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Journal of Stress Physiology & Biochemistry, Vol. 8 No. 2 2012, pp. 145-151 ISSN 1997-0838 Original Text Copyright © 2012 by Tripathi and Singh
ORIGINAL ARTICLE
Photoperiodic stress on nitrite production by splenic macrophages in fresh-water snake Natrix piscator
Tripathi Manish Kumar and Singh Ramesh
Department of Zoology, Udai Pratap Autonomous College, Varanasi -221002, Uttar Pradesh, India Phone: +91 9415687648 Fax: +91 542 2282399 E-mail: [email protected].
Received March 21 2012
Changes in day length enhance or suppress component of immune function in individuals of several species. The purpose of the present experiment was to study the role of photoperiodic manipulation on the nitric oxide production by splenic macrophages in the fresh-water snake, Natrix piscator. To study effect of photoperiod, animals were subjected to 24 hour continuous light and continuous dark for 30 days. Animals kept in natural day length served as control. At termination of experiments, animals were sacrificed, and spleen was excised. Macrophages were incubated for 24 hours and nitric oxide production was measured by measuring the nitrite concentration. Nitrite production was significantly decreased to the cultures obtained from the animals kept under continuous light. No change in nitrite concentration was found in animals kept under continuous dark, when compared to the animals kept under natural day length. The possible role of decreased melatonin synthesis in light is suggested to decrease the nitric oxide production.
Key words: nitric oxide/ photoperiod/snake/ splenic macrophage
ORIGINAL ARTICLE
Photoperiodic stress on nitrite production by splenic macrophages in fresh-water snake Natrix piscator
Tripathi Manish Kumar and Singh Ramesh
Department of Zoology, Udai Pratap Autonomous College, Varanasi -221002, Uttar Pradesh, India Phone: +91 9415687648 Fax: +91 542 2282399 'E-mail: [email protected]
Received March 21 2012
Changes in day length enhance or suppress component of immune function in individuals of several species. The purpose of the present experiment was to study the role of photoperiodic manipulation on the nitric oxide production by splenic macrophages in the fresh-water snake, Natrix piscator. To study effect of photoperiod, animals were subjected to 24 hour continuous light and continuous dark for 30 days. Animals kept in natural day length served as control. At termination of experiments, animals were sacrificed, and spleen was excised. Macrophages were incubated for 24 hours and nitric oxide production was measured by measuring the nitrite concentration. Nitrite production was significantly decreased to the cultures obtained from the animals kept under continuous light. No change in nitrite concentration was found in animals kept under continuous dark, when compared to the animals kept under natural day length. The possible role of decreased melatonin synthesis in light is suggested to decrease the nitric oxide production.
Key words: nitric oxide/ photoperiod/ snake/ splenic macrophage
Evidence that photoperiod can regulate important immune cell functions in mammals led us to study the photoperiodic influence on nitric oxide production by splenic macrophages in fresh-water snake Natrix piscator. Spleen is a secondary lymphoid organ having meshwork of reticular cells and rich supply of macrophages, and it is supposed to serve mainly as a filter for antigenic particles, parasitized blood cells and immune complexes in blood and subsequent phagocytosis. Splenectomy suppresses the humoral response totally or partially, demonstrating the functional importance of the spleen in reptiles (Kanakambika and
Muthnukkaruppan, 1972; Hussein et al., 1979). Nitric Oxide (NO), produced endogenously from L-Arginine by nitric oxide synthetases, plays an important role in many physiological processes including vascular regulation, immune responses, and neural communication. NO is extremely unstable and undergoes repaid oxidative degradation to nitrite (NO2") and nitrate (NO3"), which can be spectrophotometrically determined. The cell-mediated innate immune responses in reptiles has been addressed in literature, with reference to phagocytosis and cytotoxic response of splenic macrophages (Mondal and Rai, 1999a, b,
2GG1, 2GG2a, b), mixed leucocyte reaction and lymphocyte proliferation (Farag and El Ridi, 1985, 1986; Munoz et al., 2GGG; Cray et al., 2GG1; Work et al., 2GG1; Munoz and Fuente 2GG3; Burnham et al., 2GG5; Keller et al., 2GG5, 2GG6). Some reports are also available on seasonal variation in cell-mediated innate immune responses in reptiles (Garcia and Fuente, 1991; Zapata et al., 1992; Munoz et al., 2GGG; Munoz and Fuente, 2GG1). Study regarding photoperiodic manipulation and nitric oxide production is lacking in fresh-water snake, hence, present study was undertaken to study the effect of photoperiodic manipulation on nitric oxide production by splenic macrophages in this species.
MATERIALS AND METHODS
Animals
Male fresh-water snakes, weighing 8G-12Gg, were obtained from a local supplier who collected these animals in the suburbs of Varanasi (28° 18’N; 83° 1’E). Animals were brought to the
unconditioned laboratory. Animals were housed in vivarium (wood and wire net cages; size 5Gx3Gx3G cm). Each cage had an earthen bowl (4L capacity) filled with water and accommodate 4-5 snakes. Snakes were fed on small fishes once a week. Cages were cleaned, and bowl water was changed next day following feeding. The guideline of the committee for the purpose of control and supervision of experiment on animals (CPCSEA), Ministry of Statistics & Programme Implementation, Government of India, were followed in maintenance and sacrifice of animals. Experiment
Animals were divided into three groups: Group one animals were maintained in natural light dark cycle (1GL:14D) and served as control, group two animals, in continuous light (24L), and group three animals, in continuous dark (24D) for 3G days. Animals were sacrificed and spleen was isolated
aseptically. Spleen was macerated through a nylon strainer of pore size <100 ^m into complete culture medium (2 ml per spleen) to get single cell suspension under a sterile laminar flow hood. Spleno-somatic index (SSI: spleen weight per 100 g body weight) was calculated. Spleen cellularity (number of cells mg-1 tissue) was determined with help of hemocytometer and light microscope.
Nitrite Assay
100 ^l of spleen cell suspension or standard KNO3 was added to 96 well culture plate in triplicate and incubated for two hours at 25 0C. After incubation, adhered macrophages were washed twice with phosphate buffered saline and 100 ^l freshly prepared culture medium was added in each well. Plate was again incubated for 24 hours in CO2 atmosphere at 25 0C. Plate was centrifuged at 200g for 10 minutes. 50^l of supernatant and 50 ^l of Griess Reagent (25 ^l of 1% sulphanilamide prepared in 3 N HCl and 25 ^l of 0.1% N-naphthyl ethylenediamine prepared in distilled water) was mixed in microplate and after 10 minutes absorbance was measured at 540 nm with the help of ELISA plate reader (Thermo).
Statistical analysis
Nitrite concentration (^M) was calculated for each sample. Data are presented as mean ± SEM. Means were compared, and statistical difference between means was determined by Student’s t-test. RESULTS
Spleno-somatic index was increased in the animals kept under continuous dark, though insignificantly. Spleen cellularity was significantly increased in the animals kept under 24 hour dark. Surprisingly cellularity was also increased in the animals kept in continuous light (Fig. 1 and 2). Nitric oxide production, as measured by nitrite concentration, was significantly decreased (p<0.05) to the cultures obtained from the animals kept under
continuous light. There was no significant difference in nitric oxide production by macrophages obtained from animals kept under
continuous dark, when compared to animals kept under natural day length (Fig. 3).
Figure 1. Effect of photoperiod on Spleno-somatic index in the fresh-water snake, Natrix piscator. (LD - Natural daylength; DD - Complete dark; LL - Complete light).
Figure 2. Effect of photoperiod on spleen cellularity in the fresh water-snake, Natrix piscator. (LD Natural daylength; DD - Complete dark; LL - Complete light) (* p < 0.05).
Figure 3 Effect of photoperiod on nitric oxide production by splenic macrophages in the fresh-water snake, Natrix piscator. (LD - Natural daylength; DD - Complete dark; LL - Complete light) (* p < 0.05).
DISCUSSION
Macrophages play a critical role in the induction and expression of many innate and acquired immune responses and provide a front-line host defense against invading microbes and newly formed tumor cells (Adams and Hamilton, 1984). Their primary functions are destruction of pathogens either directly by phagocytosis or indirectly by secretion of a variety of non-specific cytotoxic substances such as reactive nitrogen/oxygen intermediates (RNI/ROI) and cytokines, TNF-a and IL-1. Macrophages also play an important role in controlling the proliferation of intracellular micro-organisms. This function is important during the onset of the immune response, when macrophages, alone or in collaboration with natural killer (NK) cells, are able to inhibit the proliferation of and can kill pathogenic intracellular micro-organisms at an early stage of the infection (Bancroft et al., 1991). Reactive nitrogen intermediates (RNI), produced by macrophages and other cell types in response to IFN-y, or IFN-y plus tumour necrosis factor-a (TNF-a) and interleukin-1
(IL-1), have been shown to play an important role in killing of pathogens (Flesch and Kaufmann, 1991). In the present study splenosomatic index was insignificantly higher in animals kept under continuous dark but spleen cellularity was increased in both groups kept either under dark or light. The change in spleen mass and cellularity is possibly due to changes in melatonin secretion by pineal gland because melatonin has been suggested to have been immunomudulatory roles (Pelham et al., 1972; Pang and Ralph, 1975). Nitric oxide production was significantly reduced in the animals kept under continuous light which might be result of reduced synthesis of melatonin in light. In summary, the result of the present study shows that continuous light exposure to fresh-water snake reduces nitric oxide production by splenic macrophages. ACKNOWLEDGEMENT
Financial grant (SR/SO/AS-15/2005) from DST, New Delhi to RS is gratefully acknowledged. MKT is indebted to CSIR, New Delhi for award of JRF.
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