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ThesisItem Open Access Clinicopathological studies of nanoalumina in wistar rats(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Shodhan, K.V.; Agarwal, SeemaThe present study was designed to examine the clinicopathological responses of nanoalumina in Wistar rats in a repeated dose study for 90 days. The experiment design consisted of 35 Wistar randomly divided into 2 groups; GI as control group with 20 rats and GII as treatment group with 15 rats. Rats of GII group were gavaged with 6 mg/kg BW of nanoalumina per day from day 0 till 90 DPT. Upon routine observation of rats, rats were found to be active with good feeding response. There was decrease in the weight gain in the treated group. Haematological examination demonstrated increase in total erythrocyte count (TEC), packed cell volume (PCV), total leukocyte count (TLC) and absolute lymphocyte count (ALC) and decrease in haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Biochemical examination demonstrated increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, creatinine and Blood Urea Nitrogen (BUN) whereas there was decrease in total protein (TP), albumin, globulin and cholesterol value. Immunological examination demonstrated increase in T cell blastogenesis, HI titre and ELISA. Grossly, treatment group revealed cyst on the surface of liver, emphysematous lungs and brain was congested. Histopathologically, in nanoalumina treated rats, liver showed congestion in central vein, sinusoidal dilatation and congestion, mononuclear cells infiltration, hepatic disarray, degeneration and coagulative necrosis of hepatocytes. Kidney revealed segmentation of glomerulus, glomerulitis with loss of bowman’s space, mononuclear cell infiltration, necrosis and narrowing of tubular lumen. Spleen revealed lymphoid depletion initially and lymphoid hyperplasia in later stages. Thymus revealed congestion, necrosis and depletion of lymphoid tissue. Brain revealed gliosis, perivascular cuffing and neural degeneration and presence of vacuolation. Lungs revealed emphysema, atelectasis and thickening of interalveolar septa with mononuclear cell infiltration. Heart revealed haemorrhage and necrosis of muscle fibres. Intestine revealed catarrhal inflammation and sloughing of villous epithelium. Testis revealed haemorrhage, distortion of seminiferous tubules and abnormal spermatid. Upon transmission electron micrography, liver and kidney revealed nanoalumina deposition in mitochondria and lysosomes with swelling and degeneration of mitochondria with loss of cristae and thickening of basal lamina of kidney tubules. Results of present study concluded that nanoalumina had no observable effect on rat behaviour and rats had normal appetite. It caused an elevation in RBC count with reduction in the average size and haemoglobin content of the erythrocytes. The increase in the biochemistry parameters indicated hepatotoxicity and nephrotoxicity which was further supported by the histopathological changes in the organs. Nanoalumina has immunopotentiating action on T and B cell population. Pathomorphological studies indicated that nanoalumina gets accumulated in various organelles and induces structural alterations in the various organs. Thus it can be said that nanoalumina at the dose of 6mg/kg BW when gavaged for 90 days causes pathological alterations in the exposed rats.ThesisItem Open Access Immunopathology of copper nanoparticles in Winstar rats(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Mewari, Neeraj Singh; Chauhan, R.S.The present study was carried out for a period of 90 days to study the immunopathology of copper nanoparticles in Wistar rats. For this study, a total of 35 rats of both the sexes were randomly divided into two groups viz. group 1 and group 2. Group 1 was kept as control and consisting of 20 rats. Group 2 was nanocopper treated and comprising of 15 rats. The rats in group 1 were provided with standard diet and water while the rats in group 2 along with standard recommended feed and RO water, were additionally given copper nanoparticles mixed in distilled water orally at NOAEL dose rate of 100 mg/kg body weight/day from 0 day of experiment till 90th day post treatment. Immunopathological, gross and histopathological studies were carried out at 0, 30th, 60th and 90th DPT. Transmission Electron Microscopy of liver, kidney and spleen was done at 90th DPT. No apparent behavioural and clinical signs were recorded in rats of control and treated groups over a period of 90 days. Mean body weight showed increase in nano-copper treated group as compared to control group. Haematological parameters showed decrease in the mean haemoglobin, mean packed cell volume, mean total leucocyte count, mean absolute lymphocyte count and mean lymphocyte count in nano-copper treated group as compared to control group. There was an increase observed in absolute neutrophil count and mean neutrophil count. Mean thickness of the skin of rat measured in delayed type hypersensitivity reaction through DNFB and mean NBT positive cells in macrophage function test were found to be decreased in nano-copper treated group as compared to control group. Biochemical parameters like total serum protein, serum albumin, serum globulin and serum gamma globulin showed decrease in values in nano-copper treated group as compared to control group. Mean HI titre and mean ELISA values of experimental rats were found to be decreased in nano-copper treated group as compared to control group. There was an elevated level of serum creatinine and liver function enzyme i.e., Aspartate aminotransferase in nano-copper treated group as compared to that of control group. The mean values of delta optical density of experimental rats in lymphocyte stimulation test using Con-A, PHA-M and LPS were found to be increased in nano-copper treated group as compared to control group. In treated group, liver showed cyst and nodule formation on gross examination. However, no observable gross lesions were noticed in other organs of treated group and in control group. Histopathological lesions were observed in various organs. Liver showed congestion in central vein, degenerative and necrotic changes in hepatocytes, sinusoidal dilatation and infiltration of mononuclear cells in intercellular spaces and around central vein. Kidneys of treated group showed degeneration and necrosis of tubular epithelium, glomerular shrinkage, congestion and haemorrhage in glomeruli, congestion in blood vessels, haemorrhage in interstitium and mononuclear cells infiltration in glomeruli and interstitial spaces. Microscopically, the lung sections of treated group demonstrated congestion, emphysema and atelectasis at places, thickening of interalveolar septa and infiltration of mononuclear cells at many places in lung parenchyma. Brain showed perivascular cuffing, necrosis, vacuolations in brain parenchyma and congestion in blood vessels. Spleen sections of treated group microscopically revealed congestion, necrosis and depletion of lymphoid cells at some places. Intestinal sections of treated group microscopically showed lesions of catarrhal enteritis and infiltration of mononuclear cells. Testis sections of treated group microscopically revealed gap between the seminiferous tubules and degenerative and necrotic changes in spermatids. Heart sections of treated group microscopically showed congestion in blood vessels and necrosis. Microscopically, thymus sections of treated group revealed congestion and lymphoid cell depletion at places. However, no microscopic lesions were observed in tissue sections of control group. Transmission electron microscopy of lung, kidney and spleen revealed electron dense areas of copper nanoparticles as aggregates, agglomerates and individual small size forms. The present study can be concluded that the nano-copper in NOAEL dose is exerting its deleterious effects on vital organs/tissues/systems of the body that may lead to immunopathology, hepatopathy, nephropathy, neuropathy and may adversely affect the reproductive health of animals. Further studies should be carried out in different animal models using varied doses and increased duration of copper nanoparticles to exactly find out the immunopathological alterations. Moreover, future research plans should be done regarding the interaction of copper nanoparticles with DNA as some studies suggested that they can cause DNA damage and cytotoxicity in various cell lines. Also, future studies should be carried out regarding comparative effects of copper nanoparticles in male and female rats so as to assess its pathological effects on reproductive organs besides, sterility, genotoxicity and teratogenic effects.ThesisItem Open Access Pathological studies on nanosilica administered Wistar rats(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-07) Neha; Batra, MunishThe present study investigated the toxicopathological effects of nanosilica at NOAEL dose in Wistar rats for a period of 90 days. A total of 35 rats of 6 weeks age were divided randomly into group I (control) and group II (treatment). Group II rats were administered nanosilica orally at NOAEL dose (2000mg/kg). A significant time dependent decrease in body weight, relative weight of liver and growth rate were observed in group II rats as compared to group I rats. There were non- significant variations in haemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC) and erythrocytic indices between the groups. PCV and MCH varied significantly within the group. The total leucocyte (TLC) decreased significantly in group II rats as compared to group I rats. In differential leucocyte count, lymphocytes, neutrophil, monocyte, eosinophil and basophil did not vary significantly neither between the groups nor within the groups. In group II rats, serum total protein, serum albumin and globulin revealed no significant change when compared to group I rats. Within the group II rats, serum total protein, albumin and globulin varied significantly. Serum aspartate aminotransaminases (AST) and alanine aminotransaminases (ALT) increased significantly both between groups I & II and within the group II. Cholesterol increased significantly in group II. Creatinine and blood urea nitrogen (BUN) were significantly increased in group II. Immunological studies revealed insignificant variations in humoral and cell mediated immunity (CMI) in group II. Grossly cysts were present on surface of liver of group II rats. Histopathologically, liver of group II rats exhibited vascular changes, mononuclear cells infiltration, dilated sinusoidal spaces, kupffer cells hyperplasia, degeneration and necrosis of hepatocytes. Lungs in group II revealed atelectasis, emphysema, congestion and infiltration of mononuclear cells and thickening of interalveolar septa. Kidneys in group II revealed mononuclear cell infiltration in interstitium, alterations in glomeruli, necrosis and sloughing of tubular epithelial cells and obliteration of tubular lumen. In group II, intestine revealed increased goblet cells, necrosis and desquamation of epithelium. Spleen in group II revealed decreased in white pulp and necrosis of lymphoid cells at few places leading to depletion of lymphoid tissue. Heart of group II rats revealed vascular changes and necrosis of cardiomyocytes was observed. In group II thymus vascular changes, mild necrosis and depletion of lymphoid tissue were observed. In brain of group II rats, degeneration, necrosis of neurons and neuronophagia was observed. Ultrastructural studies in liver and kidney revealed accumulation of nanosilica in lysosomes and mitochondria, apoptosis of hepatocytes and mitochondrial damage. Collectively, these observations indicated that nanosilica have toxic effects as hepatopathy, nephropathy and immunopathy at NOAEL dose.ThesisItem Open Access Immunopathology of calcium nanoparticles in Wistar rats(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-07) Kandpal, Diksha; Chauhan, R.S.The present experimental study was conducted in Wistar rats for 90 days for evaluating immunopathological effect of calcium nanoparticles. The rats were administered nanocalcium at NOAEL dose rate of 1000mg/kg b.wt. The experimental rats were keenly observed for presence of clinical signs and behavior. However, no alterations were recorded. The clinico-pathological parameters of the present study such as hemoglobin showed no alteration. Total leucocyte count (TLC), lymphocytes count and Absolute lymphocyte count (ALC) was found to decrease in nanocalcium treated rats. The decrease might be attributed to the lymphocytotoxic action of nanocalcium leading to lymphocytopenia and leucopenia. Neutrophil count and absolute neutrophil count (ANC) was found to increase and the increase; may be attributed to their compensatory increase against the lymphocytes in nanocalcium treated rats. Serum total protein, serum albumin and serum globulin was found to decrease whereas; serum gamma globulin was increased. The increase or decrease in protein levels may be due to the inflammatory conditions induce by nanoparticles administration. Serum creatinine and serum calcium were increased, although slight, may pose a serious chronic kidney disease risk in long run. Serum cholesterol and serum ALT were increased. Increased serum ALT increases serum cholesterol and thus calcium nanoparticles over a long durationpossess a threat for subsequent development of atherogenesis. Lymphocyte stimulation assay was performed and was found to decrease for all the mitogens used. Thus, immunotoxic effect of calcium nanoparticles on CMI and HMI was evident. The delayed type hypersensitivity was examined using DNFB and results were suggestive of manifestations of CMI which can be considered as to be down regulated due to nanocalcium. Histopathologically, there were areas of coagulative necrosis, vacuolar degeneration and infiltration of inflammatory cells in liver. Kidneys showed hemorrhages and congestion, atrophy of glomeruli, degenerative and necrotic changes of the kidney tubular epithelium. Spleen revealed wide areas of hemosiderosis, lymphoid depletion and increase in the red pulp region. In lungs emphysema, increased interalveolar septa, infiltration of mononuclear cells was present. Intestines revealed mild lesions of catarrhal enteritis. In heart, congestion and necrosis of the myofibers in were recorded. TEM showed degeneration and swelling of the mitochondria with loss of cristae along with rough endoplasmic reticulum (rER) degeneration with areas of electron dense particles in hepatocytes and kidney. TEM for kidney also recorded thickening of the basal lamina of the kidney tubule. TEM of spleen showed phagocytic process of various leucocytes with electron dense particles. The nanoparticles work through mechanism of protein corona and reactive oxygen species formation. These mechanisms assist nanoparticles to increase their circulation time in the living system and subsequently accompany the changes. Calcium being the component of the living system is degradable and less inflammatory and thus finds its usage more as compare to other nanoparticles. Present study however of short duration had recorded the ill effect of calcium nanoparticles on NOAEL dose. This paves the way for conducting a study on large number of animals for longer duration to further conclude the potential immunotoxic, hepatotoxic, nephrotoxic and other effects of calcium nanoparticles.