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ThesisItem Open Access Isolation, propagation and characterization of bone marrow derived mesenchymal stem cells and their differentiation into cardiac myocytes in canines(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Khalid Mubarik; Kandpal, ManjulMesenchymal stem cells are valuable source of multipotent cells which have the ability to differentiate into various types of tissue cells and contribute to the regeneration of a variety of mesenchymal tissues including bone, cartilage, muscle, and adipose. Bone marrow-derived mesenchymal stem cell (MSC)-based therapy is an emerging approach for the treatment of both acute and chronic heart failure. The aim the current study was to focus on canine mesenchymal stem cells and to analyse their differentiation capacity into cardiomyocyte phenotype in vitro. An adult mongrel dog of 5 years of age weighing 25 kgs was used in this study. Bone marrow was aspirated from iliac crest after anaesthesia using xylazine and ketamine. Mesenchymal stem cells were harvested using density gradient centrifugation from the mononuclear cells of bone marrow. The cells were seeded in tissue culture flasks and allowed to grow in monolayer till the attainment of 80-90% of confluency. At this confluency passaging was done using trypsin-EDTA. The cells were subcultured upto four passages. These cells were characterized at their baseline for morphology, culture behavior and also by scanning electron microscopy. At passage three, the cells were exposed to medium containing 5-Azacytidine (10µl/ml) to induce differentiation into cardiac myocyte. The media was changed into media without 5-Azacytidine after 24 hours. The cells were cultured for 21 days in an incubator maintained at 5% CO2 and 37oC. The differentiation of these cells was then analysed using cellular morphology, gene expression and scanning electron microscopy. PCR was carried out for expression of genes Nkx 2.5, Cardiac troponin-T (cTnT), β-Cardiac myosin in differentiated cells. SEM was also done for analyzing the morphology of differentiated cells. Canine MSCs demonstrated morphological changes towards cardiomyocyte like phenotype during differentiation. Cells assumed a stick-like morphology and were connecting with adjoining cells forming myotubelike structures. The cells demonstrated increased expression of cardiac specific markers. On the basis of results obtained in this study it was concluded that Mesenchymal stem cells possess potential for differentiating into cardiac myocyte like cells in vitro. The exploitation of these in vitro harvested cardiac myocyte like cells may prove an effective therapy for cardiac repair under various pathological conditions.ThesisItem Open Access Isolation, expansion and characterization of bone marrow derived mesenchymal stem cells and their differentiation into skeletal myocytes in canines(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Sandhu, Ramanpreet Singh; Jadon, Narendra SinghThe adult stem cell therapy is a blooming area of clinical research and relevance. The therapeutic benefits of mesenchymal stem cells in veterinary regenerative medicine are numerous as they are in human medicine. The characterization of canine derived MSCs is poorly mentioned in literature. The mesenchymal stem cells in canines can be potentially used for cell based therapy to regenerate damaged or lost muscle cells. The motive behind this study was fixed on the differentiation potential of canine mesenchymal stem cells into skeletal myocytes in vitro. An adult male mongrel dog of 3 years of age and 24kg weight was used for this study. The bone marrow aspirate was aseptically collected from ileac crest of the dog after administration of general anaesthesia by 2.5% thiopentone sodium till effect and atropine sulphate and diazepam as pre-anaesthetics. The bone marrow aspirate was processed in laboratory within 4 hours of its collection. Mesenchymal stem cells were isolated by density gradient centrifugation technique and cells were seeded in T-25 tissue culture flasks. After attainment of 80-90% confluence, first passage was performed by using Trypin-EDTA to expand the cell population. Cells were passaged till 4 passages and then grown in skeletal myogenic differentiation media for 21 days in CO2 incubator at 37 ° C and 5% CO2. The morphology of cells was observed at regular intervals and the cells were characterized by use of Hoechst 33342 stain after 21 days with positive result which coloured the nuclei blue. The RT-PCR for the genes Myogenin and Myo-D revealed bands on days 7 and 14 confirming the differentiation process as early differentiation factors. The striations were observed when the sample on gelatin scaffold was subjected to scanning electron microscopy and close association of parallel arranged cells with striations when the pellet was subjected to electron microscopy. On the basis of this study it was concluded that MSCs possess potential to trans differentiate to skeletal myocytes when cultured in skeletal myogenic media in vitro and the identification techniques are successful. The meticulous exploitation of MSCs in tissue engineering and regenerative therapy in veterinary medicine is the need of the hour.ThesisItem Open Access Clinical study on canine cystic endometrial hyperplasia complex(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Pushpa; Das, Arup KumarThe objectives of the study were to evaluate the effect of CEH-P on clinical, haematobiochemical and hormonal profile of dogs and changes after OH. Cystic endometrial hyperplasia is a pathological complex which incorporates conditions like cystic ovary, endometrial hyperplasia and accumulation of mucoid material in the lumen of the uterus which might be invaded by opportunistic bacteria and may gradually proceed to pyometra. This study was conducted on 18 female dogs and divided in to two groups in group A 12 female dogs with symptoms of endometrial hyperplasia complex were diagnosed and in group B rest of 6 female dogs were operated for elective ovarianhysterectomy and such animals were kept as control group. Clinical parameters, haematobiochemical parameters, hormonal profile and histo-pathological examination were observed for all the 18 animals just before the operative procedure and after 45 days of the ovarian-hysterectomy. The hormonal profile (estradiol, progesterone, LH and FSH) of the animals revealed that significant differences in the levels of steroid hormones mainly in progesterone and estrogen in female dogs suffering from cystic endometrial hyperplasia complex as compared to normal female dogs and most of the animal suffering from CEHP complex were having either history of pervious hormonal treatment or they were not bred several years before showing symptoms so there is disturbances in level of steroidal hormone mostly progesterone, which causes increases sensitivity of animals for development of pyometra and CEH. Levels of these hormones after ovarian hysterectomy procedure reduced significantly (109.67 pg/ml, 4.25 ng/ml 17.76pg/ml to, 0.17ng/ml estrogen and progesterone pre and post operatively respectively). This study concludes that in this pathological condition of the uterus the levels of steroid hormones increases, change in their receptors and exogenous administration of theses hormone may play an important role in pathogenesis of CEH-P complex. The main hormone responsible for CEH-P Complex is Progesterone. FSH and LH are not play important role in development of CEH-P complex. The ovariohisterectomuy is choice of treatment for pyometra in female dogs.ThesisItem Open Access Isolation, propagation and characterization of bone marrow derived mesenchymal stem cells and their differentiation into chondrocytes in dogs(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Joshi, Pratibha; Jadon, Narendra SinghThe intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells are contemplated as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The leitmotif of this study was differentiating cBM-MSCs into chondrocytes in vitro. The aspiration of bone marrow was done aseptically from ileac crest of the mongrel dog. The procedure was done after administration of general anaesthesia. The aspirate from bone marrow was processed within half an hour of its collection. Pellet of MSCs along with mononuclear cells were isolated by density gradient centrifugation technique. Mesenchymal stem cells were isolated on the basis of plastic adherence and elongated spindle –shape. At 80-90% confluency, cells were passaged using Trypin-EDTA to amplify the cell population. Passaging of cells were done upto 4 passages and then MSCs were grown in chondrogenic differentiation media in CO2 incubator at 37 ° C and 5% CO2 for 21 days. The morphology of cells was transitioned from spindle elongated shape to round/ spherical shape. The cells were characterized at regular intervals by staining with Toluidine blue at 7th and 21st days with positive result which coloured the extracellular matrix purple. The RT-PCR for the genes SOX9 and COMP at 10th day supported with Type II collagen and Aggrecan at 22nd day revealed bands confirming the differentiation process. The sample on gelatin scaffold and on pellet when subjected to scanning electron microscopy revealed round shape cells with cell aggregates. It was concluded with the support of this study that MSCs possess potential to differentiate into chondrocytes when cultured in chondrogenic medium in vitro and techniques for identification are affluent. It had nevertheless paved the way for the establishment of regenerative and gene therapy for repair and regeneration of damaged and diseased tissue.ThesisItem Open Access Immunity related gene expression during progression and regression of canine transmissible venereal tumour(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Tiwari, Kanika; Das, Arup KumarThe present study was conducted on clinical cases of twenty four dogs of either sex or age affected with naturally occurring canine transmissible venereal tumour (CTVT) to evaluate the gene expression during progression and regression of tumour and to evaluate the T cell response toward mitogen Concanavalin A. Animals were randomly divided into four groups consisting of 6 dogs each. Group 1 consists of TVT affected dogs in which vincristine sulphate were not administered. Group-2 consists of normal dogs in which vincristine sulphate were administered. Group-3 consists of TVT affected dogs in which vincristine sulphate were administered. Control group consists of normal dogs in which vincristine sulphate were not administered. The assessment were made on the basis of clinical examination, gross regression after chemotherapy, haemato-biochemical, lymphocyte proliferation assay and expression of gene were studied before and after administration of vincristine sulphate in CTVT affected dogs. On the basis of these parameters, it is concluded that transforming growth factor beta 1 (TGF-β1) was secreted by CTVT cells during progression phase which suppressed the lymphokine activated killer (LAK) cytotoxicity, which is suppressed by CTVT cells secreted IL-6 during the regression phase of the tumour. T- lymphocytes are also associated with the regression phase of CTVT suggested that tumour immunity also plays a major role in tumour regression.ThesisItem Open Access Comparative evaluation of external skeletal fixator, hybrid tie-in fixator and intramedullary Titanium pinning for repair of induced humerus fracture in fowl (Gallus gallus)(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-08) Mir Tufail Mohd.; Das, Arup KumarThesisItem Open Access Studies on effects of thiopental sodium with and without dexmedetomidine and its reversal with yohimbine in dogs(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-07) Saini, Rashmi; Jadon, N.S.ThesisItem Open Access Studies on effect of dexmedetomidine, ketamine and etomidate anaesthesia in dogs(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-07) Arun Kumar; Kandpal, ManjulThe study was conducted on twenty four clinically healthy dogs of either sex, divided into four groups with the objective to evaluate a safe anaesthetic protocol that could lead to fast and smoother recovery. Ketamine and etomidate alone or in combination with dexmedetomidine were used for this purpose. In group 1 ketamine was administered intravenously @ 10 mg/kg body weight. In group 2, dexmedetomidine was administered @ 10 µg/kg body weight, intravenously followed by ketamine to the effect (3.17±0.15 mg/kg). In group 3, etomidate 3 mg/kg body weight intravenous was administered and in group 4 dexmedetomidine was administered @ 10 µg/kg body weight, intravenously and followed by etomidate to the effect (1.32±0.03 mg/kg). The efficacy of different anaesthetic protocol was accessed by determining various clinicophysiological, cardiopulmonary and haematobiochemical parameters before and after anaesthesia. Dexmedetomidine prior to administration of ketamine or etomidate results in quicker induction, better analgesia for more duration and better muscle relaxation as compared to use of ketamine and etomidate alone. Heart rate increased significantly in group 1 and 2 after anaesthesia. In group 3 non-significant increased and in group 4 significant decrease in heart rate was observed. Respiration rate decreased significantly in all four groups except in group 1. Rectal temperature was non significantly decreased in all groups. Haemoglobin level was decreased non significantly in the animals of all groups. The PCV level was decrease significantly in group 1, 2 and 3 whereas in group 4 PCV level decrease non significantly. The TEC level was decreased significantly in group 1, 2 and 4 whereas it decreases non significantly in group 3. Non significant change were observed in TLC level. In group 4 a non significant and in other 3 groups significant increase in glucose level was observed. The creatinine level increase significantly in group 3 and non significantly in group 1, 2 and 4. Blood urea nitrogen showed a non-significant increase in group 2 and 4 and significant increased in group 1 however it decreases non significantly in group 3. The total proteins showed a non-significant decrease in the animals of all groups. ALT and AST level was non significantly increase in all the groups. Insignificant change were also observed in serum electrolyte level.ThesisItem Open Access Studies on physiological, haematobiochemical and clinical effects of etomidate, propofol, ketamine and sevoflurane anaesthesia in dexmedetomidine premedicated dogs(G.B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand), 2017-01) Bisth, Devender Singh; Jadon, Narendra SinghThirty six adult dogs (requiring various clinical procedures) used in this study were divided randomly into six subgroups (A1, A2, B1, B2, C1 and C2). All the dogs were subjected to the administration atropine sulphate at the dose rate of 0.04mg/kg s/c followed by dexmedetomidine given as preanaesthetic at the dose rate of 10 µg/kg iv in groups A1, B1 and C1 and at the dose rate of 15 µg/kg iv in groups A2, B2 and C2. The animals of group A1 and A2 were induced with etomidate at the dose rate of 1.66±0.11mg/kg iv and 1.58±0.19mg/kg iv, animals of group B1 and B2 with propofol at the dose rate of 2.46±0.19mg/kg iv and 2.45±0.20mg/kg iv whereas the animals of group C1 and C2 were induced with ketamine at the dose rate of 7.70±0.17 iv and 7.60±0.20 mg/kg iv respectively. Anaesthesia was maintained in all the groups of animals with sevoflurane. The minimum alveolar concentration (MAC) of sevoflurane was determined in all the animals by the tail clamp method. The level of anaesthesia was determined by observing various clinical (Induction time, duration of anaesthesia, muscle relaxation, pedal reflex, palpebral reflex, recovery time, sternal recumbency time, standing time, complete recovery time, required doses of different drugs and sevoflurane MAC reductions (%), physiological (rectal temperature, heart rate, respiration rate, blood pressure, capillary refill time, haemoglobin oxygen saturation-SpO2, electrocardiography), haematological (haemoglobin, total erythrocyte count, erythrocyte sedimentation rate, total leucocyte count, differential leucocyte count, packed cell volume, erythrocytic indices MCV, MCH, MCHC and biochemical (serum glucose, serum insulin, serum cortisol, total protein, serum albumin, serum urea nitrogen, serum creatinine, alanine amino transferase and aspartate amino transferase) parameters. The mean values of induction time were decreased in the groups receiving a higher dose of dexmedetomidine (10µg/kg vs. 15µg/kg). The duration of anaesthesia in the present study was dependent on the responses of the animals to the tail clamp. All anaesthetic combinations tested in the study were able to provide adequate muscle relaxation. The pedal reflex and palpebral reflex were totally lost after administration of the induction agents in all the groups barring the palpebral reflex in group C. The recovery time, strernal recumbency time, standing time and complete recovery time were increased in the groups administered with higher dose of dexmedetomidine as compared to the groups receiving lower dose of dexmedetomidine. An increase in the dose of dexmedetomidine resulted in a decrease in the required dose rate of induction agents (etomidate, propofol and ketamine) and the MAC of sevoflurane. The rectal temperature, heart rate and respiration rate showed significant decrease at various time intervals and reached to the base level values by 24 hours post anaesthesia. The systolic, diastolic and mean arterial pressures showed significant decrease in its values in the groups A and B but not in the group C, and returned to their base values by 3 hours post anaesthesia. In all the groups of animals the capillary refill time (CRT) and haemoglobin oxygen saturation (SpO2) were always less than 2 seconds and more than 95% respectively. ECG changes observed in the animals of group A1 were increased P-R and Q-T interval and inverted T waves, in the animals of group A2 sinus arryhythmia, in the animals of group B1 biphasic and inverted T wave, in the group B2 bradyarrhythmia, in the animals of group C1 elevated T waves whereas in the animals of group C2 biphasic T waves were observed. The haemoglobin, TLC and TEC values of the animals in various groups showed significant decrease in its values whereas the ESR showed a non-significant increase. No significant changes were observed in differential leucocyte count and erythrocytic indices. Serum glucose level increased significantly along with decrease in serum insulin levels. Serum cortisol level showed a significant decrease in its values in group A and a significant increase in groups B and C. Decrease in total protein and albumin level along with slight increase in urea nitrogen, creatinine, ALT and AST levels was observed in all the groups of animals. Clinical efficacy of the anaesthetic combinations was determined by performing various surgical operations. On the basis of above mentioned parameters it was concluded that the anaesthetic combinations used in the study have least deleterious effects on the different body systems. The most efficacious anaesthetic combination was atropine sulphate (0.04mg/kg im)dexmedetomidine (15 µg/kg iv)-ketamine (5mg/kg iv)-sevoflurane (group C2) which caused a 30.28% reduction in MAC of sevoflurane.
