L-ARGININE ACCENTUATES SICKNESS BEHAVIOR INDUCED BY LIPOPOLYSACCHARIDES IN MALE ALBINO RATS

Assiut University web-site: www.aun.edu.eg

L-ARGININE ACCENTUATES SICKNESS BEHAVIOR INDUCED BY LIPOPOLYSACCHARIDES IN MALE ALBINO RATS

HAMDY M. EMBARK

Animal Physiology Department, Faculty of Vet. Medicine, South Valley University, Qena 83523, Egypt

Received: 31 December 2016;       Accepted: 31January 2017

INTRODUCTION

The term "sickness behaviour" (abnormal illness behavior) is an organized and systematic strategy of the organism(s) to fight off the infection (Dantzer et al., 2001). This strategy is often triggered by the proinflammatory cytokines that are produced by activated neutrophils and monocytes/ macrophages in contact with invading microbes (Hart, 1988; Kent et al., 1992). These cytokines include mainly interleukin 1 (IL-1α and IL-1β), interleukin 1 (IL-6) and tumour necrosis factor (TNF) (formerly known as TNF-alpha or cachectin) (Larson and Dunn, 2001).

Increased proinflammatory cytokines cause a coordinated set of adaptive psychological changes such   as   depression   and   anhedonia,   which    are

Corresponding author: HAMDY M. EMBARK

E-mail address: h.embark@vet.svu.edu.eg; atutohm@yahoo.com

Present address: Animal Physiology Department, Faculty of Vet. Medicine, South Valley University, Qena 83523, Egypt.

recognized as emotional aspects of sickness behavior, in patients with infection and cancer (Dantzer et al., 2008; DellaGioia and Hannestad, 2010). Behavioral effects have been well-studied in animal models of inflammation induced by the bacterial endotoxin, lipopolysaccharide (LPS) (Frenois et al., 2007; de Paiva et al., 2010).

LPS is the major component of the outer membrane of Gram-negative bacteria, contributing greatly to innate immune responses from normal animal immune systems (Miyake, 2003; Kirsten et al., 2013). LPS is considered a potent sickness behavior and proinflammatory cytokine-inducer (Bluthe et al., 1994; Aubert, 1999).

LPS-induced behavioral changes can be divided into transient changes such as decreases in locomotor activity and food intake, and persistent changes such as depressive-like behavior and exploratory behavior deficit (Frenois et al., 2007; Haba et al, 2012).

L-arginine (L-Arg) is an important natural amino acid in the body that is used in nutritional supplements (Hedin, 1895; Wu and Morris; 1998 and Gad, 2010). The kidney plays a major role in endogenous L-Arg synthesis (Peters et al., 1999; Osowska et al., 2004), where L-Arg is formed from L-citrulline produced by enterocytes (Dhanakoti et al., 1990). The liver is also capable of synthesizing certain amounts of L-Arg (Watford, 1991).

L-Arg is the only physiologically significant substrate for the biosynthesis of nitric oxide (NO) (Hecker et al., 1990). NO generated enzymatically from L-Arg by nitric oxide synthase (NOS) (Wood and Garthwaite, 1994). NO is produced by all tissues of the body and plays important roles in a broad range of physiological processes including cardiovascular homoeostasis, neurotransmission, vasorelaxation, macrophage cytotoxicity and immunity (Nahrevanian, 2009).

The discovery of NO as a neurotransmitter in the brain raised the issue of its role in the function of the central nervous system (CNS) (Garthwaite et al., 1988, Knowles et al., 1989; Snyder and Bredt, 1991). The release of NO strictly bound with the stimulation of N-methyl-D-aspartate (NMDA), a subtype of excitatory amino acid receptor, suggested an important role in psychomotor behavior (Wolf, 1998).

Several studies in recent years have investigated the versatile multiplex action of NO Using the non-selective NOS inhibitor L-N^G–nitro-L-arginine methyl ester (L-NAME) which exhibit antidepressant-like properties in the forced swimming test (Harkin et al., 1999). Several human and experimental animal studies have indicated that NO could be involved in the modulation of depressive behaviors (Peng et al., 2012).

In an attempt to understand the role of the NO in the modulation of depressive behaviors in rats, the present study aimed to investigate the effects of      L-Arg on modulation of depressive behaviors in LPS-treated rats using open field test.

MATERIALS AND METHODS

Animals

Adult male albino rats were originally obtained from animal house, Faculty of Medicine, Assiut University, Assiut, Egypt, and were maintained in the Animal Care Facility at the Faculty of Veterinary Medicine, South Valley University. The animals were housed in cages (55 x 40 x 20cm), under standard laboratory conditions (22°C temperature, 60 ± 5% humidity, in a 12 h light/dark cycle), with food and water ad libitum. Environmental factors (such as cage type and size, colony size, bedding, and environmental enrichment) were standardized between cages. They were kept for two weeks under this condition to adapt the laboratory conditions before the start of the experiment.

Drugs and chemicals

LPS (from Escherichia coli 0111:B4) and L-Arg were purchased from Sigma chemical company (Aldrich, USA). All drugs were dissolved in saline 0.9% NaCl. and administered i.p. in a volume of 0.1 ml/100 g body weight (b.wt). Drugs were administered three hours prior to testing with the exception of L-Arg which, was administered 7 days prior to testing day.

Experimental Design

The experiment was carried out on twenty adult male albino rats (200-250 g) distributed in 4 groups of 5 animals each treated intraperitoneally as follows:

Group I (Control): saline solution 0.1 ml/100 g b.wt.

Group II (LPS): LPS 1 mg/kg b.wt.

Group III (L-arginine): rats pretreated with L-Arg (10 mg/kg b.wt) for 7 days before saline injection.

Group IV (LPS+L-arginine): LPS-treated rats pretreated with L-Arg (10 mg/kg b.wt) for 7 days before LPS injection.

Behavioral studies

Measurement of locomotor activity was performed as previously described (Prut and Belzung, 2003; Araki et al., 2015). Briefly, Locomotor and behavioral activity were assessed using an infrared LE8811 Actimeter system (Panlab, Barcelona, Spain). The apparatus consisted of a 45 cm (width) x 45 cm (depth) arena of black plexiglass enclosed with four clear acrylic walls (35 cm in height) (Fig. 1). A square frame mounted outside of the arena created a 16 x 16 grid of intersecting infrared beams used to track the horizontal movement of each mouse in real-time. A second frame placed above the lower frame was used to track the vertical movement of each rat, set to detect hind-leg rearing. Locomotor measures were recorded using ActiTrack software (Panlab), which uses infrared beam data to calculate parameters such as overall locomotor activity (total number of beam breaks), distance traveled, and number of rears. The recorded data are easily exported in a format compatible with the Excel program for Windows, through a SeDaCom electronic software.

In the 8^th day, three hours after injection of the saline/LPS, testing was conducted during the light phase between 07:30 h and 12:30 h for all rats. Rats were brought into the testing room in their home cage a half hour prior to testing. Each rat was then placed in the corner of the arena and allowed to freely explore it for 5 min. During testing, the lighting of the arena was 100 lx so as not to inhibit normal exploratory behaviour. At the end of testing, the rat was removed and returned to its home cage and the surface and walls of the arena were wiped clean with 30% isopropanol.

Fig. 1: Infrared LE8811 Actimeter (Panlab) apparatus for motor activity tracking in rats. Lower frame is used to track horizontal movements, while the upper frame tracks vertical movements (rearing).

Statistics

Statistical analysis was carried out using the Origin program for Windows version 6.0 for IBM and compatible computers. Statistical comparisons were made by student t test. The obtained data were expressed as mean ± standard error. P-values less than 0.05 were considered statistically significant and P-values less than 0.001 were considered statistically highly significant.

RESULTS

The effects of L-Arg pretreatment on the spontaneous total activity (total motor activity) in the LPS-treated rats

The treatment with LPS in a dose of 1 mg/kg b.wt did not significantly influenced the spontaneous total activity (216.60±127.86), compared to the control (saline) group (348.00±199.30) in a novel open-field test (Fig. 2).

Intraperitoneal administration of L-Arg 10 mg/kg b.wt was associated with an increase of the total motor activity (524.20±64.08), but statistically not significant, compared to the group treated with saline (348.00±199.30), in this behavioral experimental model in rats (Fig. 2).

The administration of L-Arg was associated with an important (p<0.001) increase of rat spontaneous total activity (524.20±64.08), compared to LPS+L-arginine group (105.20±44.36) in this experimental behavioral model in rats (Fig. 2).

Fig. 2: Spontaneous total activity of rats in a novel cage environment. Data shown represent the mean (± SEM) infrared beam breaks by control and treated rats 3 hours after saline/LPS injection (n= 5 rats per group). *, p < 0.001; L-arginine vs LPS+L-arginine.

The effects of L-Arg pretreatment on the number of horizontal movements (locomotor activity) in the LPS-treated rats

The use of LPS did not notably modify the number of horizontal movements (locomotor activity) (200.40±118.06) compared to the saline group (321.20±182.03) (Fig. 3).

Also, the use of L-Arg did not notably modify the number of horizontal movements (locomotor activity) (492.80±60.93) compared to the saline group (321.20±182.03) (Fig. 3).

The administration of L-Arg was associated with an important (p<0.001) increase of rat horizontal movements (locomotor activity) (492.80±60.93), compared to LPS+L-arginine group (98.60±42) in this experimental behavioral model in rats (Fig. 3).

Fig. 3: Horizontal movements (locomotor activity) of rats in a novel cage environment. Data shown represent the mean (± SEM) infrared beam breaks by control and treated rats 3 hours after saline/LPS injection (n= 5 rats per group). *, p < 0.001; L-arginine vs LPS+L-arginine.

The effects of L-Arg pretreatment on the number of stereotype movements in the LPS-treated rats

Regarding the number of stereotype movements, no considerable variations were observed between LPS (16.20±9.82), L-Arg (31.40±4.99) and the saline group (26.80±17.46) in this experimental behavioral model in rats (Fig. 4).

The treatment with L-Arg in LPS-treated rats (LPS+L-arginine group) was associated with a statistically significant (p<0.01) decrease of the stereotype movements (6.60±1.97), comparing with the L-arginine group (31.40±4.99), during the session of experimentation (Fig. 4).

Fig. 4: Stereotype activity of rats in a novel cage environment. Data shown represent the mean (± SEM) infrared beam breaks by control and treated rats 3 hours after saline/LPS injection (n= 5 rats per group). *, p < 0.01; L-arginine vs LPS+L-arginine.

The effects of L-Arg pretreatment on the number of vertical movements (rearing) in the LPS-treated rats

Intraperitoneal injection of LPS did not determined considerable modifications in the number of vertical movements (rearing) (10.00±7.89) comparing with the control group (8.40±6.33), in the experiment (Fig. 5).

The animals treated with L-Arg showed a significant (p<0.01) increase of the vertical movements (rearing) (9.80±2.01), compared to LPS+L-arginine group (1.40±0.75), in this behavioral test (Fig. 5).

Fig. 5: Rearing activity of rats in a novel cage environment. Data shown represent the mean (± SEM) infrared beam breaks by control and treated rats 3 hours after saline/LPS injection (n= 5 rats per group). *, p < 0.01; L-arginine vs LPS+L-arginine.

The effects of L-Arg pretreatment on the total distance travelled in the LPS-treated rats

Intraperitoneal injection of LPS did not determine considerable modifications in the total distance travelled (302.32±194.88) comparing with the control group (564.42±364.24), in the experiment (Fig. 6).

The animals treated with L-Arg showed a significant (p<0.05) increase of total distance travelled (897.74±156.46), compared to LPS group (302.32±194.88), in this behavioral test (Fig. 6).

The animals treated with L-Arg in LPS-treated rats (LPS+L-arginine group) showed a significant (p<0.01) diminution of the total distance travelled (128.46±73.50), compared to L-arginine group (897.74±156.46), in this behavioral test (Fig. 6).

Fig. 6: Total distance travelled (cm) by rats in a novel cage environment. Data shown represent the mean (± SEM) infrared beam breaks by control and treated rats 3 hours after saline/LPS injection (n= 5 rats per group). *, p < 0.01; L-arginine vs LPS+L-arginine. #, p < 0.05; L-arginine vs LPS.

The effects of L-Arg pretreatment on the mean velocity/Speed in the LPS-treated rats

Intraperitoneal injection of LPS did not determine considerable modifications in the mean velocity/Speed (1.00±0.65) comparing with the control group (1.88±1.20), in the experiment (Fig. 7).

The animals treated with L-Arg showed a significant (p<0.05) increase of the mean velocity/Speed (2.98±0.53), compared to LPS group (1.00±0.65), in this behavioral test (Fig. 7).

The animals treated with L-Arg in LPS-treated rats (LPS+L-arginine group) showed a significant (p<0.01) decrease of mean velocity/Speed (0.42±0.25), compared to L-arginine group (2.98±0.53), in this behavioral test (Fig. 7).

Fig. 7: Mean velocity/Speed of rats in a novel cage environment. Data shown represent the mean (± SEM) infrared beam breaks by control and treated rats 3 hours after saline/LPS injection (n= 5 rats per group). *, p < 0.01; L-arginine vs LPS+L-arginine. #, p < 0.05; L-arginine vs LPS.

DISCUSSION

Using an automated open field or an actimeter test, we estimated the spontaneous cage "motor" activity in rats (individual)by recording the horizontal and vertical activity (lower and upper photobeam breaks or counts, respectively), number of stereotypical movements, in a single-session of testing (Kas et al., 2008; Lynch et al., 2011).

The horizontal and vertical activities indirectly signify the animal state of fear and anxiety of the new environment, in which it was placed (Hart et al., 2010). On the other hand, the stereotype movements suggest the self-maintenance of animal personal hygiene (paw-licking activity, hair combing and nose cleaning, auto-grooming action) (Hart et al., 2010). The number of total movements represents an element that offer the information regarding the general rat behavior, during a definite interval of time, being a consistent tool for assessing the biological significance of obtained data (Lynch et al., 2011).

Previous studies indicate that intraperitoneal injection of LPS in rats induced robust sickness and depressive-like behavior in rats (Kirsten et al., 2015). In contrast, ourresults demonstrated that acute treatment with LPS produced slight sickness and depressant-like effects in the open field test in rats. These results diverge from previous studies may be due to the fact that LPS-induced sickness behavior and L-Arg antidepressant effects were dose dependant (Martin et al., 2013).

In addition, the present study seems to be the first to demonstrate that pretreatment with L-Arg alone produced non-significant increase in motor activities compared with rats treated with saline and/or LPS alone. Moreover, pretreatment with L-Arg in LPS-treated rats accentuates endotoxin-induced sickness behavior in rats. These results were surprise for us, as we suspected that L-Arg has antidepressant effect. These accentuated effects of L-Arg are likely to occur through stimulation of inflammation and neural activation in the brain to secrete different types of cytokines (Dantzer et al., 1999).

CONCLUSIONS

The present study demonstrate that NO precursor    L-Arg possesses significant depressant-like features that are LPS-dependent. Moreover, L-Arg may have a potential therapeutic value for treatment of anxiety symptoms in patients with sustained and systemic inflammation.

Conflicts of interest

The author declares that there are no conflicts of interest regarding publication of this paper.

ACKNOWLEDGEMENTS

The author gratefully acknowledges all members and professors of the department of animal physiology, faculty of veterinary medicine, south valley university, Qena, Egypt, and of animal physiology department, faculty of veterinary medicine, Aswan university, Aswan, Egypt, for their constant encouragement and kind help. This work was supported by grant No. 2505 from the Science and Technology Development Fund (STDF), Egypt.

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