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Birsa Agricultural University, Ranchi

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2015 - 20192
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Subject: Veterinary Pathology × End date: 2019 × Start date: 2013 × Type: Thesis ×
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    ThesisItemOpen Access
    Studies on Pancreatic Pathology and Its Correlation with Different Wasting Disease Conditions of Poultry
    (Birsa Agricultural University, Ranchi, 2019) KUMAR, BRAJESH; Gupta, M. K.
    3796) birds examined showed definite pancreatic pathology. Maximum incidence of pancreatic pathology was observed in fungal (83.33%) and metabolic (82.86%) diseases whereas least pancreatic pathology was registered in viral diseases (16.41%). The overall incidence of pancreatic pathology in viral disease other than RD has been observed to be 66.95%. Disease wise incidence of pancreatic involvement is significantly higher in cases of IBD (94.74%) followed by IB (93.33%) and pasteurellosis (90.48%). Contrary to rest of the viral diseases the pancreatic pathology was extremely low (13.61%) due to RD though mortality of birds registered was highest, followed by pneumonia of bacterial origin ( 16.58% ). Incidence of pancreatic pathology in different poultry varieties showed that broiler birds were most susceptible (75.34%) to develop pancreatic pathology whereas Vanraja (14.29) variety showed significantly low susceptibility. Age group wise maximum pancreatic pathology was registered in grower birds (33.69%) followed by chicks (20.06%) and adult birds (19.54%). A significant variation was also observed in susceptibility to increased pancreatic pathology under different disease conditions between the three age groups. It was observed that highest percentage of pancreatic pathology was observed in monsoon season (28.84%) followed by summer (22.57%) and least was observed in winter season (12.92%). Though the incidence of pancreatic pathology was less in summer season the percentage of pancreatic involvement was significantly higher for most of the disease conditions.
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    ThesisItemOpen Access
    USE OF ACRIDINE ORANGE STAINING FOR SIMULTANEOUS ASSESSMENT OF PROLIFERATIVE AND APOPTOTIC ACTIVITY IN N-NITROSO-N-METHYLUREA INDUCED TUMOURS IN RATS
    (Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2015) Kumar, Kaushal; Singh, K.K.
    Biological activity of a tumour is determined by a balance between proliferative and apoptotic activity of its neoplastic cells. Histopathological typing and grading has remained a gold standard to assess proliferative aggressiveness of a tuomour which relies mostly on anaplastic and apoptotic changes shown by neoplastic cells.But many times these changes do not reveal true proliferative behavior of a tumour. These problems motivated the augmentation of histopathological methods to improve the accuracy of assessment of proliferative and apoptotic activity of tumours. Proliferative behavior of a tumour is determined by mean growth fraction and mean cycle time of their neoplastic cells. Growth fraction of tumour in a tissue section is assessed by measurement of proliferation associated antigens like PCNA, Ki-67 etc. But no technique is Abstract……… Page 2 available to see the speed of proliferation of neoplastic cells particularly in terms of mean cycle time instantaneously in tissue section of tumours. This highlights the importance of identification and quantification of cells in different phases of cell cycle to throw more insight on proliferative speed and fraction of neoplastic cells. Different protocols of flow cytometry for identification and quantification of neoplastic cells in different phases of cell cycle in isolated cell system are available using acridine orange (AO) staining. These methods can not reveal complete picture of location, migration, differentiation and relation of dividing cells with other cells in their true histological architecture. So in this study an attempt was made to establish a protocol to identify, localize and quantify such cells in microscopic sections of different NMU induced tumours in Wistar rats. Moreover, AgNORs are the only parameter used to assess proliferative fraction as well as proliferative speed of a tumour on a tissue section indirectly. It has the advantage that their numbers are increased only in actively and fastly dividing cells. The major problem in acceptance of this method as an indirect marker of proliferative speed in terms of mean cycle time is deposition of innumerable fine dots in nuclei in background of large AgNOR dots which has been greatly variable from cell to cell depending upon their metabolic and proliferative status, most possibly in different phases of cycle. Hence, critical analysis of variation in pattern of deposition of such small dots in nuclei has scope to evolve as a system for identifying cells in different phases of cycle. This motivated to evolve one more new system of identifying and localizing dividing cells in different phases of cell cycle on the basis of variation in pattern of deposition of such Non-AgNOR dots in nuclei in histological sections after AgNOR staining. The procedure for optimally demonstrating highest intensity of fluorescence of DNA and RNA along with cellular characteristics for necrotic, apoptotic and neoplastic cells passing through different phases of cell cycle as described and accepted by the method of flow cytometry was accepted and being described as follows. For acridine orange staining of formalin fixed paraffinzed tissue sections of tumours were treated with 0.025% AO in 1 % acetic acid for one minute and then were washed in M/15 Phosphate Buffer (pH 6.0) for few second and sections were mounted in a drop of phosphate buffer and used for examination under fluorescent microscope to identify and count different types of cells. Abstract……… Page 3 The characteristic feature accepted for identification of cells in different phases of cell cycle was based on variation in the colour of fluorescence given by nucleus and cytoplasm of the cell. Dull green fluorescing nuclei and cytoplasm was characterized as cell in G1 phase whereas dull green nuclei with red cytoplasm was characterized as late G1 phase of cell cycle. The cells with bright green to white yellow nuclei with orange cytoplasm were identified as S phase. S/G2 phase of the cell cycle was characterized by bright green to yellow-white nuclei with red fluorescing cytoplasm. The cells with bright green nuclei with mild or no red cytoplasm were identified as M phase of the cell cycle. Apoptotic cells were characterized by condensed orange to red nuclei with red cytoplasm whereas cells with homogeneously fragmented red fluorescing nuclei were identified as necrotic cell. These characteristic feature was accepted for identification of cells by AO staining was highly comparable with those well accepted for flow cytometry taken as standard for identification of such cells in this study. However identification of cells in histological section by AO staining have advantage over flow cytometry that it gave complete picture of apoptotic activity of tumours in terms of their number, location, migration, differentiation and architectural arrangements of dividing cells. One more new system to identify and localize proliferating cells in histological sections was also evolved using AgNOR staining after a critical perusal of variation in pattern of formation and distribution of non- AgNORs along with their nature of persistent reproducibility and ability to be dissolved in sodium thiosulphate solution in AgNOR stained sections of different tumours. The characteristic features incorporated in this system were based on formation and distribution pattern of AgNOR dots and non-AgNOR dots in the nucleus. G1 phase was characterized as cells having large AgNOR dots with few small or medium sized non- AgNOR dots. The Cells with innumerable very fine and small non-AgNOR dots with or without large AgNOR dots inside nucleus was in S phase. G2 phase was characterized as cells having small to medium sized innumerable non-AgNOR dots with brown backgrounds whereas cells with black dense irregular hairy nucleus was considered to be in M phase. The cells in S, G2, and M phases of cell cycle were collectively considered to be in proliferative phase. The accuracy of both these systems to identify, localize and quantify neoplastic cells in different phases of cell cycle was further verified by seeing a correlation between counts of such Abstract……… Page 4 cells in histologic sections of different types of NMU induced tumours after AgNOR as well as AO staining. Keeping these facts in mind, both these systems were further validated by seeing a correlation between counts of cells in different phases of cycle after AgNOR staining and percentage count of quiescent, proliferative and apoptotic cells after AO staining with pathomorphological aggressiveness of different types of NMU induced tumours in wistar rats assessed in terms of tumour volume, multinodularity, total cell count and their impact on survival period. Out of 60 female virgin Wistar rats of about 35 days of age were intoxicated with NMU at the dose rate of 50mg/kg body weight intraperitoneally at 50th, 57th, 64th and 71st days of their age; In order to obtain complete pathomorphological behavior of NMU induced tumour, all the experimental animals were allowed to survive till they died either due to growth of tumour or due to NMU toxicity which prolonged for one year. A total of 44 tumours could be induced in 32 tumour bearing rats. Tumour incidence was found to be 53.33%. The average number of tumours per animal (tumour frequency) was found to be 1.33. The average survival period was 276 days. The mean value of nodularity was found to be 3.25 among the NMU induced tumours. The average volume of the tumour was found to be 26.18 cm3. These induced tumours were further classified into five histologycally distinct types which were mucinous adenocarcinoma (10), tubular adenocarcinoma (7), stromal cell sarcoma (8), mammary ductular adenocarcinoma (12) and gastric squamous cell carcinoma (7) which revealed almost their characteristic histopathological features. Count of cells in different phases of cell cycle after AO and AgNOR staining in histologic sections of almost all these different types of NMU induced tumours were found to be highly and positively correlated and thus comparable with one another. It supported the validity of both these systems to identify cells in different phases of cell cycle. Sensitivity of both these systems were further verified indirectly by correlating the count of cells in different phase of cell cycle with that of proliferative aggressiveness of all these different types of NMU induced tumours as assessed by their volume, total number of cells, multinodularity, grading and survival period which were mostly found to be positively and significantly correlated favouring the ability of this system to identify cells in different phases of cycle. Abstract……… Page 5 A critical perusal of variation in number of cells in different phases of cycle and thereby relative timing taken by neoplastic cells in different phases failed to show a general trend of variation in different histologic types of tumours. Rather, number of cells in different phases of cycle was greatly variable in individual tumour to tumour. It warrants the count of such cells in individual tumour, the critical analysis of which may throw more insight on their proliferative behavior. Higher count of cells in S-phase, G2 phase as well as in M-phase indicated presence of more actively proliferative population and actively growing tumour. Such findings can also suggest that such tumours will give more response with chemotherapy since these agents act mostly on replicating cells A simultaneous count of cells undergoing apoptosis along with count of cells in different phases of cycle will help not only in assessing proliferative behavior of tumour rather it may be of great help in assessing sensitivity or resistance of tumour to a particular drug because different drug acts through different mechanism, which involved inhibition of cells in different phases of cycle or by inducing apoptosis. Mutational changes in these loci may be responsible for a refractory of a tumour to different therapeutic agents. These protocols have a potential to evolve as a test to help in selection of most appropriate chemotherapeutic agent against a particular tumour. Thus it could be concluded that both these systems are helpful in identifying and counting of cells in different phases of cell cycle along with number of cells in apoptosis and can help in throwing more insight on proliferative fraction as well as proliferative speed of a tumour and relative timings in different phases of their cell cycle. Such assessment has got immense importance in prognostic, diagnostic and therapeutic decisions on tumour. However, both these systems of counting cells in different phases of cycle or undergoing apoptosis have still many scopes for improvements as well as exploration to make it more accurate reliable and usable in other areas of tumour and cell biology.