effect of infection of the filarial parasite brugia malayi - Pondicherry ...

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4. EXPERIMENTS CHAPTER 4.1 CHANGES IN ANTIOXIDANT ENZYMES AND ANTIOXIDANTS IN MASTOMYS NATALENSIS AFTER BRUGIA MALAY1 INFECTION AND ITS RELATION TO 4.1.1. MATERIALS AND METHODS DEC TREATMENT Male animals of the age of 6-8 weeks, weighing 35-40 gms were used for the study. A group of 20 animals was inoculated subcutaneously with 200 infective larvae of B. malayi subpenodic strain, while another group of 20 animals was kept as control. The animals were maintained for a maximum period of 120 days. Four animals from each group were necropsied on 0, 30, 60,90 and 120 days post inoculation and assayed for antioxidants and antioxidant enzymes as per the methods given below. Organs such as liver, testes, bwin, heart and lungs were excised out, washed thoroughly with saline and blotted dry. A 10% (wlv) homogenate was prepared in Tris-HCI buffer (0.01 M, pH 7.4) containing 0.25 M sucrose using a potter elvehjem homogeniser fined with a power driven teflon pestle. For assays of mitochondnal and cytosolic enzymes, subcellular fractionation was done by centrifugmg the 10Ph homogenate in a Sorval (RC5C) refrigerated (4'C) centrifuge. The nuclear fiaction was obtained by centrifuging at 800g for ten minutes and the mitochondrial fiaction was obtained by centrifuging at 8000g for 20 minutes. The post mitochondria1 supernatant was centrifuged at 100,000g (Sorval-OTD-50) for I hr to isolate the mimsomes. The soluble supmatant served as cytosol. 4.1.1.1 Superoxide dismutase Superoxide dismutase is estimated by the method of Marklund and Marklund (1974). 'he dtgnt of inhibition of autoxidation of pyrogallol at an alkaline pH by SOD was pa a measwe of the enzyme activity.
Ragenb 1. Tris-HCI buffer . : 0.1 M, pH 8.2 containing 2 mM of diethylene hiamine penta acetic acid. 2. Tris- HCI : 0.05 M, pH 7.4. 3. Pyrogallol stock solution : 25.2 rng of pyrogallol was dissolved in 1 ml of 0.05 M Tris- HCI buffer, pH 7.4 in a test tube stoppered and wrapped with an aluminum foil. 4. Pyrogallol working solution : At the time of assay 0.5 ml was diluted to 50 ml with 0.05 M TricHCl buffer, pH 7.4 to give a 2 mM solution and shielded from exposure to light. 5. Absolute ethanol. 6. Chlomfonn. Partially purified SOD was prepared as described by McCord and Fridovich (1969). To 1 ml of the tissue homogenate, 0.25 ml of absolute ethanol and 0.15 ml of chloroform was added. After 15 minutes of shalung in a mechmcal shaker, the suspension was centrifuged and the supematant obtained constituted the enzyme extract. The reaction mixture for autoxidation consisted of 2 ml of the buffer containing DETAPAC, 0.5 ml of 2 rnM pyrogalbl and 1.5 ml water. Irutially, the rate of autoxidation of pyrogalbl was noted at an interval of one minute to three minutes. The assay mixture for the enzyme contained 2 ml of 0.05 M Tris-HCI buffer, 0.5 ml pyrogallol, aliquots of the homogenate and water to give a final volume of 4 ml. The rate of inhibition of pyrogallol autoxidation after the addition of the enzyme was noted. Iron accelerates pyrogallol autoxidation even in trace amounts. DETAPAC acts as a chelator and thus prevents the interference from ~ e as ~ well ' as from CU" and hIn2*. The enzyme activity was expressed in terms of unitsimg protein in which one unit corn& to the amount of enzyme that mhibited the autoxidation reaction by 50%. The activity of catalase was assayed by the method of S~nha (1972). The caFakPc myme pnparation was allowed to split hydrogen peroxide for ~ifFcmnt paiod of time. The reaction was stopped at specifibdgme intervals by adding
Page 1 and 2: EFFECT OF INFECTION OF THE FILARIAL
Page 4 and 5: I wish to express my deep sense of
Page 6: 1. INTRODUCTlON 2. REVIEW OF LlTERA
Page 9 and 10: As stated earlier, the host immune
Page 11 and 12: 13 Testicular changes Testosterone
Page 14 and 15: 2. REVIEW OF LITERATURE Filariasis
Page 16 and 17: 2.2 Clinical signs Inflammalation o
Page 18 and 19: the tegument by the release of thei
Page 20 and 21: the compound kills the filarial wor
Page 22: 3. MATERIALS AND METHODS COLONY MAI
Page 27 and 28: 3. Reduced glutathione : 30 mM To I
Page 29 and 30: 4.1.1.6 Glucose-6-phosphate dehydro
Page 31 and 32: absorbance of the clear supernatant
Page 33 and 34: -eats I. Lowry's reagent :Solution
Page 35 and 36: 4.1.2.1 Changes in antioxidant enzy
Page 37 and 38: Fig.3: Effect of B.malayi on SOD in
Page 39 and 40: Fig.9: Effect of B.rnalayi on GST i
Page 41 and 42: Fig.12: Effect of 6.rnalayi on GPx
Page 43 and 44: Fig. 18: Effect of B.malayi on G6PD
Page 45 and 46: 12- 3 m- 15- lor, Fig.21: Effect of
Page 47 and 48: Fig.27: Effect of B.malayi on Ascor
Page 49 and 50: 4.1.2.3 Change in the antioxidant e
Page 51 and 52: Fig.35: Effect of B.malayi on CAT i
Page 53 and 54: m 20 0) - 15 9 g 10 il! 5 0 Fig.39:
Page 55 and 56: 30 25 8 - a 20 2 15 C I 10 I 5 0 35
Page 57 and 58: Fig.45: Effect of B.maiayi on GSH i
Page 59 and 60: Fig.49: Effect of B.malayi on Vit-C
Page 61 and 62: 4.1.3 DISCUSSION 4.1 3.1.1 Toxic ox
Page 63 and 64: 1973). The two major systems of LPO
Page 65 and 66: 1981), and their increase in human
Page 67 and 68: 1 0 Fig.58:Relationship between cha
Page 69 and 70: 1 - 0- ' 1 5 - 6 - 7 - Fig.63:Relat
Page 71 and 72: Catalase had been reported to be re
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- Fig.68:Relationship between chang
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esults in its reduced activity (Con
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g 20- :I-. F1g.70:Relationship betw
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12, - I 0 U) Q 4 2 0 - + - Fig.75:R
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$ Fig.80:Relationship between chang
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Fig.85:Relationship between change
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V) (3 :; - Fig.9O:Relationship betw
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A reduction in the total thiol cont
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2 5- Fig.98:Relationship between ch
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Fig. 103:Relationship between chang
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4.2.1.1 N ay ATPase Reagents Nay AT
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Reagents 1. Acetylcholine bromide :
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To 1.5 ml of carbonate buffer, 1.5
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4.2.1.10 Differential leucocyte cou
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increase in the control animals (Fi
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- Fig.110: Effect of 6. malayi on H
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efflux and thereby alter the membra
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with i n d lymphocytes and decrease
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Tubes were labeled for calibrators,
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Fig.114: Effect of B, malayi on GTP
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433 DISCUSSION Testosterone is a se
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CHAPTER 4.4 HISTOPATHOLOGICAL CHANG
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Platc 2 Plate 3 Plate 4 Plate 5 Pla
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Platc X Plate 9 Plate 10 Plate 11 P
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Plate w: Section of Testes of norma
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4.4.2.1.5 Brain The control animals
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Plate ICt Section of Kidney of infe
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4.4.2.2 Histopathological changes i
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Plate 32
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The mf could not be seen in spleen
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ain, it decreased from 0 day itself
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inflammatory nature indicative of c
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BIBLIOGRAPHY Ackerman, S.J., Gleich
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Case, T., Leis, B., Witte, M., Way,
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Drabkin, D.L.R. and Austin, J.H. (1
Page 147 and 148:
Fukuota, M., Zhou, Y. and Tanaka.(l
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Jacob, H.S. and Lux, S.E. (1968). D
Page 151 and 152:
Linda, I. and Mayeda, B. (1974). Th
Page 153 and 154:
Murray, H.W. (1981). Susceptibility
Page 155 and 156:
Santiago- Stevenson, D., Oliver-Gon
Page 157 and 158:
Tate, S.S., Thomson, G.A. and Meist
Page 159 and 160:
Zaini, M., Ramachandran, C.P. and E
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