CHAPTER. V SUMMARY AND CONCLUSIONS. during postnatal period and to provide elasticity during prenatal and early

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1 CHAPTER. V SUMMARY AND CONCLUSIONS The present study was conducted on 40 samples, each of thymus and Spleen of goat from prenatal to four months and above age. The small pieces from each thymus and spleen were processed separately for histomorphological, histochemical and imunohrstological study' The age wise histomorpological status of thymus and spleen was studied from the sections stained with Haematoxylin and eosin, Van Gieson'S, MaSson'S trichrome, Verhoeff's elastic tissue stain, Gomori's reticulin, Toluidin blue, Periodic acid schiff's, Alcian blue periodic acid schiff's, Von kossa's in thymus and pearl's prusian blue method in spleen. The histoenzymic observations of the thymus and spleen were recorded from the frozen sections to ascertain the presence of acid phosphatase and alkaline phosphatase. The immunohistological method such as Alpha Naphthyl Acetate Esterase (ANAE) was performed to locate T 1ymphocytes in thymus and spleen and Peroxidase antiperoxidase (PAP) to locate B lymphocytes in the spleen. The general histoarchitecture of spleen showed that it was composed of connective tissue capsule, trabaculae, white pulp and red pulp' The splenic capsule was found to be composed of outer fibrous layer and inner muscular layer. The thickness of capsule was gradually increasing due to increase in thickness of muscular layer with the advancement of age. The components of outer fibrous layer were wavy, intermingled collagen, reticular and elastic fibers' A progressive decrease in size and density of reticular fibers with increase in density of collagen and elastic fibers was noticed with the increase in age from group I to group lv. The elastic fibers however, became short and fragmented during postnatal stage in group lv. This change in the concentration of these fibers in capsule was attributed to provide firmness to the organs from outside during postnatal period and to provide elasticity during prenatal and early postnatal stage. The connective tissue trabeculae radiated from capsule in to splenic pulp and supported the blood vessels. The trabeculae were few in early prenatal age,

2 but showed increase in their size and amount and became branched during advancement of age. The components of trabeculae were similar to that of capsule. The cellular and fibrous reticulum was present throughout the splenic pulp in all age groups. The white pulp and red pulp were the two major components of splenic parenchyma in all groups of animal. During early to mid prenatal age in group l, the tissues of white pulp were in the form of diffused lymphoid patches scattered throughout the red pulp. The major cellular population of these patches was formed by lymphocytes. Later on, by the migration of the lymphocytes, these patches organised around the blood vessels, to form periarterial lymphatic sheath of white pulp during mid to late prenatal age in group l. The periarterial lymphatic sheath formed nodules. lt consisted of centrally or eccentrically placed white pulp artery. The formation of white pulp was completed without any patches of lymphocytes in red pulp towards the late prenatal stage. The white pulp nodules were round to ovoid in shape in all age groups. These nodules showed highly significant increase in size with the advancement of age. ln all age groups, the tissue of white pulp was delimited from surrounding red pulp by a marginalzone. The cellular population of periarterial lymphatic sheath and nodules of white pulp in group I was chiefly constituted by densely packed small and medium lymphocytes, reljcular. cells with few plasma cells, macrophages and granulocytes. However, from group lt, few mast cells were observed in the nodules near by the vicinity of white pulp artery. The number of macrophages in white pulp was increasing with the advancement of age. The macrophages were found more towards the periphery of nodules to facilitate the phagocytosis and initial immune response. The number of plasma cells in white pulp was more in group lll and lv. The presence of two or three white pulp arteries within the same nodule was recorded in group ll, lll and lv. The germinal centers were not observed in any of the splenic nodules in group I and ll, but were found in group lll and lv. More numbers of plasma cells in white pulp of group lll and lv may be attributed to the blastic transformation of lymphocytes in the germinal center to form the plasma cells for producing high immunity levelwith advancement of age. 75

3 The reticular fibers formed loose network throughout the tissue of white pulp in group I and ll. However, in group lll and lv, the reticular network was absent in the germinal centers of the nodules, but was present in the periarterial lymphatic sheath The reticular fibers were found condensed around the white pulp arleries and at the marginal zone in all age groups. The irregularly anastomised splenic cords and sinuses were present in red pulp. The cellular masses of cords were supported by framework of reticular tissue in ali age group of animals. The red pulp consisted lymphocytes, reticular cells, erythrocytes, granulocytes, macrophages and few mast cells in all age groups under study. ln addition, the megakaryocytes were found scattered throughout the red pulp in group I but were absent in group ll, lll, and lv. Fairly more number of plasma cells and macrophages were noted in group lll and lv, which indicated increased phagocytic activity and maximum immunity level of organ. The amount of muscle fibers in red pulp was found increased with the advancement of age. This increase was considered to provide help in contraction of splenic parenchyma. The branching of white pulp artery in the red pulp formed penicillar artery was observed in all age group animals. Although, the sinuses of red pulp was poorly developed in early to mid prenatal age, but were found well developed and anastomised in group ll, lll and lv. The sinuses were lined by fenestrated elongated endothelial cells. These sinuses were encircled and supported by reticular fibers in all age groups during the present study. The Periodic acid schiff's reaction for the presence of glycogen was varying from moderate to intense in all the components at different age groups in spleen. This is suggestive of enhanced metabolism of carbohydrates necessary for major physiological functioning during development of spleen. The presence of acid mucopolysaccharides in Some components of spleen may be attributed to its necessity for providing optimum condition for gromh and development of germinal center. The acid phosphatase activity was moderate to intense in various component of spleen at different stages of development. High level of acid phosphate was considered to demonstrate the release of lysosomal hydrolyses, which was associated with modification of splenic parenchyma during growth. 76

4 High to intense alkaline phosphates activity was noted in group lll and lv and may be ascribed to increase metabolic activity during this age. The presence of iron revealed high activity in the macrophages throughout the splenic parenchyma in group l. lron content of macrophages was noticed only in red pulp in group ll, lll, and lv. The presence of iron in spleen may be attributed to the fact that iron is present in the haemoglobin as ferritin or haemosiderin. The frequency of occurrence of B rymphocytes in germinal center was found more than the peripheral part of splenic nodule. There was increase in number of T lymphocytes in peripheral part of splenic nodule and periarterial lymphatic sheath, while it was decreased inside the nodule with the advancement of age. Thymus of goat showed that it was composed of capsule, trabeculae, cortex and medulla. The capsule was varying in its thickness with the advancement of age. The interlobular septa radiated from the capsule and divided the thymic parenchyma in to irregular lobules of different sizes. The progressive vascularization with increase in size of vessels and peri - vascular cellular infiltration was observed in interlobular connective tissue septa with advancement of age. The thickness of capsule showed non significant. statistical difference detween the age groups, while the diameter of lobule showed highly significant statistical difference with the advancement of age. The differentiation of cortex and medulla was completed at the end of late prenatal stage. The mitotic activity in cortical cells was more in group l, which reduced with the advancement of age. A significant statistical difference was noted in the thickness of cortex with the advancement of age. The cellular population of medulla was chiefly constituted by number of epithelial reticular cells. The number of hypertrophied epithelial reticular cells rn the medulla was increased with the advancement of age. The Hassall's corpuscles were found to appear at medullary differentiation in group l. Two types of Hassall's corpuscles were found viz. unilamilar and multilamilar. The unilamilar Hassall's corpuscles were found more in group l. However, it turned in to multilamilar with the advancement of age in group ll, lll and lv. 77

5 The presence of Periodic acid schiff's reaction for the presence of glycogen was varying from moderate to intense in all the components of thymus at different age groups. This was suggestive of higher energy required for overall enhancement of carbohydrate metabolism for extracting tissue proliferation during thymic development. The alcinophilic activity for the presence of acid mucopolysaccharides was found in group l, ll and lll but it was negative in group lv. The positive ABPAS activity in group I may be correlated with growth and physiologicat requirement during prenatal stage. Similarly, decrease in alcinophilic activity in Hassall's corpuscles with advancement of age may be considered as indicative of initiation of process of regression in Hasbail's corpuscres. Moderate to intense acid phosphatase activity was noticed in all the components of thymus in all groups during the present study. This positive acid phosphatase reaction may be attributed to lysosomal activity in growing phase of thymus due to continuous tissue alteration during advancement of age. / The alkaline phosphatase activity was found negative in group I and ll, but positive activity'in group lll and lv during present work was considered as sign of increased metabolic activity in thymus. The presence of calcium was found negative in almost all the components of thymus during different age groups. The number of T lymphocytes was found increased in the cortex with the advancement of age. The thymic medulla, however, showed few lymphocytes in all age groups under study. ln general the histoarchitecture of spleen and thymus in goat was found similar to that of other mammalian species. Although the thymus was found in its structural and functional growing stage, but the involutionary process in some of the lobules during present study was found to start during early postnatal life. 78