O Abdel Salam, A Baiuomy
buspirone, gastric mucosa, inflammation, nociception
O Abdel Salam, A Baiuomy. Effect Of Buspirone On Inflammation, Pain And Gastric Injury In Mice. The Internet Journal of Pharmacology. 2007 Volume 6 Number 1.
Buspirone, a 5HT1A receptor partial agonist, was evaluated in various acute nociceptive pain models, on the carrgaeenan-induced paw oedema, in Porsolt's forced-swimming test, on haloperidol-induced catalepsy, on the indomethacin-induced gastric mucosal damage and on gastric acid secretion in mice. Buspirone (2, 4 or 8 mg/kg, i.p.) significantly increased the response latency in the mouse hot plate test. The anti-nociceptive activity was observed with 2 mg/kg and a maximal increase in hot-plate latency by 41.3%. Buspirone in addition markedly increased the nociceptive thresholds in the acetic-acid-induced writhing assay, in the capsaicin-induced chemogenic pain and in electrically-induced pain in mice. It displayed no antidepressant activity in the Porsolt's forced-swimming test, but at doses of 8 or 16 mg/kg, buspirone decreased the duration of catalepsy induced by haloperidol by 28.1 and 58.3%, respectively. When given 30 min prior to subplantar carrageenan injection, the drug at 8, 16 mg/kg, inhibited paw oedema response 4 h post-carrageenan by 26.3, 41.6%, respectively. Buspirone (2, 4 or 8 mg/kg, i.p.) significantly inhibited the development of indomethacin gastric mucosal lesions and gastric acid secretion in a dose-dependent manner. In conclusion, Buspirone increased pain threshold in models of thermal, chemogenic, electrical and visceral pain and displayed anti-inflammatory and gastric protective properties. The drug might prove of value for the management of inflammatory painful conditions.
5-Hydroxytryptamine 1A (5-HT1A) receptors are important targets for the treatment of mood disorders (1,2) . Buspirone, is a partial 5-HT1A receptor agonist buspirone which is widely used for treating anxiety (3) . 5-HT1A receptors are located presynaptically on cell bodies in the raphe nuclei (somatodendritic receptors) and postsynaptically in 5-HT forebrain projecting areas. By activating somatodendritic receptors, 5-HT and 5-HT1A receptor agonists decrease the firing of 5-HT neurons in the raphe, and, consequently decrease 5-HT terminal release (4) . Drugs acting on 5-HT receptors and enhancing serotonergic neurotransmission e.g., the serotonin reuptake inhibitors clomipramine (5,6) and fluoxetine (7,8,9) have been shown to exhibit analgesic and anti-inflammatory activity in animal models. Data suggest that a tonic release of serotonin in the spinal cord may occurs during ongoing peripheral inflammation, modulating the neurogenic component of oedema either by an inhibitory action on 5-HT1 receptors or by a stimulatory action on 5-HT2 receptors (10) . Serotonin reuptake inhibitors, however, displayed gastric irritant properties and increased gastric acid secretion in experimental animals (11,12) , while reports of increased gastric bleeding events have been reported in humans (13) . In the present study we describe the effects of buspirone in animal models of pain and acute inflammation. In addition, the effects of the compound in Porsolt's forced-swimming test, on haloperidol-induced catalepsy, on gastric acid secretion and on gastric mucosal damage caused by indomethacin, a non-steroidal anti-inflammatory drug (NSAID) commonly used in many arthritic and inflammatory conditions was evaluated.
Materials and methods
Swiss albino mice weighing 20-25 g of body weight (National Research Centre, Cairo) were used. They were housed under standardized conditions with free access to food and water. All animal procedures were performed in accordance with the recommendations for the proper care and use of laboratory animals. Equal groups of 6 mice/group were used in all experiments. The doses of buspirone employed in the study were based upon the human dose after conversion to that of rat according to Paget and Barnes conversion tables (14) .
Tests of nociception
The hotplate test was performed on mice by using an electronically controlled hotplate (Ugo Basile, Italy) heated to 52°C (± 0.1°C) (15) . The cut-off time was 30s. Groups of mice (n = 6 / group) were given buspirone at doses of 2, 4 or 8 mg/kg, i.p. , saline (control), or indomethacin (18 mg/kg, i.p.) 30 min prior to testing. The experimenter was blind to dose. Latency to lick a hind paw or jump out of the apparatus was recorded for the control and drug-treated groups. Nociceptive thresholds were measured sequentially before and at 60 min post-treatment.
Tail electric stimulation test
Groups of mice (n = 6 / group) were given buspirone (2, 4, 8 or 16 mg/kg, i.p.), indomethacin (18 mg/kg, i.p.) or saline (control). The minimum current required to elicit vocalisation upon electrical stimulation of the tail was determined for the control and drug-treated groups (16) . Electrical stimulation of the tail was applied by means of Pulse generator 57800-001 (Eugo Basil EXT Unit) (Frequency 50 pulse/sec, shock duration 2 sec).
Capsaicin-induced hind paw licking
Buspirone (2, 4, 8 or 16 mg/kg) or saline was given i.p., 30 min before injection of capsaicin (1.6 µg/paw; 25 µl) under the skin of the dorsal surface of the right hind paw of mice. The observation period started immediately after the capsaicin injection and lasted for 5 min. The time the animals spent licking the injected paw was evaluated by using a stopwatch (17) .
Acetic acid-induced writhing
Separate groups of 6 mice each were administered saline, Buspirone (2, 4, 8 or 16 mg/kg, 0.2 ml, i.p.) or indomethacin (18 mg/kg, i.p.). After 30 min interval, an i.p. injection of 0.2 ml of 0.6% acetic acid was administered (18) . Each mouse was then placed in an individual clear plastic observational chamber, and the total number of writhes made by each mouse was counted for 30 min after acetic acid administration.
Tests of inflammation
Carrageenan-induced paw oedema
The effect of systemic administration of buspirone (2, 4, 8 or 16 mg/kg, i.p., 0.2 ml, n = 6/ group) or indomethacin (18 mg/kg, i.p., 0.2 ml) given as a 30 min pretreatment was studied. The control groups received saline (0.2 ml, n = 6 per group; i.p.). Paw oedema was induced by sub-plantar injection of 25 µl of 1% sterile carrageenan lambda in saline into the right hind paw (19) . Contralateral paw received an equal volume of saline. Paw volume was determined immediately before carrageenan injection and at 4h later using a plethysmometer (Ugo Basile, Milan, Italy). The oedema component of inflammation was quantified by measuring the increase in paw volume (ml) at before carrageenan injection and at 4h after carrageenan injection with respect to the pre-injection value for each animal. Oedema was expressed as a percentage of change from control (pre-drug) values.
Porsolt's forced-swimming test
Each mouse was placed individually in a glass cylinder (diameter 12 cm, height 24 cm) filled with water at a height of 12 cm. Water temperature was maintained at 22–23°C. The animal was forced to swim for 6 min and the duration of immobility was measured. The mouse was considered as immobile when it stopped struggling and moved only to remain floating in the water, keeping its head above water. The floating time, which was the measure of despair (20) , was recorded after treatment with buspirone (2, 4, 8 or 16 mg/kg, i.p.), saline or imipramine (15 mg/kg, i.p.).
Catalepsy induced by haloperidol, defined as a reduced ability to initiate movement and a failure to correct posture, was measured by means of the bar test. To test for catalepsy, mice were positioned so that their hindquarters were on the bench and their forelimbs rested on a 1 cm diameter horizontal bar, 4 cm above the bench. The length of time the mice maintained this position was recorded by stopwatch to a maximum of 180 s. This procedure occurred 30 min after haloperidol (2 mg/kg, i.p.) administration (21) . Buspirone (4, 8 or 16 mg/kg) was i.p. co-administered with haloperidol. Mice were judged to be cataleptic if they maintained this position for 30 s or more.
Gastric ulcerogenic studies
Gastric mucosal damage was evoked in mice by the administration of indomethacin (20 mg/kg, i.p.). The effect of buspirone (4, 8, or 16 mg/kg, i.p.) administered at time of indomethacin injection was studied. Food and water were provided ad libitum. Mice were killed 24 h after drug administration, stomachs excised, opened along the greater curvature, rinsed with saline, extended on a plastic board and examined for mucosal lesions. The number and severity of mucosal lesions were noted and lesions were scaled as described before (22,23) .
In a separate set of experiments, the effect of buspirone on gastric acid secretion induced by 1 ml of distention was examined in mice. Mice were deprived of food for 18 hours before the experiments, but allowed free access to tap water. After brief ether anaesthesia, the abdomen was opened through a midline incision, the pylorus ligated, stomach was then distended by filling with 1 ml of saline. Mice received either saline (0.2 ml, i.p., n = 6) (control) or buspirone (2, 4, 8 or 16 mg/kg, 0.2 ml i.p., n = 6 each). Two hours later, gastric juice was collected and gastric acid output determined by automatic titration to pH 7.0 with 0.01N NaOH and H + output expressed as µEq/2h
Buspirone (Bristol Global Napi Pharmaceutical Co. Cairo, A.R.E.), carrageenan, capsaicin, urethane (Sigma, USA), indomethacin (Kahira Pharm & Chem. IND Co., Cairo, A.R.E.), imipramine hydrochloride (Sigma, USA) were used. Stock solutions of capsaicin (10 mg/ml) contained 10% ethanol, 10% Tween 80, 80% saline solution. Analytical-grade glacial acetic acid (Sigma) was diluted with pyrogen-free saline to provide a 0.6% solution for i.p. injection. All drugs were dissolved in isotonic (0.9% NaCl) saline solution immediately before use, except indomethacin which was dissolved in a 5% solution of sodium bicarbonate.
Data are expressed as mean ± S.E. The results of carrageenan-induced paw oedema experiments are expressed as a percentage of change from control (pre-drug) values. Data were analyzed by one-way analysis of variance, followed by a Tukey's multiple range test for post hoc comparison of group means. When there were only two groups a two-tailed Student's t test was used. For all tests, effects with a probability of P < .05 were considered to be significant.
Effect of buspirone in tests of nociception
The mean reaction time on the hot plate was significantly delayed at 1h after the administration of buspirone or indomethacin, compared with basal values, denoting decreased nociception. The anti-nociceptive activity was observed with 2 mg/kg and a maximal increase in hot-plate latency by 41.3%. Meanwhile, hot-plate latency increased by 26.8% after indomethacin treatment(Fig. 1).
Tail electric stimulation test
The administration of 4, 8 or 16 mg/kg buspirone increased the threshold for vocalisation during stimulation in a dose-dependent manner compared with the basal level of nociceptive reaction. The anti-nociceptive activity was observed with 4 mg/kg (25.9% 1h post-dosing) and a maximal increase in hot-plate latency by 32.5% after 16 mg/kg buspirone 1h post-dosing. Meanwhile, a significant rise in nociceptive thresholds by 22.2% was obtained with indomethacin 1h following administration (Fig. 2).
Capsaicin-induced hind paw licking
The duration of paw licking following capsaicin injection was significantly shortened by 29.2, 38.5 and 60.6% after pretreatment with buspirone at 2, 4 or 8 mg/kg, respectively (Fig. 3).
Acetic acid induced writhing
Acetic acid-induced writhing was significantly reduced in mice receiving buspirone in a dose-related manner with a maximal reduction of the writhing score of 95.1% by 16 mg/kg buspirone compared with 51.9% reduction of the writhing score by indomethacin (Fig. 4).
Effect of buspirone on the carrageenan-induced paw oedema
In the control group, paw volume increased by 85.6 ± 3.2% at 4h after injection of carrageenan. Buspirone at 2 or 4 mg/kg administered i.p., 30 min prior to carrageenan had no significant effect on the paw oedema, whereas oedema was significantly inhibited by 8 or 16 mg/kg 26.3 and 41.6%,respectively. Meanwhile, indomethacin markedly and significantly inhibited the paw oedema response by 57.9% (Fig. 5).
Effect of buspirone in Porsolt's forced-swimming test
Buspirone administered at doses of 2, 4, 8 or 16 mg/kg did not alter immobility time in contrast to the tricyclic antidepressant drug imipramine which significantly reduced immobility time by 27.1% (Fig. 6).
Effect of buspirone on the duration of haloperidol-induced catalepsy
Haloperidol administered i.p. at the dose of 2 mg/kg produced a signifcant cataleptic response. The duration of haloperidol-induced catalepsy was significantly reduced by 28.1 and 58.3% by 8 and 16 mg/kg buspirone, respectively (Fig. 7).
Effect of buspirone on gastric mucosal lesions caused by indomethacin
Buspirone (4, 8 or 16 mg/kg) administered at time of indomethacin injection inhibited the development of gastric lesions in a dose dependent manner (Fig. 8).
Effect of buspirone on gastric acid secretion
In pylorus-ligated mice, gastric acid secretion induced by distention was dose-dependently increased by the administration of buspirone (76.8, 80, 86.8 and 89.5% inhibition by 2, 4, 8 or 16 mg/kg buspirone, respectively) (Fig. 9).
The present study provides evidence that buspirone, an 5HT1A receptor agonist, exerted antinociceptive effects in different pain models. The drug increased the nociceptive threshold to thermal or electrically induced pain. In the writhing test in mice, a widely used model for visceral pain, which involves the local release of prostaglandins, buspirone markedly inhibited the nociceptive behavioral response, with maximum 95.1% inhibition of the number of abdominal constriction being observed. In addition, chemogenic pain evoked by intraplantar capsaicin was markedly reduced by buspirone. The drug at doses which caused effective anti-nociception, did not impair mouse performance as evaluated by the rotarod test, thus ruling out the confounding influence of a possible sedative effect. Observations in the present study are thus consistent with data reported by other researchers. Buspirone has been shown to induce thermal antinociception in the hot-plate test in mice (24) . Visceral analgesic properties have been also reported for buspirone which attenuated the abdominal withdrawal response and antagonized the changes in arterial pressure in rats subjected to colorectal distension (25) . The analgesic effects of buspirone are likely to be mediated through an action on 5-HT1A receptors (25) , but might involve ATP-sensitive K+ channels as well (24) or a non-opioid-adrenally mediated mechanism, since buspirone (5 mg/kg, i.p.) produced elevation in plasma norepinephrine and corticosterone (26) . In contrast, buspirone decreased the pain threshold studies in the foot pressure test (27) or did not affect pain perception and stress-induced analgesia in rats (28) .
Buspirone also exerted moderate anti-inflammatory effect when tested in the carrageenan assay of acute inflammation in the present study. The degree of paw oedema inhibition by buspirone was less than that of indomethacin. Dahu et al. (10) reported that buspirone injected intrathecally in rats decreased the inflammatory paw oedema caused by subcutaneous carrageenan injection.
The forced swimming test in mice is a commonly used tool for screening of potential antidepressants (19) . In the present study, buspirone did not alter the immobility time. Other investigators reported that buspirone (5.0, 10.0 mg/kg)reduced (29) , while at lower dose of 1 mg/kg, prolonged (30) or failed to influence (31) immobility time in the rat forced swimming test. Furthermore, Redrobe and Bourin (32) , found that low dose buspirone (0.06 mg/kg, i.p.) enhanced the activity of subactive doses of SSRIs in the mouse forced swimming test, while a high dose of buspirone (0.5 mg/kg, i.p.)attenuated the antidepressant-like effects of active doses of these drugs.
Catalepsy occurs following high dopamine D2 receptor blockade by the typical antipsychotic drug haloperidol. The haloperidol-induced catalepsy, is a behavioural predictor of extrapyramidal symptoms liability (33) . Serotonergic pathways in the central nervous system are intimately involved in the modulation of the basal ganglia and in the pathophysiology of human involuntary movement disorders (34) . In rats, endogenous 5-HT acting on 5-HT2C receptors tonically inhibits basal dopamine release in the prefrontal cortex, while stimulation of 5-HT2C receptors with an exogenous agonist preferentially inhibit stimulated release (35) . Blocking 5-HT2C attenuated haloperidol-induced catalepsy in a dose-dependent manner (36) . In the present study, buspirone decreased the duration of catalepsy in a dose-related manner. This effect was observed at a dose of 8 mg/kg. In rats, buspirone (1 mg/kg) was reported to reverse haloperidol-induced catalepsy (37) . Several investigators have also shown a role for buspirone in decreasing extrapyramidal movements in rats. In parkinsonism induced by unilateral 6-hydroxydopamine injection, buspirone lessened L-dopa-induced dyskinesia and improved L-dopa related motor performace due to an action on 5HT1A receptor (38,39) . In addition, tacrine-induced tremulous jaw movements in rats, a putative model of parkinsonian tremor, was decreased by injection of 5-HT(1A) agonists 8-OH-DPAT and buspirone (40) . These data suggest that 5HT1A receptor agonists might be of value in the treatment of parkinsonism.
Observations in the present study also indicates that buspirone possesses gastric protective effects. The acute gastric mucosal lesions evoked by indomethacin in the rat were decreased in a dose-depedenat manner by co-administration with buspirone. Buspirone has also been shown to suppress the stress-induced gastric ulcerations in the rat (41,42) . In accordance with findings in the present study, 5HT1A agonists inhibited gastric acid secretion in rats (43) .
In conclusion, the present study describes antinociceptive, anti-inflammatory, anti-cataleptic, gastric protective and gastric acid inhibitory effects of buspirone.