Study Animals

Sexual dimorphism is reported in innate immune responses of lizards (Mondal and Rai, 1999) and turtles (Keller et al., 2005); therefore, we used only male individuals in this study. Freshwater snakes, weighing 80–120 g, were obtained from a local supplier who collected these animals in the suburbs of Varanasi (28°18′N; 83°1′E), India during February and March (max. temp. 27–32°C, min. temp.14–18°C; photoperiod 1100–1200 h; relative humidity 55–68%), the reproductively inactive period (Haldar and Pandey, 1989). Animals were brought to the unconditioned laboratory and housed in vivaria (wood and wire net cages; size 50 × 30 × 30 cm). Each cage had a wooden floor and frame with wire net sides, one side being a window. Each cage had an earthen bowl (4-L capacity) filled with water to accommodate 4–5 snakes. Snakes were fed on small fishes once a week. Cages were regularly cleaned, and bowl water was changed the next day following feeding. Animals were acclimated to the laboratory conditions for two weeks prior to experimentation. The guidelines of the committee for the purpose of control and supervision of experiment on animals (CPCSEA), Ministry of Statistics and Programme Implementation, Government of India, were followed in maintenance and sacrifice of animals.

Chemicals

Tetrazolium dye, NBT (Nitroblue Tetrazolium), and MTT (3-[4, 5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide), mitogens (concanavalin A [Con A], phytohemaglutinin [PHA], and lipopolysaccharide [LPS]), and melatonin were purchased from Sigma Chemicals. Culture medium (RPMI-1640), lymphocyte separation medium (HiSep), L-glutamine, gentamycin, fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and other chemicals were purchased from Himedia Laboratories Pvt. Ltd. (India). The culture medium was supplemented with 1 μL/ml gentamycin, 10 μL/ml of 200 mM L-glutamine, 10 μL/ml anti-anti (Gibco), and 5% FBS and referred to as complete culture medium.

Experimental Procedure

Captivity lasted 2 weeks before experimentation to reverse the capture-related stress and to acclimate the animal to the laboratory conditions. Following acclimation, snakes were divided into 6 groups. Animals of groups 1 and 2 received intraperitoneal (i.p.) injection of melatonin (5 μg/g body weight) daily; groups 3 and 4 received i.p. injection of melatonin (10 μg/g body weight) daily; and groups 5 and 6 served as control received i.p. injection of vehicle saline daily. Stock solution of melatonin was prepared by dissolving it first in a few drops of ethyl alcohol and then diluting with phosphate buffered saline (PBS). The dose of injection chosen was based on earlier observations in fish, birds, and mammals (Brennan et al., 2002; Hriscu 2004; Cuesta et al., 2008). Champney et al. (1998) have reported time dependent effects of injected melatonin; evening injections of melatonin were reported more effective than morning injections. Hence, in this study, all injections were given during evening hours between 1600 and 1700.

On the day after expiry of the treatment, as stated above, blood was isolated through cardiocentesis in heparinized tubes using 30 G-needle. Only large adult males (>100 g, N = 5) were blood-sampled to avoid possible detrimental effects of the procedure on smaller individuals. Blood was kept at 4°C and soon after used to study total leukocyte count (TLC), differential leukocyte count (DLC), leukocyte phagocytosis, nitroblue tetrazolium (NBT) slide assay, quantitative NBT reduction assay, nitrite assay, and blood lymphocyte proliferation.

Total Leukocyte Count (TLC)

For TLC, 20 μL of blood was diluted 20 times with Turk's fluid (0.2% gentian violet solution in 3% acetic acid). The diluted blood was applied on Haemocytometer Neubauer counting chamber (ROHEM, India), cells from the four chambers of a square millimeter were counted under the microscope, and number of leukocytes/mm³ was determined.

Differential Leukocyte Count (DLC)

For DLC, a uniform blood film was smeared on a clean glass slide. The blood smear was air dried and stained in a mixture of Giemsa and Leishman stain. After washing under tap water, slides were dried, dehydrated, cleared in xylene, and mounted in DPX. DLC established the relative percentage frequency of each cell type. Stained slides were observed under an oil immersion objective from the upper edge of the smear to the extreme lower edge. One hundred leukocytes were identified and counted. Once the relative percentage frequency of each type of cells was obtained, their number was calculated in mm³ blood from total leukocyte count.

Phagocytic Assay

For phagocytic assay, the yeast cells were used as target cell. The yeast cell suspension was prepared by mixing 20 mg of commercial baker's yeast (Saccharomyces cerevisae) in 10 mL of 0.2 M PBS. The suspension was kept at 80°C for 15 min. The cells were washed three times in PBS and finally suspended in culture medium to get a concentration of 1 × 10⁸ cells/mL. Equal amounts of blood and yeast cells were mixed and incubated for 30 min and 60 min. After incubation, a thin smear was made on a clean glass slide, air dried, fixed in methanol, stained with Giemsa, and examined under oil immersion. For each slide, a total of 100 phagocytes were examined haphazardly without any predetermined sequence. The phagocytic index was determined by calculating the average number of yeast cells engulfed by a single phagocyte. The percent phagocytosis was calculated by dividing the number of phagocytes showing phagocytosis by 100.

NBT Slide Assay

NBT solution (0.1%) was prepared in PBS (pH 7.2) and stirred at room temperature for 1 h. The solution was stored at 4°C. For the assay, 20μL of blood was mixed with 50 μL of NBT solution. The mixture was applied on clean cover glass and incubated for 1 h at room temperature in a moist chamber. Following incubation, the cover glass was washed with PBS and mounted on a slide with PBS. NBT reduction results in formation of blue formazan deposits in leukocytes. The number of blue cells was scored haphazardly in 20 optic fields at 4 × 10 times magnification.

Isolation of Leukocytes

Peripheral blood leukocytes (PBL) were collected from the buffy coat (the layer of PBLs between the plasma and RBCs) using a slow spin technique as described by Keller et al. (2005). The tubes were centrifuged at 42 × g for 25 min at 8°C. The PBLs were collected by gently swirling the buffy coat into the plasma and transferring the cells in a new tube. Following centrifugation at 200 × g for 10 min, the plasma was removed and the cell pellet was gently resuspended in 1 mL of complete culture medium.

Quantitative NBT Reduction Test

NBT assay was performed following the methods of Berger and Slapnickova (2003). Leukocytes were counted and adjusted to 2 × 10⁶ cells/mL in complete RPMI. Cell viability was checked through trypan blue exclusion test, which exceeded 95%. Fifty microliters leukocytes (1 × 10⁵ cells) were mixed with 50 μL of RPMI containing NBT (1 mg/mL) in 96 well culture plates in triplicate from each animal. One well with culture medium and without cells served as a blank. Plates were then incubated in CO₂ atmosphere at 25°C for 2 h, centrifuged at 700 × g, washed with PBS and fixed in 70% methanol. Twenty microliters of 0.1% triton X-100 was mixed in each well. The formazan crystals, present inside the cells, were dissolved by mixing 120 μL KOH (2 M) and 140 μL DMSO in each well. Optical density was measured at 620 nm with the help of ELISA plate reader (Thermo Multiscan).

Nitrite Assay

Nitric oxide (NO) is a major effector molecule of cellular immune response. It is a highly unstable compound produced from L-arginine by the enzyme nitric oxide synthase (NOS). Soon after production, nitric oxide decomposes to other nitrogen oxides such as nitrate (NO₃^–), and nitrite (NO₂^–), popularly known as reactive nitrogen intermediate (RNI) (Jorens et al., 1995). Therefore, nitrite was assayed as a marker of cytotoxicity. Nitrite content was measured by the method of Ding et al. (1988). Briefly, 100 μL of leukocytes (1 × 10⁶ cells mL⁻¹) was added to each well of a 96 well culture plate. The plate was incubated in CO₂ atmosphere at 25°C for 24 h, then centrifuged at 200 × g, and the supernatant collected. Equal volumes of supernatant and Griess reagent (1% sulfanilamide in 3N HCl and 0.1% naphthylenediamine dihydrochloride in distilled water) were mixed, and optical density of the solution was measured at 540 nm with an ELISA plate reader (Thermo Multiscan). Culture medium alone without any cells served as a blank. All the samples were taken in triplicate from each experimental animal. Following blank subtraction, the mean OD values for each set of triplicates were used in statistical analysis.

Lymphocyte Proliferation Assay

Blood lymphocytes were isolated by density gradient centrifugation using HiSep (Density 1.077 g/mL). Whole blood (1 mL) was overlaid on 1 mL HiSep in RIA vials and centrifuged at 400 × g for 30 min with the brakes off at 8°C. Using a clean glass Pasteur pipette, the lymphocytes layer (at the interface between medium and HiSep), was carefully aspirated, washed three times with PBS, counted and assessed for viability on a haemocytometer by trypan blue exclusion. Viable cells (> 95%) were adjusted to 2 × 10⁶ cells/mL with culture medium.

The lymphocyte proliferation was assessed using colorimetric assay based on tetrazolium salt (MTT) following the methods of Berridge et al. (2005). The colorimetric method, using tetrazolium salts, has been an advantageous alternative method measuring lymphoproliferation (Mosmann, 1983). Basal as well as mitogen-induced lymphocyte proliferation was assessed in vitro. Stock solution (1 mg/mL) of mitogens was made in a 0.2M PBS and further diluted with culture medium. Lymphocytes, isolated from control as well as melatonin-injected animals, were seeded at 1 × 10⁵ lymphocytes in 50 μL of medium/well in a flat-bottom 96 well culture plate. Fifty microliters of mitogens was added: Con A at final concentration of 5, 10, and 20 μg/mL; LPS at final concentration of 10 and 20 μg/mL; PHA at final concentration of 5 and 10 μg/mL in the well. One hundred microliters of culture medium was added to make a final volume of 200 μL/well. Fifty microliter cell suspensions, with 150 μL of mitogen-free culture medium per well, represented unstimulated basal or spontaneous proliferation. Additional wells containing 200 μL of culture medium served as a blank. All assays were made in triplicates from each experimental animal. Following incubation in humidified CO₂ atmosphere at 25°C for 48 h, 20 μL of MTT reagent (5 mg/mL) was added to each incubation well, and plates were again incubated overnight. Tetrazolium salts are reduced into a colored formazan product in mitochondria of metabolically active cells. The quantity of formazan product as measured by the amount of absorbance at 570 nm light is directly proportional to the number of living cells in culture (Cory et al., 1991). After incubation, the supernatant was aspirated, and the reaction product blue formazan was dissolved in 100 μL of DMSO. Absorbance was measured at 570 nm in an ELISA plate reader. Following blank subtraction, mean OD values for each set of triplicates were used in statistical analysis. Stimulation index (SI) was calculated (SI = OD of stimulated culture/OD of unstimulated culture).

Effect of In Vitro Melatonin on Lymphocyte Proliferation

Lymphocytes (50 μL) from in vivo melatonin-injected snakes were seeded separately with mitogens Con A, LPS, and PHA (50 μL) as described above. Fifty microliters of different concentrations of melatonin (100, 200, and 500 pg/mL; final concentration) and 50 μL of culture medium were added to cultures to make a final 200-μL volume. Both mitogens and melatonin were added to the plates before the addition of cells. The concentrations of melatonin added were selected based on earlier reports in mammals (Kriegsfeld et al., 2001) and birds (Kliger et al., 1999). Control cultures contained only lymphocytes and mitogens. Treated cultures contained lymphocytes, mitogens, and melatonin. In addition, cultures with no melatonin and no mitogen were prepared to provide basal proliferation. All assays were made in triplicate from each experimental animal. Following blank subtraction, triplicate cultures were averaged.

Following incubation in humidified CO₂ atmosphere at 25°C for 48 h, 20 μL of MTT reagent (5 mg/mL) was added to each incubation well, and plates were again incubated overnight. After incubation, the supernatant was aspirated, and the reaction product blue formazan was dissolved in 100 μL of DMSO. The optical density (OD) of each well was determined with a microplate reader (Thermo multiscan) equipped with a 570-nm filter. The OD values from basal cultures (no mitogen and no in vitro melatonin) were subtracted from treatment OD (with mitogen and in vitro melatonin) or from control OD (with mitogen and no in vitro melatonin). The OD values for control cultures were considered to be the baseline and assigned a value of 100%. Percentage cell proliferation in melatonin-treated cultures was calculated relative to proliferation in control cultures (with mitogen and no melatonin) as below:$$\text{Percentage cell proliferation = ([treatment OD − control OD] / control OD) × 100 + 100.}$$

Statistical Analysis

Data are presented as mean ± SE. Data were analyzed by nonparametric Mann–Whitney test and ANOVA followed by the post-hoc test, Newman-Keul's multiple range test (α = 0.05).

Effect of In Vivo Melatonin

Compared to the respective control, total leukocyte count was increased insignificantly in animals receiving 5 μg/g body weight melatonin for 10 days (Fig. 1). Concerning differential counts, no consistent change in heterophil count was observed in animals receiving in vivo melatonin; however, lymphocytes, monocytes, and eosinophils had a tendency toward an increase in animals receiving in vivo melatonin. Monocytes and eosinophils significantly increased in animals receiving melatonin 10 μg/g body weight for 20 days. Number of basophils significantly increased in animals receiving melatonin 10 μg/g body weight for 10 or 20 days (Fig. 1). In NBT slide assay, number of blue cells (NBT positive cells) was increased in the animals treated with 10 μg/g body weight dose of melatonin for 10 days and 20 days, as compared to control animals, but 5 μg/g body weight dose of melatonin had no effect. After completing the quantitative NBT test, however, we found that both doses of melatonin had significant stimulatory effects on the NBT reduction at 10th and 20th days. The effect was more pronounced in leukocytes obtained from snakes treated with melatonin 5 μg/g body weight for 10 and 20 days (Fig. 2). Percent phagocytosis decreased significantly in snakes administered with melatonin 5 and 10 μg/g body weight for 10 days; however, the effect was not significant in snakes receiving 10 μg/g body weight melatonin for 20 days. No consistent change was obtained in phagocytic index (Fig. 2). A highly significant increase in nitrite release was observed in cultures obtained from snakes receiving both doses of in vivo melatonin, as compared to the respective control. This effect of melatonin was concentration dependent. Higher doses of 10 μg/g caused more profound increases in nitrite release as compared with 5 μg/g dose of melatonin on the 10th day (Fig. 2). Melatonin administration (in vivo) significantly enhanced the Con A–induced lymphocyte proliferation in animals treated with either dose of melatonin for 10 days, whether the lymphocytes in culture were stimulated by 5 or 10 or 20 μg mL⁻¹ of Con A. The average proliferative response of lymphocytes obtained from animals injected with 5 μg/g melatonin for 20 days, however, was unaffected when lymphocytes were stimulated with any Con A concentration, but lymphocyte cultures obtained from animals injected with 10 μg/g melatonin for 20 days showed an increased proliferative response (Fig. 3). PHA-induced proliferative response of lymphocyte cultures obtained from animals injected with 5 or 10 μg/g melatonin for 10 days was significantly enhanced at both concentrations of mitogen, as compared to the respective control. There was no effect of 5 μg/g in vivo melatonin injected for 20 days (Fig. 3). We found differential effects of in vivo melatonin administration on LPS-induced proliferative response where LPS-induced proliferative response increased significantly in lymphocyte cultures obtained from animals injected with 5 μg/g melatonin for 10 days but not in lymphocyte cultures obtained from animals injected with 5 μg/g melatonin for 20 days. Melatonin at the dose of 10 μg/g caused significant proliferation when treated for 20 days (Fig. 3).

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Effect of In Vitro Melatonin

Effects of in vitro melatonin on proliferative response is presented as percentage cell proliferation. When the overall effect of in vitro melatonin is reported, the percentage cell proliferation for both doses of in vivo melatonin is combined. Melatonin in vitro (100, 200, and 500 pg/mL) invariably had significant stimulatory effects on mitogen-induced proliferation of lymphocytes obtained from snakes treated with in vivo melatonin for 10 and 20 days except that in vitro melatonin had no significant stimulatory effect on PHA-induced (10 μg/mL)-induced proliferation of lymphocytes obtained from snakes treated with in vivo melatonin for 20 days (Fig. 4).

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