Stress response modulating effects of lactic acid in mice

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1 RESEARCH ARTICLE Stress response modulating effects of lactic acid in mice Naveen Shivavedi 1, Shyam Sunder Chatterjee 2, Vikas Kumar 1 1 Neuropharmacology Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi , Uttar Pradesh, India 2 Stettiner Straße 1, D 76139, Karlsruhe, Germany Correspondence: Vikas Kumar vikas.phe@iitbhu.ac.in Received: November 12, 2014 Published online: December 01, 2014 Lactic acid is commonly found in sour milk and is often consumed with food, which plays a role in various biochemical processes and used for trauma, surgery, burn injury and as a neuroprotective. Present pilot study was designed to investigate its putative stress response modulating effects in male mice. A battery of behavioural test models viz. stress-induced hyperthermia, tail suspension test and pentobarbital-induced hypnosis was used to assess the adaptogenic activity of lactic acid. Doses of lactic acid (5, 25, 125 and 625 mg/kg) as solution (10 ml/kg) was prepared in distilled water and administered orally for 11 consecutive days. The body weight and body temperature were recorded daily as a measure of stress induced changes. Stress induced hyperthermia test was performed on 1st, 5th, 7th, and 10th day of the treatments. On 11th day, they were subjected to tail suspension test and on day 12th to pentobarbital induce hypnosis test. Stress-induced hyperthermia was reduced by lactic acid in dose dependent manner and also compensated the changed in body weight and basal rectal temperature due to daily handling and intermittent foot-shock stress. Test drug was also reduced the immobility period in tail suspension test and showed considerable effect on onset and duration of sleep in pentobarbital-induce hypnosis test when compared to control, the significant (p<0.05) activity of lactic acid was found at dose 25 mg/kg onwards. These observations indicate that lactic acid has potential for adaptogenic activity, suggesting its use against varied spectrums of psychopathologies associated with stress. Keywords: Foot-shock stress; body weight, hyperthermia; rectal temperature; immobility; pentobarbitone induced sleep To cite this article: Naveen Shivavedi, et al. Stress response modulating effects of lactic acid in mice. Ther Targets Neurol Dis 2014; 1: e418. doi: /ttnd.418. Introduction Lactate has long been regarded as a waste product of cell metabolism. However, various researches in last two decades have revealed that lactate may be act as an energy substrate for skeletal muscle. Data that have come into view during the last couple of years have also been shown that neurons can use lactate to generate energy [1]. Knudsen et al., have been reported that, the transportation of lactate into the brain takes place via blood-brain barrier (BBB) [2]. Additionally, in brain ischemia, ph of tissue and local plasma ph change are responsible for transportation of lactate. However, in certain pathological conditions lactate possesses neuroprotective function, such as diabetes, prolonged starvation and cerebral ischemia [3-7]. Although, it is still uncertain that, the neuroprotective role of lactate is indirect (via increased cerebral blood flow caused by hyperlactemia) [8] or can serve as a direct neuronal energy substrate. The kinetics for lactate transportation through the human BBB has so far not been explained. In preclinical trials, L-lactate has been shown to cross the rodent s BBB through facilitated diffusion mechanism operated by a stereo specific monocarboxylic acid carrier [9]. This carrier allows the flowing out of lactate from the brain if there is excess, and then brain normally Page 1 of 6

2 exhibiting a small amount of lactate efflux [2, 10]. Study on metabolism of lactate has also revealed that, lactate taken orally may be used as an energy substrate [11], either by the oxidation or by conversion into glucose. Lactate clearance by oxidation, and gluconeogenesis utilizes protons and could temporarily augmented the blood bicarbonate levels which cause increase in blood ph. These characteristics revealed that lactate utilization has worked as an energy substrate and as a buffering agent. Lactate consumption has also been responsible for increase in blood ph via bicarbonate levels and increases time to exhaustion in high intensity work [12]. The brain requires continuous supply of oxygen and energy substrates involving glucose [13]. In this connection, recent researchers have been reported that lactate plays an important role in the supply of energy to the brain, especially during acute neural activation [14, 15]. According to the astrocyte-neuron lactate shuttle hypothesis [16-18], astrocytes supply lactate to neuron to facilitate the function of neuronal activity [15, 16, 19, 20]. Therefore, according to recent concept stated here, lactate is a preferentially used energy substrate over glucose in various conditions including acute neuronal condition [21]. In view of that, the present study was designed to investigate the brain modulating effects of lactic acid. Materials and methods Animals This study was conducted on Swiss albino male mice of 20±5 g. They were procured from the Central Animal House of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India (Registration Number: 542/AB/CPCSEA). The mice were acclimatized for two week under laboratory conditions. The mice were housed in groups of six in polypropylene cages and maintained at temperature of 25±1 C and 45-55% relative humidity, under 12 h light/dark cycle. Animals were always provided with commercial food pellets and water ad libitum. The waste and fecal matter in the cages was renewed daily to ensure hygienic condition and maximum comfort for mice. Ethical clearance for handling the animals was obtained from the Central Animal Ethical Committee of the University (CAECU) which an approval no. Dean/2014/CAEC/609, dated , prior to the beginning of the experimental works. Animal grouping and drug treatments Male mice were equally divided in six experimental groups (n=6). Orally administered volume of lactic acid (Sigma Aldrich Chemicals Pvt. Ltd., Bangalore) treatment was 10 ml/kg, and the control animals treated accordingly with the vehicle. In pilot experiments, effects of single and repeated daily doses of lactic acid 5, 25, 125 and 625 mg/kg/day, (p.o.) were evaluated in the mice groups on 1st, 5th, 7th, 10th, 11th and 12th day of the experiment. Diazepam (Lupin Ltd., Jammu) 5 mg/kg was taken as standard drug. All tests were conducted 60 min after the treatments except last day of experiment for estimation of prior treatment effects. Experimental procedure Effects of test agent in this pharmacological model described in the following were quantified after their single and repeated daily doses. Stress induced hyperthermia On 1st, 5th, 7th and 10th day of experiment the body weight and rectal temperatures of all the animals were measured before drugs administration. 60 min after the treatment with samples to their respective groups, individual mouse from each group was transferred into a black box (24 x 29 x 40 cm) with a electric steel grid floor for a period of 1 min. Electric foot shock through the grid floor (2 ma, 50 Hz of 2 ms duration) was delivered to each animals for inducing transient stress sensations. Five consecutive foot shocks with 10 s intra-shock intervals were given. At the end of a minute the animals were transferred in their home cages. After 10 min of exposure in electric foot shock stress body temperature was measured by rectal thermometer [22, 23]. A change in rectal temperature as an indicator of stress induced hyperthermia was calculated. Tail suspension test On the 11th day of the experiment body weights and rectal temperatures of all the animals were measured before and 60 min after the drugs administrations, immediately thereafter individual mouse from each group was hung on a wire in an upside down posture. After initial vigorous movements, the mouse assumed an immobile posture and the period of immobility during 5 min observation were recorded [24]. Potentiation of pentobarbital induced hypnosis On the 12th day (i.e. 24 hr after the last treatment) body weights and rectal temperatures of all the animals were measured again. Immediately, after that individual mouse from each group was administered with pentobarbital (40 mg/kg, i.p.) for sleep test. Onset of sleep (loss of righting reflex), and duration of sleep were recorded [22, 25]. Page 2 of 6

3 Figure 1. Effect of daily handling and intermittent foot shock stress on mean body weight of mice treated with Lactic acid. Values are mean±sem, n=6. *p<0.05 versus control group (Two way ANOVA followed by Bonferroni post tests). LA- Lactic acid. Statistics and data analysis Mean±standard error of mean (SEM) was calculated for the observed values in each experimental group. Statistical analysis was performed by one way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test, two way ANOVA followed by Bonferroni post test. GraphPad Prism- 5 software (GraphPad Software, Inc. CA, USA) was used for statistical analysis. Origin-Pro 8 (OriginLab Corporation, MA, USA) software was used for graph representation. p<0.05 was considered to be statistically significant. Results Mean body weights: Result of mean body weight of mice treated with graded oral doses of lactic acid and with a sub-anxiolytic dose of diazepam and vehicle treated control groups are summarized in Figure 1. It was found that mean body weight of control group mice were consecutive decreased significantly (p<0.05) during 12 observational days. However, data analysis (not shown) of vehicle treated control mice clearly revealed that the mean differences of their body weights recorded on the first day were always significantly higher than those recorded on the fifth and subsequent experimental days and that such reduction increased in magnitude with increasing test days. Since continuous reductions in body weights of vehicle treated mice were always observed in numerous other earlier experiments conducted and using analogous bioassay systems, they seem to be due to daily handling and intermittent foot shocks treatment during the course of the experiments. On repeated dose administration with 5 mg/kg Figure 2. Effect of Lactic acid on basal rectal temperature in mice on day 1, 5, 7, 10, 11 and 12. Values are mean±sem, n=6. =p<0.05 versus day 1 values of the same group (One way ANOVA followed by Student-Newman-Keuls multiple comparison test) and *p<0.05 versus control group (Two way ANOVA followed by Bonferroni post tests). LA- Lactic acid. lactic acid decreased in body weight was observed like diazepam. Moreover, significant (p<0.05) protective effects against daily handling and foot shock were recorded on continuous administration of lactic acid at dose levels of 25, 125 and 625 mg/kg/day. In these doses of lactic acid treated mice, mean body weights were increased as consecutive days. Basal rectal temperatures: Mean basal rectal temperatures of different treatment groups of mice are summarized in the Figure 2. On daily handling and foot shock induced stress, mean basal rectal temperatures of vehicle treated control mice were increased slightly on the 5th and subsequent observational days and remained almost constantly elevated on the 7th, 10th, 11th, and 12th days of the experiments. On critical observations made during these results were showed that as in similar experimental conditions, the mean basal rectal temperatures of lactic acid with 5 mg/kg/day treated groups were increased but somewhat lower as compared with vehicle treated control groups. However, these similar experimental conditions lactic acid doses groups with 25, 125 and 625 mg/kg showed somewhat decrease in rectal temperature as compared with control groups. More precise significant (p<0.05) effect on basal rectal of these doses were found on day 7th to 12th as subsequent test drug treatment when compared with control mice. No statistically significant effects on basal temperatures of the standard drug as diazepam were observed on the first day of the experiments. Stress induced hyperthermia (SIH): Stress induced Page 3 of 6

4 Figure 3. Effect of Lactic acid on stress induced hyperthermia in mice on day 1, 5, 7 and 10. Values are mean±sem, n=6. *p<0.05 versus control group (Two way ANOVA followed by Bonferroni post tests). LA- Lactic acid. hyperthermia by vehicle treated control group, lactic acid and diazepam treated mice groups were summarized in Figure 3. Stress induced hyperthermia of all control group mice were increased on subsequent days but within 0.7ºC to 0.9ºC. On repeated daily oral dose administration with lactic acid (5 mg/kg) showed somewhat increased in SIH effect but 10th day this effect was decreased but not as significantly. Effect of standard drug as diazepam treated group showed significant decreased in SIH were seen from day 5th. However, lactic acid with doses groups with 25, 125 and 625 mg/kg were showed decreased in SIH effects from day 5 as non-significant way. No statistically significant effects of any of the tested lactic acid doses on SIH were observed on the 1 st and 5th days of the experiment. But on continuous oral administration with lactic acid (25, 125 and 625 mg/kg) were showed significantly (p<0.05) decreased in SIH effects from 7th and 10th observational days. Except for standard drug as diazepam showed significant decreased in SIH effect from day 5th to last observation days. Tail suspension Test: Result on immobility period of mice with lactic acid, diazepam and vehicle treated groups were summarized in Figure 4. On continuous 11 daily oral administrations of all doses of lactic acid were showed decreased in immobility period of mice as compared with control group like diazepam treated mice groups. Pentobarbitone induced sleep test: In pentobarbital induced sleep test, effect of lactic acid and diazepam treated groups on onset and duration of sleep were summarized in Figure 5. All tested doses of lactic acid and standard drug were significantly decreased or increased the onset and duration of sleep period as dose dependent manner when compared to vehicle treated control mice. Since this test was conducted 24 hours of the last administered doses of the test agents, their observed effects seem to be due to their longer lasting physiological effects after their repeated daily oral doses. Discussion The present study demonstrated that lactic acid is effective when administered orally. Adaptogenic are the substances which promote longevity, revitalizing the body and enhance body s capacity to adapt in stressful environmental condition [26]. However, literature search did not reveal any systematic study defining the pharmacological dose and treatment regimen of lactic acid necessary to quantify its adaptogenic potential. Moreover, lactate present in blood stream is an important energy source for the brain [27], and such efficacy of a lactic acid was reconfirmed during the pilot study cum dose finding experiments. Therefore, to evaluate lactic acid as an adaptogenic substance battery of behavioural experiments i.e. stress induced hyperthermia, pentobarbital induced sleep time and tail suspension test were used to estimate the pharmacologically active dose range and Page 4 of 6

5 Figure 4. Effect of Lactic acid on tail suspension test on day 11. Values are mean±sem, n=6. *p<0.05 versus control group (One way ANOVA followed by Student-Newman-Keuls multiple comparison test). LA- Lactic acid. treatment regimen of lactic acid in mice commonly used for treatment of psychopharmacological associate disorders. Basal rectal temperature increased due to psychological stress [28]. Stress-induced hyperthermia is regulated by autonomic nervous system and occurs both prior to and during exposure to stressful or noxious stimulus like noise, heat, handling or pain [29]. Results of the experiments summarized in Figure. 2 reveal that even the highest single oral dose of lactic acid do not alter basal core temperature of mice. However, significant and dose dependent inhibitory effects of lactic acid tested against foot shock induced transient hyperthermia was apparent after its repeated oral doses between 25 and 625 mg/kg. These observations strongly suggest that lactic acid possess anxiolytic in this model conventionally used for identifying such efficacies for adaptogenic and other psychoactive agents. After daily oral doses the observed effects of lactic acid treatments against foot shock triggered hyperthermia were more pronounced than those observed after its similar acute doses. It must be noted though that the basal rectal temperature of the control group on the last treatment day was significantly higher than observed for the same group on the first day of the experiment (Figure 2). Such daily handling and treatment triggered elevation in basal core temperature were less pronounced in high doses of lactic acid treated groups, unlike 5 mg/kg. This effect of lactic acid treatments increased with its increasing daily oral doses. It was interesting to note that although after acute oral doses no diazepam like sedative effects of lactic acid was observed in the pentobarbital test, after its repeated daily doses such significant and dose dependent effects of the lactic acid became apparent. These observations could indicate that repeated daily treatments with lactic acid Figure 5. Effect of Lactic acid on pentobarbital induced sleep test on day 12. Values are mean±sem, n=6. *p<0.05 versus control group (One way ANOVA followed by Student-Newman-Keuls multiple comparison test). LA- Lactic acid. sedative or tranquilizing effects, or that the duration pentobarbital action is shortened by induction of pentobarbital metabolizing enzymes by daily lactic acid treatments. In any case, these results of the pilot study cum dose finding experiments clearly reveal that repeated daily administration of lactic acid is necessary to observe therapeutically interesting brain function modulating activities of lactic acid, and that lactic acid is one of the adaptogenic substances. In the tail suspension test, decrease in immobility period was observed which may be due to enhancement of central 5 HT and catecholamine neurotransmission in the brain. in reality decrease in locomotory activity sign was found after repeated daily oral doses of lactic acid, which proves its antidepressant like activity. The observations made in the pentobarbital sleep test also suggests that mechanisms may be involved GABA A modulating effect on sleep [30] its other than those involving benzodiazepine receptors could also be involved in its hypnosis observed in behavioural models. Results of the pilot study cum dose finding experiments not only revealed that repeated oral doses of lactic acid are well tolerated by experimental rodents, but also strongly suggested that its 25 mg/kg oral doses are maximally effective ones in modulating the functions of the central nervous system and that its psychopharmacological efficacy increases after its repeated daily doses. In addition, they indicate also that efficacy of its daily 25 mg/kg/day doses and higher can be quantitatively less than those observable after its lower ones. Therefore, for further experiments, daily graded oral doses between 25 and 625 mg/kg/day of lactic acid were considered to be appropriate for more detailed studies. Present study finding confirms that lactic acid exerts an adaptogenic like efficacy in mice behavioral models. In addition, our findings explored the effectiveness of lactic Page 5 of 6

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