%), 18 (66.7%) and 17 (63.0%) postmenopausal-HCBCa exhibited ER-, PR- and HER2- tumour expressions, respectively. Endocrine disruptors could have contributed to adiposity and be associated with negative receptor expression, which may induce cell proliferation and probably cause derangement in thyroid metabolism. A thesis in the Department of Chemical Pathology submitted to the Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy, University of Ibadan, Ibadan, Nigeria.

EFFECTS OF ACUTE CHRONIC ADMINISTRATION OF ETHANOL EXTRACT OF Cola nitida SCHOTT AND ENDLICHER (KOLANUT) AND CAFFEINE ON GLUCOSE METABOLISM IN RATS

Tue, 01 Dec 2015 00:00:00 GMT

EFFECTS OF ACUTE CHRONIC ADMINISTRATION OF ETHANOL EXTRACT OF Cola nitida SCHOTT AND ENDLICHER (KOLANUT) AND CAFFEINE ON GLUCOSE METABOLISM IN RATS AFOLABI, O.A. Cola nitida is known for its high caffeine content. Caffeine administration has been shown to cause glucose intolerance and insulin resistance. However, few studies on the effect of kolanut and decaffeinated kolanut on glucose metabolism. This study was designed to investigate the effect of acute and chronic administration of Ethanol Extract of Kolanut (EEK) and Decaffeinated Ethanol Extract of Kolanut (DEEK) on glucose metabolism in Wistar rats. Kolanut was obtained from Ilobu, Osun State and authenticated at Department of Applied Biology, LAUTECH, Ogbomoso with herbarium number LHO 442. The seeds (500 g) were air-dried, powdered and macerated in cold ethanol. Decaffeination was achieved by soaking equal amount of the seeds in distilled water and adding dichloromethane. Caffeine content of EEK and DEEK was analysed using Gas Chromatography-Mass Spectrometry (GC-MS). In acute studies, 24 Wistar rats (235-300 g) divided into 4 groups were orally administered single dose of caffeine (6 mg/Kg), EEK (6 mg/Kg), DEEK (6 mg/Kg), and 0.3 mL distilled water (control). Sample collection commenced 30 minutes after administration. In chronic studies, the same doses were orally administered daily for eight weeks to another set of 24 rats (240-310g). Samples were collected a day after the final dose was given. In both phases, oral glucose tolerance test was carried out using standard procedure and the Areas Under Curve (AUC) calculated. Blood samples (0.2 mL) and liver biopsies were taken from each animal to determine insulin levels. Liver Glycogen Synthase (LGS), and phosphorylase activities using ELISA. Plasma glucose was determined using glucose oxidase method. Glycogen level was determined spectophotometrically by anthrone method. Data were subjected to descriptive statistics and analysed using ANOVA and Student t-test at α0.05. The GC-MS revealed caffeine content of 17.5% and 3.3% in the EEK and DEEK respectively. In the acute study, AUCᵍˡᵘᶜᵒˢᵉ. AUCᶦⁿˢᵘˡᶦⁿ increased by 19.0% and 11.8% for caffeine and EEK, respectively while DEEK had no effect on AUCᵍˡᵘᶜᵒˢᵉ. The AUCᶦⁿˢᵘˡᶦⁿ increased by 66.6%, 21.5% and 10.6% for caffeine, EEK, and DEEK, respectively. The liver glycogen content significantly decreased from 2.4±0.1 (control) to 1.1±0.1 and 1.3±0.2g/100g for caffeine and EEK, respectively while DEEK showed no effect. Acute administration of caffeine and EEK significantly decreased the activities of LGS and phosphorylase while DEEK had no effect. Chronic caffeine treatment increased AUCᵍˡᵘᶜᵒˢᵉ by 9.2% while EEK reduced AUCᵍˡᵘᶜᵒˢᵉ by 3.2% and DEEK had no effect. Caffeine increased AUCᶦⁿˢᵘˡᶦⁿ from 131.4±5.2 to 157.1±6.9 ng.min/mL, while EEK reduced AUCᶦⁿˢᵘˡᶦⁿ to 114.5±5.6 ng.min/mL, DEEK had no effect on AUCᶦⁿˢᵘˡᶦⁿ. Caffeine and DEEK had no significant effects on LGS and phosphorylase activities. On the contrary, EEK increased liver glycogen content from 1352.0±253.1 to 1987.8±304.9 mg/100g, and LGS activities from 1270.0±154.4 to 2470.0±229.9 ng/mg protein while the phosphorylase activities reduced from 21.3±1.1 to 17.6±0.9 ng/mg protein. Chronic administration of ethanol extract of kolanut enhanced glucose tolerance and insulin sensitivity while its acute administration resulted in glucose intolerance and insulin resistance. Decaffeination had no effect on insulin sensitivity and glucose tolerance. A Thesis in the Department of Physiology submitted to the Faculty of Basic Medical Sciences, College of Medicine in partial fulfillment of the requirements for the award of degree of Doctor of Philosophy of the University of Ibadan, Nigeria.

REVERSAL OF SODIUM ARSENITE-INDUCED DELAY IN GASTRIC ULCER HEALING IN RATS BY KOLAVIRON, VITAMIN E AND ZINC

Thu, 01 Jun 2017 00:00:00 GMT

REVERSAL OF SODIUM ARSENITE-INDUCED DELAY IN GASTRIC ULCER HEALING IN RATS BY KOLAVIRON, VITAMIN E AND ZINC ADEBAYO, G. I. Arsenic acid is a major contaminant of various water sources used for human consumption and industrial activities in most developing countries. It has been reported to cause degenerative inflammation and oxidative damage in many tissues. However, there is a paucity of information on its effect on various gastrointestinal tract conditions of exposed individuals. A mechanistic study in rats on the ameliorative activities of antioxidants (kolaviron, zinc, and vitamin E) on the effects of Sodium Arsenite (SA) exposure during acetic acid - induced gastric ulcer healing was undertaken. Wistar rats (n= 125,150-200g), randomly divided into five groups were treated for two weeks as follows: control (distilled water), SA (5 mg/kg, p.o), SA+ kolaviron (100 mg/kg, p.o), SA+ vitamin E (100mg/kg, p.o), SA + zinc sulphate (20mg/kg p.o). Kolaviron was obtained from Garcinia Kola using soxhlet extraction process. Gastric ulceration was induced by administration of acetic acid (0.06 ml, 40 % v/v). Indices of ulcer healing determined on days 3, 7,14 and 21 post induction were ulcer score and area using planimetry, total gastric acidity by titration, and neutrophil/inflammatory cell infiltration using histomorphometry. Blood cells were quantified using haemoeytometry and activities of Superoxide Dismutase (SOD), Catalase, protein level, Malondialdehyde and Nitric oxide (NO) concentration were determined by spectrophotometry. Stomach sections were immunostained for CD 31 and Factor VIII (angiogenesis), p53 (apoptosis), Epidermal Growth Factor Receptor (EGFR) and Ki-67 (cell proliferation). Stomach tissue was also stained with H&E, and viewed under light microscope. All these variables were evaluated by days 3,7,14 and 21 post-induction. Data were analysed using ANOVA at alpha 0.05. In the ulcerated control animals, the ulcer areas were 0.74 ± 0.01cm², 0.57 ± 0.08cm², 033 p ± 0.12cm² and 0.24 ± 0.04 cm² while SA exposure significantly increased ulcer areas for 81.0%, 100.0%, 65.0%, 33.0% by days 3, 7, 14 and 21, respectively. By day 21 post-ulcer induction, zinc, Kolaviron and vitamin E had reduced ulcer areas relative to SA-treated group by 21.0%, 0.5% and 2.0%, respectively. Sodium arsenite decreased gastric mucosal thickness and parietal cell mass but increased lipid peroxidation, malondialdehyde levels and Neutrophil-Lymphocyte ratio. These affects were reversed by vitamin E and kolaviron. Total gastric acidity reduced while the levels of SOD, catalase, total protein and NO increased as healing progressed in all groups, but at a higher rate in the SA exposed group. The expression of CD31, factor VIII, Ki67 and EGFR proteins were significantly reduced by sodium arsenite, co-treatment with the antioxidants increased the labelling indices towards control values in the order zinc>kolaviron>vitamn E. The p53 expression was increased in SA treated animals but was reduced by the antioxidants in the same order. Gastric tissue necrosis, hyperplasia and haemorrhage observed in the SA-treated animals were reduced by the antioxidants. Sodium arsenite delayed gastric ulcer healing in rats via oxidative stress, inflammation, alteration in proliferative and apoptotic activities and impaired angiogenesis in the stomach. A Thesis in the Department of Physiology submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements for the Degree of Doctor of Philosophy of the University of Ibadan, Nigeria.

PROTECTIVE EFFECT OF Pterocarpus mildbraedii HARMS EXTRACT ON PROPANIL-INDUCED HEPATOTOXICITY AND ALTERATIONS IN APOPTOTIC RELATED PROTEINS IN WISTAR RATS

Mon, 01 May 2017 00:00:00 GMT

PROTECTIVE EFFECT OF Pterocarpus mildbraedii HARMS EXTRACT ON PROPANIL-INDUCED HEPATOTOXICITY AND ALTERATIONS IN APOPTOTIC RELATED PROTEINS IN WISTAR RATS OTUECHERE, C. A. One of the probable causes of liver diseases is exposure to environmental chemicals. Agrochemicals containing propanil are known to induce hepatic toxicity. Pterocarpus mildbraedii leaf is used in traditional medicine to treat various disorders without scientific justification. This study was designed to investigate the protective tole of extract of Pterocarpus mildbraedii against propanii-induced hepatotoxicity in rats. Pterocarpus mildbraedii leaves, purchased from Oyingbo market, Lagos Stata, were authenticated at the University of Lagos Herbarium (LUT/5913). Powdered leaf was extracted in soxhlet, using dichloromethane:method (1:1), to yield crude extracts of Pterocarpus mildbraedii (PME). Sixty-four male Wistar rats (130-160 g), comprising of eight groups (n=8) were used for these experiments. Rats were treated orally with normal saline (control), PME (100mg/kg) PME (200mg/kg), PME (400mg/kg), propanil (200mg/kg), PME (100mg/kg) + propanil (200mg/kg), PME (200mg/kg) + propanil (200mg/kg) and PME (400mg/kg) + propanil (200mg/kg) for seven consecutive days. Hepatic tissues and serum were assayed for markers of hepatic damage, oxidative stress, inflammation, and apoptosis. Aspartase aminotransferase (AST), lactate dehydrogenase (LDH), bilirubin (BIL), superoxide dismutase (SOD), catalase (CAT), redused glutathione (GSH), malondiadehyde (MDA), myeloperoxidase (MPO) and nitric oxide (NO) were assayed by spectrophotometry. Inducible Nitri Oxide Synthase (iNOS), Cyclooxygenase-2 (COX-2), Nuclear factor kappa B (NF-kB), Caspase 3, cASPASE 9, Bax, Bcl-2 expressions were measured using immunohistochemistry. Tumour supressor p53, Bcl-2 antagonist of cell death (Bad), NF-kB, inhibitor of total nuclear factor- KAPPA Bα(1kBα), stress activated protein kinase/C jun NH₂-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38) and signal transducer and activator of transcription 3 (STAT 3) were assessed by ELISA. Histopathology of liver was determined by microscopy and apoptosis by TUNEL assay. Data were analysed using ANOVA at α0.05. The yield of PME was 41.9%. Administration of propanil significantly increased AST (132.10±6.32 U/L), LDH (85.70±6.60 U/L), BIL (1.15±0.16 mg/dL), SOD (0.97±0.05 U/mg protein), MDA (1.03±0.08 ugMDA/mg protein), MPO (4.98±0.12 Umol/min/mg protein) and NO (0.38 umol/mg protein) relative to control (115.90 ± 8.65, 32.84±9.39, 115±0.16, 0.38±0.01, 0.40±0.11, 2.47±0.10 and 0.19 ±0.05, respectively). Pre-treatment of propanil-exposed rats with (200 mg/kg) significantly decreased LDH (83%), BIL (50%), SOD (50.5%), MDA (33.1%), MPO (63.3%) and NO (59.5%). Further, propanil administration increased the levels of GSH (2.98±0.24 ug/mg protein) and CAT (52.7±0.24 umol H₂0₂ consumed/min/g tissue) when compared with the controls (2.04±0.09 and 51.00±0.51). However, intervention with PME restored these serum biochemical indices and antioxidant parameters back to normal values. Expressions of iNOS, COX-2, NF kB, Caspase 3, Caspase 9 and Bax were higher in the propanil group relative to control. Levels of signaling mediators p38 (81.28±7.70), STAT 3 (88.80±4.40) and NF-kB (72.76± 5.30) were lower, while SAPK (125.39±9.30), 1kBα (115.83±5.60) and Bad (112.48±4.70) were higher in propanil-treated rats relative to control value set at 100. TUNEL-positive nuclei and severe periportal fibrosis were observed in tissues following propanil exposure. However, pre-treatment with PME significantly attenuated the observed propanil induced inflammation and apoptosis. Pterocarpus mildbraedii extract protected against propanil-induced hepatotoxicity via mechanisms that involved its antioxidant, anti-inflammatory and anti-apopiotic properties. A Thesis in the Department of Biochemistry submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements for the Degree of Doctor of Philosophy of the University of Ibadan, Nigeria.

CHEMOPROTECTIVE ROLE OF 6-GINGEROL IN ULCERATIVE COLITIS AND COLORECTAL CANCER IN BALB/c MICE

Wed, 01 Nov 2017 00:00:00 GMT

CHEMOPROTECTIVE ROLE OF 6-GINGEROL IN ULCERATIVE COLITIS AND COLORECTAL CANCER IN BALB/c MICE AJAYI, B.O Ulcerative colitis (UC) is an inflammatory disease of the colon that predisposes to colorectal cancer (CRC), the fourth leading cause of cancer death worldwide. Some drugs used in treatment of UC and CRC have adverse effects. Therefore, the search for new drug candidates is desirable. A bioactive component, 6-Gingerol (6-G) from ginger rhizome, has been reported to possess anti-inflammatory activity. However, there is paucity of information regarding the effect of 6-G on UC and CRC. This study was designed to evaluate the chemoprotective role of 6-G in UC and CRC in mice. Male BALB/c mice (n=138, 18.0±1.0 g) were orally treated in three different experimental models. Acute UC model consisted of 6 groups (n=7) of mice orally treated with corn oil (control), 6-G (100 mg/kg), dextran sulfate sodium (DSS) (5% w/v), DSS+6-G (50 mg/kg), DSS+6-G (100 mg/kg) and DSS+6-G (200 mg/kg). The DSS and 6-G were administered for 7 and 14 days, respectively. Chronic UC model consisted of 6 groups (n=6) of mice orally treated with corn oil, 6-G (100 mg/kg), sulfasalazine (100 mg/kg), DSS (2.5% w/v), DSS+6-G (100 mg/kg) and DSS+sulfasalazine (100 mg/kg). The sulfasalazine, DSS and 6-G were administered for 63, 21 and 63 days, respectively. The CRC model consisted of 4 groups (n=15) of mice orally treated with corn oil, 6-G (100 mg/kg), Benzo[a]pyrene (125 mg/kg)+DSS (4% w/v) [BDS] and BDS+6-G (100 mg/kg). The BDS and 6-G were administered for 28 and 119 days, respectively. Mice were sacrificed in the models and samples (blood and colon) collected. Colon myeloperoxidase activity was determined spectrophotometrically. Plasma tumour necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) were assessed using ELISA. Disease activity index (DAI) and tumour incidence (TI) were scored using standard methods. Adenocarcinoma was detected by microscopy. Cyclooxygenase-2, β-catenin, cyclin D1 (CCND1), adenomatous polyposis coli (APC) and p53 expression were assessed using immunohistochemistry, while apoptosis was detected using TUNEL assay. Data were analysed using ANOVA at α0.05. In acute UC model, DSS significantly increased DAI (5.7±0.6), myeloperoxidase (2.8±0.3 unit/mg protein), TNF-α (32.1±2.1 pg/ml), IL-1β (111.4±4.2 pg/ml) relative to control (0.0±0.0, 1.5±0.1 unit/mg protein, 19.2±1.9, 90.2±3.9 pg/ml), respectively. The 6-G (50, 100 and 200 mg/kg) significantly decreased DAI (1.3±0.1, 1.0±0.1, 1.1±0.2), myeloperoxidase (2.1±0.6, 1.7±0.2, 1.9±0.4 unit/mgprotein), TNF-α (18.1±1.1, 19.5±3.0, 24.0±2.3 pg/ml) and IL-1β (93.3±2.8, 91.5±4.8, 90.0±3.7 pg/ml). In chronic UC model, DSS significantly increased myeloperoxidase (5.2±0.3 unit/mg protein), β-catenin (91.9±6.0%) and cyclooxygenase-2 expression (81.0±1.5%) relative to control (1.8±0.2 unit/mg protein, 13.3±3.4, 7.5±0.5%), respectively. Sulfasalazine and 6-G attenuated myeloperoxidase (50.0 and 42.3%), β-catenin (53.3 and 40.0%) and cyclooxygenase-2 (33.8 and 34.0%). In CRC model, BDS induced adenocarcinoma with 78.0, 69.4 and 86.6% decrease in APC, p53 expression and number of apoptotic cells, respectively, while TI, β-catenin and CCND1 expressions were increased (97.0%, 68.4% and 89.2%, respectively). The 6-G increased APC, p53 expression and number of apoptotic cells by 71.8, 69.0 and 75.0%, respectively but reduced TI, β-catenin and CCND1 expression by 41.5, 21.0 and 41.6%, respectively. The 6-Gingerol elicited chemoprotective effect in ulcerative colitis and colorectal cancer via anti-inflammatory and anti-tumourigenic properties. A thesis in the department of Biochemistry submitted to the Faculty of the Basic Medical Sciences, in partial fulfillment of the requirements for the award of the degree of Doctor of philosophy of the University of Ibadan, Nigeria

EVALUATION OF ANTPSYCHOTIC EFFECTS OF METHYL JASMONATE IN MICE

Sun, 01 Jan 2017 00:00:00 GMT

EVALUATION OF ANTPSYCHOTIC EFFECTS OF METHYL JASMONATE IN MICE ANNAFI, O.S Psychosis is a chronic neurological disorder that impairs the quality of life of the patients and remains a major health challenge worldwide. Current drugs used to manage psychosis are expensive and only provide symptomatic relief without altering the underlying pathological derangement. Methyl jasmonate (MJ) is a bioactive compound known to have beneficial effects against a wide range of neurological disorders. However, its usefulness in treatment of psychosis has not been scientifically proven. Thus, this study was undertaken to investigate the effects of MJ on psychosis in animal models. Fifty male Swiss mice (23.5±1.5 g) were assigned to 10 groups to evaluate acute antipsychotic-like effect of MJ on bromocriptine or ketamine-induced stereotypy. Groups 1-5 received 1% ethanol (vehicle, 10 mL/kg, i.p.), MJ (25, 50, 100 mg/kg, i.p.) and haloperidol (1 mg/kg, p.o.) 60 minutes prior to bromocriptine (5 mg/kg, p.o.) treatment, while groups 6-10 received vehicle (10 mL/kg, i.p.), MJ (25,50,100 mg/kg, i.p.) and risperidone (0.5 mg/kg, p.o.) 60 minutes prior to ketamine injection. Thereafter, each mouse was placed independently in an observation chamber (20 cm × 20 cm × 23 cm) and stereotyped behaviours were observed for 2 min at 10, 15, 30, 45 and 60 min after bromocriptine or ketamine injection. Another 36 mice (n = 6) were also allotted into treatment groups. Group 1 received vehicle (10 mL/kg, i.p.) while groups 2-6 were treated with ketamine (20 mg/kg, i.p.) once daily, for 14 days. Then, from 8th to 14th day, group 2 was treated with vehicle (10 mL/kg, i.p.) while Group 3-6 received MJ (25,50.100 mg/kg, i.p.) and risperidone (0.5 mg/kg) 60 minutes after ketamine injection. Hyper-locomotion was then measured as an index of psychotic-like behaviour using the open field chamber while memory was assessed using the Y-maze. Thereafter, whole brain samples were used to assay for malondialdehyde, reduced glutathione (GSH), catalase and Superoxide Dismutase (SOD) using spectrophotometric techniques. Histology of prefrontal cortex, hippocampus and substantial nigra were viewed in ketamine-treated mice and neuronal density was determined. Data were analysed using descriptive statistics and ANOVA at α0.05. Methyl jasmonate (25, 50 and 100 mg/kg) significantly reduced stereotypy score (0.62±0.21, 0.24±0.10 and 0.06±0.04) relative to vehicle (1.84±0.15) and (0.50±0.08, 0.36±0.06, 0.26±0.07) relative to vehicle (1.52±0.10) induced by bromocriptine and ketamine, respectively. Methyl jasmonate significantly ameliorated ketamine-induced hyper-locomotion (74.67±4.70, 75.67±2.88, 78.00±4.16 s) compared to vehicle (185.0±3.63) and memory deficit (80.1±2.8, 71.2±2.9, 57.0±3.4 %) relative to vehicle (53.7±2.0%). Methyl jasmonate reduced malondialdehyde concentration (19.96±1.64, 22.84±1.16, 24.65±1.70 μmol/g tissue) relative to vehicle (33.72±2.28 μmol/g tissue) but increased GSH levels (47.43±2.22, 42.23±2.83, 37.26±1.84 μmol/g tissue) compared to vehicle (21.95± 2.69 μmol/g tissue). Methyl jasmonate also increased catalase level in the brain homogenate (86.63±4.65, 83.36±4.24, 76.06±3.22 units/mg protein) relative to vehicle (59.91±3.94 units/mg) and SOD (27.52±1.63, 24.41±1.49, 19.71±1.59 units/mg protein) compared with vehicle (13.08±1.33 units/mg protein), respectively. Brain histology revealed that MJ has protective property on neuronal cells compared to ketamine-treated mice. Methyl jasmonate demonstrated antipsychotic property via activation of antioxidation pathway in Swiss mice. A dissertation submitted to the department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, University of Ibadan in partial fulfillment of the requirements for the award of the degree of Master of Philosophy (Pharmacology) of the University of Ibadan, Nigeria.

EFFECTS OF VITAMIN C ON CARDIAC AUTONOMIC FUNCTION, OXIDATIVE STRESS AND DYSLIPIDAEMIA IN FRUCTOSE-ALLOXAN DIABETIC RATS

Thu, 01 Jun 2017 00:00:00 GMT

EFFECTS OF VITAMIN C ON CARDIAC AUTONOMIC FUNCTION, OXIDATIVE STRESS AND DYSLIPIDAEMIA IN FRUCTOSE-ALLOXAN DIABETIC RATS FABIYI, T. D. Cardiac Autonomic Neuropathy (CAN) is characterised by cardiac autonomic dysfunction and is the principal cause of mortality in type-2 diabetes. Oxidative stress has been implicated in the aetiology of CAN, thus antioxidants are possible therapeutic agents for CAN. Although, Vitamin C (VC) is a readily available and water soluble antioxidant, there is a dearth of information on its effects on CAN in diabetes. Therefore, this study was designed to investigate effects of vitamin C on cardiac autonomic function in fructose-alloxan diabetic rats. Thirty rats were grouped equally into Control, Fructose (20% w/v), Fructose +150 mg/kg (i.p.) alloxan. Fructose solution was freely administered via gavage for 2-weeks before a single alloxan injection. Thereafter, rats were observed for fourteen weeks. Response to metformin and Insulin tolerance test were done. Fasting Plasma Insulin (FPI) and Fasting Blood Glucose (FBG) were measured using ELISA and spectrophotometry. Homeostasis Model Assessment (HOMA) was used to evaluate Insulin Resistance (IR), Insulin sensitivity and Beta cell function. Further, 72 rats were grouped equally into: control, fructose (20% w/v), diabetic (fructose-alloxan), diabetic + VC, diabetic + VC+metformin and diabetic metformin. The VC (1g/kg/day) and metformin (500 mg/kg/day) were administered orally for 6-weeks. Each group was further divided into two (n=6). Cardiac autonomic function tests were done on one sub-group using electrocardiography based Heart Rate variability (HRV) after anaesthesia with urethane (1g/kg, i.p) and non-invasive blood pressure. Rats in the other sub-group were bled and sacrificed after anaesthesia to harvest organs for histology and histomorphometry. Oxidative stress markers, haematology, apolipoproteins and atherosclerotic plaques were determined using spectrophotometry, hemocytometry, immunoturbidimetry and histochemistry. Data were analysed using ANOVA and Fishers LSD post-hoc test at α0.05. Administration of alloxan to rats pre-treated with fructose elevated FBG (378.3±40.0 vs control 60.8±2.5 mg/dL), which was not reduced significantly by insulin (351.8±30.4 mg/dL) but significantly decreased in response to metformin (259.8±38 mg/dL). Diabetic heart rate and blood pressures were significantly increased compared to control (392.6±8.9 vs 310.0±15.6 bpm, MBP 189.7±4.1 vs 93.6±3.8 mmHg) and VC group (327±22.0 bpm, 97.4±5.4 mmHg). The HRV analysis revealed reduced total power (290.4±0.5 ms²), Low Frequency (LF; 126.5±4.6m²), High Frequency (HF;108.4±7.0 ms² ), RMSSD (65.1±1.0 ms²) in diabetic group compared to control (45,159.3±7839.2, 5434.0±877.0, 19,574, 1±4019.1, 107.5± 11.5ms²) and VC group (44,927.8±7831.5, 5338.7±1191.8, 14,634.1±4865.8, 98.3±13.3 ms²). Normalised-LF and LF/HF ratio were significantly (p<0.01) greater in diabetic than control and VC group. Diabetic rats showed decrease in glutathione (47.8%), VC (35.7%) and apolipoprotein-A1 (25.0%) but these variables were increased by 29.0%, 2.1% and 34.6%, respectively upon VC administration. Diabetic group showed increase in FPI (8.2.± 1.4 vs 4.8±0.3 IU), IR (10.3±3.0 vs 0.58±0.0), SOD (82.5%), LPO (102.2%), NO (111.4%), MPO (40.5%), neutrophils (14.1%), monocytes (14.8%), apolipoprotein B (62.5%) and intima-media thickness (107.4±9 vs 78.87±2.3um), but VC group showed no significant difference from control. Atherosclerotic-amyloids were present in diabetic heart and aorta but regressed following VC administration. Vitamin C ameliorated cardiac autonomic dysfunction in diabetic rats via mechanisms related to reduction of oxidative stress, leucocyte activation, vascular inflammation and dyslipidaemia. A Thesis in the Department of Physiology submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements for the Degree of Doctor of Philosophy of the University of Ibadan, Ibadan, Nigeria.

NEUROBEHAVIOURAL AND NEUROPROTECTIVE EFFECTS OF KOLAVIRON IN LABORATORY RODENTS

Thu, 01 Jun 2017 00:00:00 GMT

NEUROBEHAVIOURAL AND NEUROPROTECTIVE EFFECTS OF KOLAVIRON IN LABORATORY RODENTS IBIRONKE, G. F. The high cost of orthodox medicines and their attendant side effects in some cases make the search for an effective and affordable alternative imperative. Kalaviron, a complex biflavanoid from Garcinia kola seeds, possesses hepatoprotective and antidiabetic properties. However, there is paucity of information on its effects on the nervous system. The antinociceptive, neurobehavioural and neuroprotective effects produced by kolaviron and its possible mechanism of action were evaluated in laboratory rodents. Garcinia kola seeds were air-dried, powdered and extracted in Soxhlet to obtain methanol fraction. The fraction was partitioned in chloroform:distilled water (3:1). The chloroform portion was concentrated to give a golden yellow substance called Kolaviron (KV). Male Wistar rats (180-220 g) and Swiss mice (18-25 g) grouped into five groups of six animals each were treated with normal saline (10 ml/kg, p.o.), kolaviron (50, 100 and 200 mg/kg, p.o.) and indomethacin (10 mg/kg, i.p). Psychoemotional stress was induced using the whisker removal technique. Antinociceptive activity was studied using the hot plate, tail flick and acetic acid-induced writhing tests. Neurobehavioural properties were observed using the openfield, elevated plus-maze, light/dark box and hole board methods. Brain tissue levels of reduced glutathione (GSH). catalase and malodialdehyde were estimated using spectrophotometric method. Histological analysis of the frontal cortex and cerebellum stained with haematoxylin and eosin were observed using light microscope. Data were analysed using ANOVA and Newman-Keul’s post-hoc test at α0.05. Kolaviron dose dependently prolonged tail flick latencies by 23.5, 45.0 and 53.5% relative to control and hot plate latencies by 41.5, 125.5 and 217.9% relative to control. Kolaviron inhibited acetic acid- induced writhings by 6.6, 14.5 and 31.8% relative to control. Reductions in frequencies of locomotion (69.0±6.0, 64.5±3.3 and 52.3±5.8 vs 76.2 ± 3.2), rearing (22.2±1.5, 15.8±3.8 and 17.5±3.9 vs 30.7±3.7) and grooming (5.7±1.0, 5.7±0.8 and 3.3±0.4 vs 13.3±0.8) were observed in the openfield, so also were reductions in head dip frequencies in the hole board (19.1±1.3, 19.6±1.5 and 15.5±1.3 vs 37.1±1.7). In the elevated plus-maze, kolaviron reduced the time spent in the open-arm (55.1±4.5, 40 ± 3.5 and 25.5 ± 4.1) compared with control (65.1±3.3s). Significant reductions were observed in the time spent in, the light arena of the light/dark box (80.8±8.5, 70.5±5.2 and 51.3 ±25.5) compared with the control (180.5±25.5s). Malondialdehyde (U/mg protein) levels in the cerebellum (6.5±0.4) and the frontal cortex (30.1± 4.7) of stressed animals were significantly higher compared with their control values (3.8±0.2 and 14.6±2.5, respectively). Catalase and GSH levels in the cerebellum and frontal cortex of the treated animals were significantly lower than their controls. Kolaviron pre-treatment significantly attenuated all these changes in Malondialdehyde, Catalase and GSH levels. Some of the stress-induced histological abnormalities like vacuolation, vascular congestion and appearance of abnormal Purkinje cells were partially reduced by kolaviron. Kolaviron exhibited positive antinociceptive and neurobehavioral effects mediated through sedation and neuroprotective effects via mechanisms that involve- inhibition of oxidative stress. A Thesis in the Department of Physiology submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements for the Degree of Doctor of Medicine of the University of Ibadan, Ibadan, Nigeria.

EFFECTS OF MATERNAL SLEEP DEPRIVATION ON REPRODUCTIVE FUNCTIONS OF MALE OFFSPRINGS IN WISTRAR RATS

Sat, 01 Jul 2017 00:00:00 GMT

EFFECTS OF MATERNAL SLEEP DEPRIVATION ON REPRODUCTIVE FUNCTIONS OF MALE OFFSPRINGS IN WISTRAR RATS AKINDELE, O.O Maternal Sleep Deprivation (MSD) has been reported to alter sexual performance in offsprings of rats. There is paucity of information on the critical period during gestation at which MSD affects reproductive functions. This study was designed to investigate the effects of MSD at different gestation periods on reproductive functions of male offsprings in Wistar rats. Sixty pregnant rats were assigned into six (Control = C; Sleep Deprived = SD) groups (n=10) at different Gestation Days (GD) as follows: GD1-7C, GD1-7SD, GD8-14C, GD8-14SD, GD15-21C and GD15-21SD. The MSD was induced using the modified multiple platform method. Caesarean section was performed on five animals from each group on GD 8, 15 and 21 for placental morphometric and biochemical analyses. The remaining five dams from each group littered naturally and only their male offsprings were studied. The offsprings were weighed on post-natal day 1 on a digital electronic balance. Reproductive functions of male offsprings were determined by assessing; testes descent, fertility index, sperm count and motility, testosterone, corticosterone, melatonin and reproductive organ histology. The testes descent day was determined by daily palpation of the scrotal sac for the presence of testes. Fertility index was determined on post-natal week 17 after mating, as percentage of female rats (of proven fertility) impregnated by the male offsprings according to the standard technique. Epididymal fluid was analysed by microscopy to determine the sperm motility and sperm count. Serum testosterone, corticosterone and melatonin were quantitated by ELISA. Histological assessment of the testes and epididymes was done using paraffin section processing and microscopy for tissue microscopic examination. Placental malondialdehyde, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were assayed by spectrophotometry to determine the redox status. Immunohistochemistry of placental Bcl2 and p53 was done to determine apoptotic status. Data were subjected to descriptive statistics and analysed using Student’s t-test at α0.05. Birth weight reduced in GD15-21SD (5.3±0.1 vs 5.7±0.2 g). Testes descent occurred late in GD15-21SD (25.8±0.4 vs 23.0±0.8 days). Fertility index of GD15-21SD was 0 %. Sperm motility and count decreased in GD15-21SD (72.0±4.9 vs 89.0±2.9 %; 64.4±14.9 vs 114.5±3.0 million/mL). Offspring’s testosterone reduced in GD15-21SD (2.9±1.1 vs 7.0±1.3 ng/mL). Offspring’s corticosterone increased in GD1-7SD, GD8-14SD and GD15-21SD (106.0±3.0, 115.0±8.2, and 131.6 ± 6.6 nmol/L) compared with their controls (57.2±19.5, 65.8±5.9, 96.6±9.3 nmol/L), respectively. Melatonin increased in GD1-7SD (482.1±33.5 vs 355.3±19.21 iii nmol/L) and decreased in GD8-14SD (237.3±15.1. vs 353.6±20.5 nmol/L). The testicular and epididymal sections of GD15-21SD showed aberrant seminiferous tubules and atrophic ducts respectively. Dam’s testosterone reduced in GD15-21SD (216.0±5.8 vs 370.0±38.2 pg/mL). Placental malondialdehyde increased in GD1-7SD (0.1±0.0 vs 0.0±0.0 nmol/mg) and GD15-21SD (0.2 ± 0.0 vs 0.1 ± 0.0 nmol/mg). Placental SOD decreased and GPx increased in GD15-21SD (62.9±5.6 vs 103.0±11.7; 53.0±5.7 vs 35.3±4.7 U/mg), respectively. Bcl2 and p53 decreased in GD15-21SD decidua (7.8±0.7 vs 16.4±0.2; 0.4±0.1 vs 9.4±0.3 %), respectively. Reproductive functions were adversely affected during the fifteenth to twenty-first days of gestation. A thesis in the department of physiology submitted to the faculty of Basic Medical Sciences i n partial fulfilment of the degree of Doctor of Philosophy of the University of Ibadan

REVERSAL OF SODIUM ARSENITE-INDUCED DELAY IN GASTRIC ULCER HEALING IN RATS BY KOLAVIRON, VITAMIN E AND ZINC

Thu, 01 Jun 2017 00:00:00 GMT

REVERSAL OF SODIUM ARSENITE-INDUCED DELAY IN GASTRIC ULCER HEALING IN RATS BY KOLAVIRON, VITAMIN E AND ZINC ADEBAYO, G.I Arsenic acid is a major contaminant of various water sources used for human consumption and industrial activities in most developing countries. It has been reported to cause degenerative inflammation and oxidative damage in many tissues. However, there is a paucity of information on its effect on various gastrointestinal tract conditions of exposed individuals. A mechanistic study in rats on the ameliorative activities of antioxidants (kolaviron, zinc, and vitamin E) on the effects of Sodium Arsenite (SA) exposure during acetic acid - induced gastric ulcer healing was undertaken. Wistar rats (n=125,150-200g), randomly divided into five groups were treated for two weeks as follows: control (distilled water), SA (5mg/kg, p.o), SA+ kolaviron (100 mg/kg, p.o), SA+ vitamin E (100mg/kg, p.o), SA + zinc sulphate (20mg/kg p.o). Kolaviron was obtained from Garcinia Kola using soxhlet extraction process. Gastric ulceration was induced by administration of acetic acid (0.06ml, 40% v/v). Indices of ulcer healing determined on days 3, 7, 14 and 21 post induction were ulcer score and area using planimetry, total gastric acidity by titration, and neutrophil/inflammatory cell infiltration using histomorphometry. Blood cells were quantified using haemocytometry and activities of Superoxide Dismutase (SOD), Catalase, protein level, Malondialdehyde and Nitric oxide (NO) concentration were determined by spectrophotometry. Stomach sections were immunostained for CD31 and Factor VIII (angiogenesis), p53 (apoptosis), Epidermal Growth Factor Receptor (EGFR) and Ki-67 (cell proliferation). Stomach tissue was also stained with H&E, and viewed under light microscope. All these variables were evaluated by days 3,7,14 and 21 post-induction. Data were analysed using ANOVA at α0.05. In the ulcerated control animals, the ulcer areas were 0.74 ± 0.01cm², 0.57 ± 0.08cm², 0.53 ± 0.12cm² and 0.24 ± 0.04 cm² while SA exposure significantly increased ulcer areas for 81.0%, 100.0%, 65.0%, 33.0% by days 3, 7, 14 and 21, respectively. By day 21 post-ulcer induction, zinc, Kolaviron and vitamin E had reduced ulcer areas relative to SA-treated group by 21.0%, 0.5% and 2.0%, respectively. Sodium arsenite decreased gastric mucosal thickness and parietal cell mass but increased lipid peroxidation, malondialdehyde levels and Neutrophil-Lymphocyte ratio. These effects were reversed by vitamin E and kolaviron. Total gastric acidity reduced while the levels of SOD, catalase, total protein and NO increased as healing progressed in all groups, but at a higher rate in the SA exposed group. The expression of CD31, factor VIII, Ki67 and EGFR proteins were significantly reduced by sodium arsenite, co-treatment with the antioxidants increased the labelling indices towards control values in the order zinc>kolaviron>vitamin E. The p53 expression was increased in SA treated animals but was reduced by the antioxidants in the same order. Gastric tissue necrosis, hyperplasia and haemorrhage observed in the SA-treated animals were reduced by the antioxidants. Sodium arsenite delayed gastric ulcer healing in rats via oxidative stress, inflammation, alteration in proliferative and apoptotic activities and impaired angiogenesis in the stomach. A thesis submitted in partial fulfillment of the requirements for the award of Doctor of Philosophy in the department of Physiology, College of Medicine, University of Ibadan, Nigeria.