INTRODUCTION:

In mammals, the suprachiasmatic nuclei (SCN) in the anterior hypothalamus is the master circadian clock that drives the daily regulation (24 h) of physiological, biochemical, endocrine rhythms [1], it also control circadian rhythms in other parts of the brain, such as the cerebral cortex, pineal gland, and in peripheral tissues such as liver, kidney and heart. The SCN consists of different neuroactive substances such as acetyl choline, glutamate, gamma amino butyric acid (GABA), aspartate, neuropeptide Y (NPY), serotonin (5-hydroxy tryptamine or 5-HT), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), arginine vasopressin (AVP), somastostatin (SOM) and gastrin releasing peptide (GRP) that play an important role in SCN function [2].

Light is the principal Zeitgeber for the generation of rhythms; photic information being relayed to the SCN from the retina via the retino hypothalamic tract (RHT) and indirectly from the intergeniculate leaflet (IGL) thalamus via the geniculohypothalamic tract (GHT) [3]. Light stimulation of the retina results in direct secretion of glutamate from the RHT into the ventral VIP-containing part of the SCN. Continuous light (LL) can cause the formation of reactive radicals [4], photo oxidation and stress mediated lipid peroxidation [5].

LL causes the release of glutamate, which initiates a signal transduction cascade in SCN neurons that ultimately result in a phase shift of the circadian system [6, 7].

Monosodium glutamate is the sodium salt of aminoacid, glutamic acid. It has another name ajinomoto. Glutamic acid is one of the most abundant amino acids exists both as free glutamate and bound with other amino acids into protein [8]. L-glutamic acid, produced by fermentation on agricultural substrates containing sugars, and its salts, mostly the ‘L’ form of monosodium glutamate (MSG), are now commonly used as palatability enhancers, both in processed foods and private home cooking, in Western as well as Eastern countries [9]. Glutamate is absorbed from the gut by an active transport system specific for amino acids. Glutamic acid in dietary protein is digested to free amino acids and small peptides, both of which are absorbed into mucosal cells where peptides are hydrolysed to free amino acids and some of the glutamate is metabolized. Excess glutamate appears in the portal blood [10], induces alterations in the concentration of intracellular ions, especially Ca²⁺Glutamate-mediated pathological increases in intracellular Ca²⁺ are thought to be mediated by either (i) prolonged activation of glutamate receptors, or by (ii) injury induced alterations in receptor functioning leading to increased Ca²⁺ influx. Ca²⁺ overload can trigger many downstream neurotoxic cascades, including the uncoupling mitochondrial electron transfer from ATP synthesis, the activation and overstimulation of enzymes such as calpains and other proteases, protein kinases, nitric oxide synthase, calcineurins and endonucleases [11]. Alterations in activity of these enzymes can lead to increased production of toxic reactive oxygen species (ROS) such as nitric oxide, activation of genetic signals leading to cell death (apoptosis) and mitochondrial dysfunction [12]. The main objective of the present study is to investigate the influence of MSG exposure on the characteristics (acrophase, amplitude and mesor) of circadian rhythm of biochemical variables and histopathological changes under LL condition.

MATERIALS AND METHODS:

Animals

Adult male Wistar rats (180-200g) were obtained from Central Animal House, Faculty of Medicine, Annamalai University. The rats were housed in polypropylene cages at room temperature (30 ± 2^○C) under semi-natural conditions. Animals were maintained in natural light-dark cycles (12:12h) in an experimental room.All animals were fed with standard pellet diet (Hindustan Lever Ltd., Bangalore, India) and water was available ad libitum. Food and water were replenished daily. The experimental protocol was approved by the Committee for Research and Animal Ethics, Annamalai University (Vide no: 587/2008) and were in accordance with the guidelines of the National Institute of Nutrition (NIN), Indian Council of Medical Research (ICMR), Hyderabad, India.

Experimental design

The animals were randomized and divided into four groups (n = 6 in each group). MSG (50mg/kg) [13] was injected subcutaneously to group III and group IV rats once in a day for 60 days.

Group– 1 Control (Light Dark (LD) 12:12).

Group– II Constant light (LL) condition.

Group–III MSG administration

(50mg/kg, subcutaneously).

Group–IV LL+MSG administration

(50mg/kg, subcutaneously).

Temporal biochemical determinations

After the experimental period, blood samples were collected from all the groups at every four hour intervals (00:00, 04:00, 08:00, 12:00, 16:00, 20:00 and 24:00h) throughout the 24 h period continuously. Minimal amount of blood (0.75 ml) was collected from the orbital sinus with great care using heparinized tubes. Levels of nitric oxide [14] and Vitamin- C [15] were estimated in plasma activities at the above mentioned time intervals.

The values of the variables (mean ± SD) were plotted versus the time of blood collection. Measurements of acrophase (f - measure of peak time of the variable studied), amplitude (A- corresponds to half the total rhythmic variability in a cycle), mesor (M- rhythm adjusted mean) and ‘r’ values were calculated by cosinor analysis using “cosinorwin” computer software program [16].

Yti = M + A Cos (ωt - f)

Where, Yti – Cosine function at the time point, M – Mesor, A – Amplitude, t – Time, f –Phase.

Histopathological examination

The brain tissues which were obtained from all the experimental groups were washed immediately with saline and then fixed in 10% buffered neutral formalin solution. After fixation, the tissues were processed and embedded in paraffin. Then, the tissues were sectioned and stained with hematoxylin and eosin (H & E) and examined under a high power microscope (40 x) and photomicrographs were taken.

RESULTS AND DISCUSSION:

Glutamate treated rats showed a significant increase in the body weight, where as in group II and IV rats showed a significant reduction in body weight when compared with control rats. 24 h rhythm of Vitamin- C activity revealed maximum activity in group I animals at 05:50 h and in groups ii, III, IV animals 04:30 h, 07:10 h, 06:00 h respectively. Decreased mesor and altered amplitude values were shown in groups II, III, IV rats when compared with group I rats were shown in the table 1. Antioxidants have been reported to play a significant role in the protection against lipid peroxidation [17]. Vitamin- C has been established biochemically as an antioxidant mops up free radicals produced in the body and shows the ability to scavenge superoxide, hydrogen peroxide, and hydroxyl radicals [18].

The level of nitric oxide was found to be 10:55 h in group I rats and maximum value of nitric oxide in group II, III, IV rats were shown at 01:30 h, 08:43 h and 11:50 h respectively. The amplitude and mesor values were altered significantly in groups ii, iii and IV when compared with group I rats were shown in the table 1. Constant light enhances the release of glutamate, which could result in excess nitric oxide production, inhibits the activities of antioxidant enzymes [19]. L-glutamate binds to the N-methyl-D-aspartate (NMDA) receptors [20] and thereby increases intracellular Ca²⁺ concentration [21] which activates nitric oxide synthase and produces nitric oxide [22], associated with an increase in brain injuries induced by oxygen deprivation or cerebral ischemia [23, 24]. Oxidative stress is an important mechanism for tissue injury. In our present study shows that the brain section of control rats shows normal architecture, where us the constant light and MSG treated rat shows mild cortical edema and microcyclic degeneration in brain were shown in the figure 1 (A, B and C). LL + MSG treated rats shows high necrotic changes necrosis of Purkinje cells of the cerebellum were shown in the figure 1 (D). Prolonged high doses of MSG produces neurodegeneration, neurotoxicity [25] and oxidative damage in different organs [26, 27].

Table 1. Temporal pattern of Nitric oxide and Vitamin-C in control and experimental rats.

Biochemical Variables	Characteristics of rhythm	Group I	Group II	Group III	Group IV
Biochemical Variables	Characteristics of rhythm	Control	LL	MSG	LL+MSG
Nitric oxide	Acrophase f (h)	10:55	01:30	08:43	11:50
	Amplitude (A)	0.9	1.6	1.8	2.1
	Mesor M (mg/dl)	2.8	3.1	3.4	3.9
	r-value	0.65 dr (p < 0.02)	0.55 dr (p < 0.05)	-0.29ns(p < 0.50)	0.30ns (p < 0.50)
Vitamin-C	Acrophase f (h)	05:50	04:30	07:10	06:00
	Amplitude (A)	0.8	0.5	0.4	0.3
	Mesor M (mg/dl)	1.7	1.3	1.4	0.6
	r-value	0.60 dr (p < 0.04)	0.50dr (p < 0.50)	-0.55ns(p < 0.05)	-0.80ns (p < 0.50)

dr- detectable rhythmicity; ns- no significant rhythmicity

Figure 1. Histopathological changes in brain. (A) Control rats shows normal architecture (H&E × 40). (B) LL exposed rats and (C) MSG treated rats shows mild cortical edema and microcyclic degeneration (H&E × 40). (D) LL + MSG treated rats shows high necrotic changes necrosis of Purkinje cells of the cerebellum (H&E × 40).

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Received on 11.12.2013 Accepted on 22.01.2014

Asian J. Res. Pharm. Sci. 4(1): Jan.-Mar. 2014; Page 01-04