Abraham Lukpata

MATERIALS AND METHOD MATERIALS EXPERIMENTAL CHEMICALS 97% Pure Hesperidin It will be bought from the TONIIQ company (USA) on Amazon. It is lab-tested, pure, and potent; and has a certificate of analysis & third-party independent lab results. It contains 90 capsules (500mg of pure hesperidin/capsule, hence, a total of 45,000mg) 0.9% Normal Saline Fluoxetine GENERAL MATERIALS 4 bag of feed, 6 bag of cage bedding, 30 adult male Wistar rats MATERIALS FOR STRESSORS 2 buckets, Rope, 30 cages (housing and isolation) MATERIALS FOR NEUROBEHAVIOURAL TEST Wood (designs will be sent to a carpenter for construction) Bottles (for sucrose preference test) Apparatus for tail suspension test. MATERIALS FOR SAMPLE COLLECTION Gloves, mask, towel, dissection kit, cotton, 23G/27G needle and blood sample collection tube (Plain sample tube). RESEARCH METHOD Ethical Clearance Experimental protocols and procedures that will be used in this study will be approved by Health Research Ethics Committee, University of Cross River State. Animal protocol and handling will be carried out in strict compliance to the National Institutes of Health guide for the use and care of Laboratory animals (NIH Publications No.8023, revised 1978). Animal procurement A total of 30, 160-180g adult Wistar Rats of body weight will be obtained from the Faculty of Basic Medical Sciences Animal House, University of Cross River State for the study. The animals will be housed and caged under normal temperatures with night and day cycle. The animals will be allowed to acclimatize for a period of one week. The animal will be handled according to the guidelines of National Institute of Health, 2011. EXPERIMENTAL DESIGN EXPERIMENTAL DESIGN STUDY MODEL: This study is an animal study using rat models. ANIMAL MODEL SELECTION: Male Wister rats (n=30). Age: 8-10 weeks. Weight: Approximately 180-220 grams. Randomly divided into 5 groups. EXPERIMENTAL GROUP ALLOCATION: Control Group (n=5): Rats without PTSD induction PTSD Group (n=5): Rats exposed to PTSD induction without any treatment. Hesperidin Treatment Group (n=25): Rats exposed to PTSD induction and treated with hesperidin. Dose-dependent treatment: Hesperidin – 10mg/kg Hesperidin – 30mg/kg Thirty (30) adult male Wistar rats with an average of 170g will be randomized into 6 groups, each consisting of 5 rats (n=5). The groups will be treated as follows (shown in the table below) PTSD INDUCTION: Method: Single prolonged stress (SPS) model. The single prolonged stress paradigm is one of the best models for eliciting PTSD-related symptoms in Wistar rats (Nahvi et al., 2019). Procedure: Rats in the PTSD and Hesperidin Treatment groups will undergo the following steps: i. Restraint stress (2 hours). ii. Forced swim (20 minutes) iii. Ether exposure (10 minutes). iv. Rest for 7 days. HESPERIDIN TREATMENT/ADMINISTRATION: Pure Hesperidin extract will be sourced and purchased. Hesperidin will be administered orally. Dosage: 10, 30 mg/kg body weight. Treatment duration: 14 consecutive days. Preparation of Hesperidin solution: Hesperidin will be diluted with 0.9% normal saline and administered intraperitoneally. Administration method: Prepared hesperidin solution will be administered via oral gavage. Table1. Experimental design and protocol EXPERIMENTAL GROUP NO OF RATS TREATMENT DOSAGE /kg/bodyweight DURATION GROUP 1 (NORMAL CONTROL) 5 Normal saline/distilled water 1ml of 0.9% normal saline 42days GROUP 2 (NEGATIVE CONTROL) 5 SPS only NIL 14 days GROUP 3 5 SPS + Fluoxetine) 10mg/ (I.p injection once daily) 14days SPS+ 14days treatment GROUP 4 5 SPS + Hesperidin (Low dose) 50mg/ (I.p injection once daily) 14days SPS + 14days treatment GROUP 5 5 SPS + Hesperidin (Medium dose) 100mg/ (I.p injection once daily) 14days SPS + 14days treatment GROUP 6 5 SPS + Hesperidin (High dose) 200mg/ (I.p injection once daily) 14days SPS + 14days treatment DRUG ADMINISTRATION/DOSAGE EXPERIMENTAL PROTOCOL Acclimatization and 1st Neuro Behavioral Study The experimental rats will be exposed to 1 week (7 days) of acclimatization in which they will be fed and reared under normal conditions. The first Neurobehavioral test and weight monitoring will be conducted on Day 7 after the one week of acclimatization. SPS Induction and 2nd Neurobehavioral Study: The treatment groups will be exposed to 3 weeks (Day 8-Day 28) of Single prolonged stress. The rats will be exposed to SPS and housed separately in different cages for social isolation and five rats per cage will be housed in the control group. During the modeling period, all groups, except the control group, will be subjected to social isolation and SPS for 14 days. The Rats will be subjected to 3 different stressors which are the regimen for SPS. 1. Restraint or immobilization for 2 hrs. 2. Forced swimming for 15 minutes. 3. Exposure to diethyl ether One of these stressors will be performed every day for a period of 3 weeks. After the 3 weeks of SPS induction, the 2nd weight monitoring and neurobehavioral test will be conducted to estimate the state of the rats. Treatment and 3rd Neuro behavioral Study: The SPS-induced groups will now be treated with hesperidin for 2 weeks (Day 29-Day 42). The standard control group will be treated with 2.5mg/kg of hesperidin every morning for the next 2 weeks. And so will each of the other treatment groups using different doses of hesperidin. The different treatment groups and the quantity of hesperidin used to treat them is shown in Table 1. After the 2 weeks of treatment, the 3rd weight monitoring and neurobehavioral test will be conducted to estimate the state of the rats. SPS REGIMEN NEURO BEHAVIORAL TESTS Anxiety Behavioral Tests Elevated Plus Maze EPM: The test will be used to examine the differences in the anxiety levels of rats across treatment groups. The rats will introduce into the elevated plus apparatus that stood 45cm tall with two open arms and two closed arms for an exploration time of 5 minutes. The open arm duration will be estimated as total time the rats spent in the open arm of the elevated plus maze for the duration of the study. The close arm duration will be estimated as total time the rats spent in the close arm of the elevated plus maze for the duration of the study. Depression Behavioral Tests Sucrose Preference Test (SPT): The SPT will be used to measure anhedonia, which is a characteristic sign of depression. We will calculate the preference for a 1% sucrose solution by using a two-bottle-choice test. The rats will be kept in cages with two bottles of water for 5 days at the adaptation stage. Then, during the test phase, rats will be housed individually in cages with two bottles of 1% sucrose and water for 12 hrs (Overstreet, 2011) The location of bottles will be switch after 6 hrs to avoid any side preference. The consumption of sucrose will be measure by the following equation: Sucrose preference % = (the milliliters consumed from the sucrose bottle/the milliliters consumed from the water bottle + the milliliters consumed from the sucrose bottle) × 100 (Sanchi-Segura, 2005) Locomotor Behavioral Tests Open Field Test (OFT): The OFT will be conducted to evaluate the locomotor activity (velocity, total distance move (TDM)) and immobility. During the test, a mouse will be placed separately in an empty arena (square test box, 45 × 45 cm) and left to move freely for 3 min. A smart video recording system will be used to monitor the velocity, TDM, and immobility. Table 2 shows the model of the neuro-behavioral tests. Neurobehavioral Test Outcome Measurement Sucrose Preference Test (Depression) The mean value of the volume of sweetened and unsweetened water.  Elevated Plus Maze Test (Anxiety) Mean value of time spent in open and closed arms. Open Field Test (Locomotors Activity) The mean value of the number of lines crossed ANIMAL SACRIFICE, TISSUE SAMPLE COLLECTION AND PROCESSING SACRIFICE: At the end of the experiments, the rats will be fasted for 24 hours to prepare them for sacrifice on day 43. Rats for histological analysis will be euthanized using 20mg/kg of ketamine (intraperitoneal). Transcardial perfusion will be used by exposing the left ventricle and injecting 50ml 0.1M PBS (pH 7.4) followed by 100ml 10% neutral buffered formalin while the rat will be suspended in an inverted position. DATA COLLECTION: Brain tissue samples will be collected for neurotransmitter analysis (hippocampus, hypothalamus, prefrontal cortex). Blood samples will be collected for inflammatory marker analysis. Neurotransmitter and inflammatory marker levels will be recorded in mg/mL or pg/mL. All samples will be collected and stored following proper protocols to maintain integrity. TISSUE SAMPLE COLLECTION: Excised brains will be weighed and then rinsed in 0.9% normal saline 3 times for 5 minutes each and then post-fixed in 10% neutral buffered formalin for 24 hours before it will be stored for further processing. Blood will be obtained through the retro-orbital vein using a capillary tube. Coronal section of the hippocampus, and hypothalamus will be obtained from the rats. Subsequently, sections will be processed routinely to obtain paraffin wax embedded blocks for histology and cresyl fast violet staining to demonstrate Nissl substances in neurons. BIOCHEMICAL ANALYSIS The blood serum collected will be subjected to biochemical analysis of enzymes (TPH, TH, DBH), and genetic markers. NEUROTRANSMITTER ASSESSMENT: Neurotransmitters: Serotonin (5-HT), norepinephrine (NE), dopamine (DA). Sampling: Blood and brain tissue samples will be collected at the end of the treatment period. Assay: ELISA (Enzyme-Linked Immunosorbent Assay) or immunohistochemistry will be used to measure neurotransmitter levels. NEUROMODULATOR ASSESSMENT: Neuromodulators: Tryptophan hydroxylase (TPH), Tyrosine hydroxylase (TH), Dopamine-β-hydroxylase (DBH). Sampling: Blood and brain tissue samples will be collected at the end of the treatment period. Assay: ELISA or immunohistochemistry will be used to measure neuromodulator levels. HISTOMORPHOLOGICAL ANALYSIS: Tissues from specific regions of the brain (Hypothalamus, Hippocampus) will be processed histologically. Immunohistochemical staining will be conducted to assess changes in neural morphology, neurogenesis, and glial activation in relevant brain regions. This will provide insights into the potential neuroprotective effects of hesperidin. Hematoxylin and Eosin Stain (H&E) and Cresyl Fast violet stain (CFV) will be used. LIGHT MICROSCOPY For light microscopic studies, the hypothalamus, pituitary, and hippocampal sections on glass slides will be captured using Olympus binocular research microscope (Olympus, New Jersey, USA) which will be connected to a 5.0MP Amscope Camera (Amscope Inc, USA). STATISTICAL ANALYSIS The results of the data to be obtained in the experiment will be analyzed using SPSS version 17 software. The statistical significance of differences between groups will be assessed using a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple comparisons of means if the ANOVA test is significant. Pearson correlations test will be used to correlate the neuromodulators and neurotransmitters. Significance will be set at p<0.05*. The outcomes would be represented in bar charts with error bars to show the mean and standard error of mean respectively. The data will be summarized using mean plot, graph etc.