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Apart from M.Sc. dissertation & Ph.D. Thesis,
I have been carrying out independent as well as collaborative
research studies with Senior Scientists at Institute of
Pathology.
Utility of semi-thin plastic section
Purification of Glutaraldehyde
Ultrastructural study of hypo-pigmentation
in leprosy and in
vitiligo
Ultrastructural studies in Donovanosis and Post Kala-azar Dermal
Leishmaniasis
Vascular Pattern of Placenta in Pre-Eclamptic Toxaemia
Studies on an Experimental Model of Indian Childhood
Cirrhosis
Diagnostic Electron Microscopy of Muscular
Dystrophies
Ultrastructural Study On Aflatoxicosis in Labeo rohita (Rohu)
Cutaneous lesions in acute
meningococcaemia
Viral particles in persistent generalised lymphadenopathy.
1. Utility of semi-thin plastic section:
This project was taken up to evaluate the role of plastic sections for use in the light microscopic pathology. The effect of different parameters like fixatives, plastic resins, and staining of semithin plastic sections were assessed on different rat tissues. A total number of 450 blocks of various tissues fixed in 4 different fixatives and embedded in 4 different resins were used to get one micron semithin sections. These were stained with 38 different staining methods using various combinations of twenty different dyes. The results provided a comparative evaluation of nuclear-cytoplasmic differentiation, colour richness and stability. Our results showed that 1 micron thick plastic sections allowed clear visualisation of fine structures like intercellular bridges, brush borders and fine lipid droplets under the light microscope and thus could be used for routine histopathology. The advantages of better resolution of tissue details and also quicker processing schedules make plastic embedding methods definitely superior to the existing paraffin embedding systems. In an another study on utility of semithin sections in the diagnosis of Donovanosis, three cases reported to be negative or doubtful for the Donavon organisms by tissue smears and paraffin sections, semithin plastic sections provided definite diagnosis.
Indian J. Med. Res. 85:212-221 (1987).
Indian J. Med. Res. 90:270-274 (1989).
2. Purification of Glutaraldehyde
Glutaraldehyde is most commonly used primary fixative during specimen processing for routine electron microscopy. It is a costly chemical and needs to be imported. Very often it is supplied in 25% concentration. Due to difficulties in frequent import of various processing chemicals for electron microscopy, investigators, very often go in for bulk purchases which is not utilised within the limited shelf life of these chemicals. The glutaraldehyde solution undergoes polymerisation reactions on storage. Its pH goes down from 5.0 to less than 3.5 and colour changes colourless to yellow. Polymerisation also causes drastic deterioration in its fixation properties rendering it useless for electron microscopy processing. In our laboratory attempts were made to distill such fixative solutions in order to retrieve still un-polymerized dimers suitable for proper fixation. Fixation properties of undistilled, distilled and freshly obtained glutaraldehyde solutions were assessed and it was observed that it is possible to retrieve such biologically active glutaraldehyde under laboratory conditions.
3. E. M. Studies on Hypopigmented lesions in Leprosy and Vitiligo:
a. Ultrastructural study of hypo-pigmentation in leprosy.
The transfer of melanized melanosomes into the adjacent keratinocytes from the melanocytes results in uniform pigmentation of the skin. Leprosy is a systemic bacterial disease. It is manifested by the appearance of light coloured skin patches. Earlier studies did not demonstrate any reduction in the number of melanocytes over hypopigmented areas. In this study an attempt was made to find a microscopic correlate and pathogenesis of hypo-pigmentation in leprosy by studying the melanosome concentration and distribution in the epidermal cells. The results showed a decrease in the number of melanosomes in melanocytes and keratinocytes in the hypopigmented area. The findings indicate that there is a decrease in melanization and the effect of disease at some regulatory mechanism may be the cause of hypo-pigmentation in leprosy.
Proc. II Internat. Symposium Electron Microscopy and Biophysics and XVII EMSI Conf., Punjab University, Chandigarh, E-B51 (abstract) (1986).
b. Ultrastructural study of hypo-pigmentation in vitiligo
The pathogenesis of pigment depletion in vitiligo associated with highly dendritic melanocytes and non-dendritic melanocytes was assessed by quantitative estimation of the pigment and enzyme levels in the melanin units and by EM studies. It was observed that highly dendritic melanocytes were large and bizarre and had numerous dendritic processes. The contained the enzyme rich but pigment less pre-melanosomes which are passed on the keratinocytes. Photo-oxidation gives rise to auto-toxicity and destruction of the dendritic processes and melanosomes thus rounded non-pigmented cells remain resulting in the vitiliginous patch. In non-dendritic marginal melanocytes it was observed that the defect was different from that in highly dendritic melanocytes. In these cases there was a defect in the replacement of melanosomes within the melanocytes, leading to a complete depletion of pigment in the affected area. The non-dendritic melanocytes showed a loss of melanosomes and vacuolation. It was hypothesised that there is a destruction of the cytoplasmic organelles of these cells leading to depigmentation.
Proc. XIV Internat. Pigment Cell
Conference, Kobe, Japan, VAP-2:149(abstract) (1990).
Proc. XIV Internat. Pigment Cell Conference Kobe, Japan,
VAP-2:150(abstract) (1990).
Proc. XIV Internat. Pigment Cell Conference, Kobe, Japan,
VAP-2:152(abstract) (1990).
4. Ultrastructural studies in Donovanosis
In an ultrastructural study undertaken on Donovanosis, the inflammatory response and the fine structure of causative organism, Calymmatobacterium granulomatis was attempted. Macrophages were seen to be activated and showed presence of numerous filopodia and increase in the number of lysosomes and rough endoplasmic reticulum. Many cells showed vacuoles of varying size in the cytoplasm, some of which contained the organisms. Other cells seen included plasma cells, polymorphonuclear cells and sparse lymphocytes. Ultrastructural study of donovan organisms revealed presence of electron dense polar material, in addition to regular bacterial structures like mesosome, ribosomes and nuclear material. Surface appendages i.e., fimbriae and blebs were studied in detail. Morphology of fimbrium at the site of its attachment to the cell membrane was described. A distinct layer of homogenous material of varying density surrounding the organism indicated the possibility of it being a capsule.
Indian J. Med. Res. 90:158-164
(1989).
Indian J. Med. Res.[A]. 93:225-231(1991)
5. Ultrastructure of Post Kala-azar Dermal Leishmaniasis
A study of the detailed structure of the skin lesions in PKDL patients with a view to understand the host-parasite relationship in this unusual parasitic disease form is in progress at the Institute. The electron microscope studies at our Institute have shown presence of Leishmania donovani as rounded or oval bodies measuring between 1.5 X 2.5 mm to 3 X 6.5 mm in the cytoplasm of macrophages. Recently, some membrane bound dense core bodies with or without presence of LD bodies have also been observed in the cytoplasm of macrophages in our studies. Such bodies have not been reported previously in PKDL cases and it is possible that these might represent some stage during life cycle of organism.
Invited Talk at XX National Conference of Electron Microscope Society of India, Calcutta.
6. Vascular Pattern of Placenta in Pre-Eclamptic Toxaemia
PET and PIH are major complications responsible for intrauterine growth retardation (IUGR) of foetus. Metabolic and trophoblastic changes have been implicated in its aetiogenesis. Placental blood vessels play an important role for interchange of material between foetus and mother. It is possible that disturbances of placental vasculature may affect the foetus, the mother or both. Our studies have revealed an increase in thickness of trophoblastic basement membrane in the placental villi from toxaemic women. Increased amount of interstitial collagen fibrils, cytotrophoblastic cells and dilated spaces in the cytoplasm of syncytiotrophblast were observed in PET placentae in comparison to control. The EM study of these cases revealed that there is probably a decrease materno-foetal gaseous exchange resulting from a reduction in density of vasculosyncytial membranes. Further, an increase in the number of syncytial knots has been observed which is considered as structural expression of placental insufficiency, ischaemia and hypoxia. Analysis of clinical data contradicted the widely believed hypothesis that pregnancy induced hypertension results in Intra-uterine growth retardation. It was observed that in more than 50% cases of PIH, the baby was healthy and weighed 2.5 Kg or more. In fact, in one case of pre-term caesarean delivery by PIH mother, the weight of the baby was 3.1 Kg. This could be the result of increasing awareness about medical care and nutrition for the pregnant women. Ultrastructural study of the walls of primary and secondary placental blood vessels revealed no difference between control and PIH or PET cases. However, endothelial cells in all types of blood vessels showed significant alterations such as bulging, dilated endoplasmic reticulum, altered mitochondria, increased number of pinocytic vesicles, and coated vesicles in PET and PIH cases showing IUGR.
Previous studies have also revealed a considerable rise in the activity of alkaline phosphatase in human placenta throughout normal pregnancy. Such elevated levels of this enzyme in blood during pregnancy have been attributed to circulating placental enzyme. It is also suggested that alkaline phosphatase plays a role in the absorptive process specifically of fatty acids and could be used as an index of placental function. An examination for alkaline phosphatase activity in normotensive placenta showed electron dense deposits representing presence of alkaline phosphatase in the trophoblastic basement membrane and along the plasma membrane lining of the syncytial microvilli. Similar deposits were also observed on the surface of syncytial microvilli and trophoblastic basement membrane in the samples of placenta of PIH patients but their concentration was markedly enhanced in the syncytial microvilli. Further studies are in progress.
Electron Microscopy 1998 : Volume IV ; Benavides H A C and Yacaman M J (eds.) Institute of Physic Publishing, Bristol pp. 647 - 648, (1998).
7. Studies on an Experimental Model of Indian Childhood Cirrhosis
This project had been initiated as a follow-up of the trace element analysis of liver biopsy specimens in which we had observed an increase in not only copper but also zinc in ICC and other allied disorders of liver. This study was taken up with a view to understand the role of iatrogenic factors in ICC related liver disorders. The results of histopathological examination of liver samples from 30 male rats (16 adults and 14 in the age group of 28 ± 3 days) over a period of 20 months showed dilatation of portal vein, marked hepatocellular ballooning and presence of focal hepatocellular necrotic lesions accompanied by focal infiltrate. Fibrosis was seen infrequently and that too superficially. Mesenchymal response seemed to be lacking. The findings indicate that onset of an early damage although late sequences are not yet observed.
8. Diagnostic Electron Microscopy of Muscular Dystrophies
Diseases of skeletal muscle constitute a significant proportion of neurological disorders. In the past not much importance was given to detection and correct diagnosis of these disorders as most of the diseases are genetically determined and have no specific mode of treatment. Recently, more emphasis is being given to diagnostic application of electron microscopy in different types of muscular disorders and we receive a large number of samples of different types of muscular myopathies for ultrastructural examination.. Electron Microscope examination showed increased number of mitochondria in mitochondrial myopathy. In samples suspected of muscular dystrophies the changes varied along with severity of the disorder. While some samples showed normal configuration with no ultrastructural changes at all, others showed presence of few muscle fibres amongst extensive collagen bundles, a variation in the size of muscle fibres with all most complete loss of basal lamina around the muscle fibres cytoplasmic vacuolation and abnormal banding patterns. These findings are being correlated with histopathological findings, CPK levels and familial history.
9. Ultrastructural Study On Aflatoxicosis In Labeo rohita (Rohu)
Aflatoxins are a group of extremely toxic fungal metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. These fungi grow on a variety of agricultural products and the contaminated feed is responsible for Aflatoxicosis in a variety of fish. Aflatoxin B1 (AFB1) is known to be hepatotoxic, cytotoxic, genotoxic, carcinogenic, mutagenic, teratogenic and nephrotoxic in animals, fishes and human beings. Toxicity of aflatoxins has been found to vary widely between different species of fish such as salmonoids, channel catfish, tilapia, common carp, etc. However, very little is known about toxic effects of this mycotoxin on Rohu, an important culturable species of Indian major carps. This study was undertaken as a short-term collaborative project between Institute of Pathology and Dr. P. K. Sahoo, (SERC visiting fellow of DST) Central Institute of Fresh Water Aquaculture, Kausalyaganga, Bhubaneswar - 751002.
Liver, kidney and gill tissues from fish exposed to acute (10 days) and chronic (90 days) doses of aflatoxin were studied at the light and electron microscopic levels. In control samples, the gills showed normal architecture. The lamellar tip showed stratified squamous epithelium covering the central capillaries. In the samples subjected to acute or chronic toxicity, the samples showed fusion of secondary lamellae, proliferation and hyperplasia of epithelial cells at lamellar base. The acute group further revealed presence of oedematous space between epithelium and central capillaries and at higher dose even a rupture of lamellar capillaries Normal glomeruli, tubules with typical brush border and all types of haemopoietic cells were observed in control kidney samples. The acute dose group revealed glomerular epithelial hyperplasia, tubular epithelial necrosis, and prominent telolysosomes in the tubular epithelium, intra-cytoplasmic desmosomes and loss of lymphocytic cells in the interstitium. In sub-chronic toxicity, the fish had hyperplasia of mesangial cells in glomeruli, atypical telolysosomes in tubular epithelium and marked reduction of lymphocytic cells with increase in neutrophilic granulocytes in the interstitium. The epithelial cells showed degenerative changes with increased number of swollen and elongated mitochondria.
In the control samples, the hepatocytes were apparently normal. Few hepatocytes showed presence of irregular electron-dense bodies (telolysosomes) in the cytoplasm. In the acute group, the hepatocytes were enlarged, separated and there was individualisation of hepatocytes. Some hepatocytes revealed degenerative and necrotic changes. The telolysosomal bodies were more prominent in the cytoplasm. Chronic toxicity group showed presence of nodules, which were well separated from surrounding hepatocytes. The telolysosomal were enlarged in size and the plasma membrane had lot of processes. These finding are indicative of nephrotoxic, cytotoxic and immuno-suppressive nature of aflatoxin in Rohu.
10. Other Studies
a. Cutaneous lesions in acute meningococcaemia
Fifty children with culture proven acute meningococcaemia were studied during the winter outbreak of the disease in 1986-87. The electron microscopy of skin lesions indicated the involvement of the coagulative mechanism in the pathogenesis of meningococcaemia.
Indian J. Med. Res. 91:27-32 (1990).
b. Viral particles in persistent generalised lymphadenopathy
In this study electron microscopy was carried out on a lymph node specimen from a heterosexually promiscuous male who presented with persistent generalised lymphadenopathy. The follicular dendritic cells were identified by the presence of a voluminous, clear, frequently indented nucleus with a thin marginal bane of heterochromatin and a distinct nucleolus. A few retroviral like particles were seen lying extra-cellularly between the dendritic cell extensions.
Indian J. Pathol. Microbiol. 35:201-208 (1992).
c. In addition I have also been associated with following studies
1. Ultrastructural study of Bhopal Gas Disaster Victims and Experimental MIC studies carried out at DRDE, Gwalior with Dr. S. Sriramachari and Dr. Ashok Mukherjee.
2. Ultrastructural studies in on liver biopsies in Indian Childhood Cirrhosis with Dr. S. Sriramachari and Dr. B. Iyengar.
3. Ultrastructural tumour diagnosis with Dr. Ashok Mukherjee and Dr. B. Iyengar.
4. Endothelial involvement in human and experimental leprosy with Dr. Ashok Mukherjee.
5. Ultrastructure of blood vessel involvement in nude mice artificially inoculated with M. leprae with Dr. Ashok Mukherjee.
6. Ultrastructural studies on the lymphoid organs of normal Indian fresh water cat fish: C. Batrachus with Prof. Kunal Saha and Dr. Ashok Mukherjee.
