Bilgin Kaygisiz

The antinociceptive effect and mechanisms of action of pregabalin in mice

BACKGROUND Pregabalin, a potent anticonvulsant agent, is used in treatment-resistant epileptic patients. It is reported that pregabalin also has analgesic effect in different pain syndromes. However, there is limited data on its antinociceptive mechanisms of action. We aimed to investigate the central and peripheral antinociceptive effects of pregabalin and the contribution of nitrergic, serotonergic, and opioidergic pathways in mice. METHODS We used tail flick, tail clip and hot plate tests to investigate the central antinociceptive effects and acetic acid-induced writhing test to assess peripheral antinociceptive effects of pregabalin (10, 30, 100mg/kg). We also combined pregabalin (100mg/kg) with, a nonspecific nitric oxide synthase inhibitor l-NAME (100mg/kg), a serotonin receptor antagonist cyproheptadine (50 μg/kg), and an opioid receptor antagonist naloxone (1mg/kg). RESULTS Pregabalin 30 mg/kg enhanced the percentage of maximal possible effect (% MPE) in tail flick test. Pregabalin 100mg/kg significantly increased % MPE in tail clip and tail flick tests and decreased the number of writhings. Pregabalin made no significant alteration in hot plate test at all doses. The combined use of pregabalin 100mg/kg with l-NAME, cyproheptadine, and naloxone showed that % MPE was reduced only in the combination of pregabalin with naloxone and solely in tail clip test while no significant difference was observed in writhing test. CONCLUSIONS We suggest that pregabalin (30 and 100mg/kg) presents central spinal but not central supraspinal antinociceptive effect and pregabalin 100mg/kg shows peripheral antinociceptive effect. The opioidergic pathway seems to mediate the central spinal antinociceptive effect of pregabalin while nitrergic and serotonergic pathways are not involved.

Effects of single and combined gabapentin use in elevated plus maze and forced swimming tests.

Background Gabapentin, a third-generation antiepileptic drug, is a structural analogue of γ-aminobutyric acid, which is an important mediator of central nervous system. There is clinical data indicating its effectiveness in the treatment of psychiatric illnesses such as bipolar disorder and anxiety disorders. Objectives We aimed to investigate the antidepressant and anxiolytic-like effects and mechanisms of gabapentin in rats. Material and Methods Female Spraque–Dawley rats weighing 250±20 g were used. A total of 13 groups were formed, each containing 8 rats: gabapentin (5, 10, 20, 40 mg/kg), amitriptyline (10 mg/kg), sertraline (5 mg/kg), diazepam (5 mg/kg), ketamine (10 mg/kg), gabapentin 20 mg/kg was also combined with amitriptyline (10 mg/kg), sertraline (5 mg/kg), diazepam (5 mg/kg) and ketamine (10 mg/kg). All the drugs were used intraperitoneally as single dose. Saline was administered to the control group. Elevated plus maze and forced swimming tests were used as experimental models of anxiety and depression, respectively. Results It was observed that gabapentin showed an anxiolytic-like and antidepressant-like effect in all doses in rats. Its antidepressant effect was found to be the same as the antidepressant effects of amitriptyline and sertraline. There was no change in the antidepressant effect when gabapentin was combined with amitriptyline and ketamine, but there was an increase when combined with sertraline and diazepam. Gabapentin and amitriptyline showed similar anxiolytic effect, whereas ketamine and diazepam had more potent anxiolytic effect compared with them. Conclusions These data suggest that gabapentin may possess antidepressant- and anxiolytic-like effects.

Evaluation of Cisplatin Neurotoxicity in Cultured Rat Dorsal Root Ganglia via Cytosolic Calcium Accumulation

BACKGROUND Calcium homeostasis is considered to be important in antineoplastic as well as in neurotoxic adverse effects of cisplatin. AIMS This study aimed to investigate the role of Ca(2+) in cisplatin neurotoxicity in cultured rat dorsal root ganglia (DRG) cells. STUDY DESIGN Cell culture study. METHODS DRG cells prepared from 1-day old Sprague-Dawley rats were used to determine the role of Ca(2+) in the cisplatin (10-600 μM) neurotoxicity. The cells were incubated with cisplatin plus nimodipine (1-3 μM), dizocilpine (MK-801) (1-3 μM) or thapsigargin (100-300 nM). Toxicity of cisplatinon DRG cells was determined by the MTT assay. RESULTS The neurotoxicity of cisplatin was significant when used in high concentrations (100-600 μM). Nimodipine (1 μM) but not MK-801 or thapsigargin prevented the neurotoxic effects of 200 μM of cisplatin. CONCLUSION Voltage-dependent calcium channels may play a role in cisplatin neurotoxicity.

The effect of Des-Arg9-bradykinin and bradykinin-potentiating peptide C on isolated rat hearts.

OBJECTIVES Des-Arg9-bradykinin and bradykinin-potentiating peptide C (BPPC) may contribute to the regulation of cardiovascular function. Therefore, we studied effects of these peptides on coronary perfusion pressure (CPP), heart rate, left ventricular developed pressure (LVDP) and maximum rate of increase of left ventricular pressure (+dP/dtmax). METHODS The isolated rat hearts were perfused with modified Krebs-Henseleit solution. RESULTS Infusion of 10, 100 and 1000 nM Des-Arg9-bradykinin decreased CPP (-13.6, -14.8 and -19.0%), LVDP (-16.5, -21.0 and -30.7%) and +dP/dtmax (-11.8, -17.8 and -23.7%), respectively (p < 0.001). Ten or 100 nM Des-Arg9-bradykinin did not alter heart rate, but 1000 nM increased it (+11.3%, p < 0.01). One, 10 and 100 nM BBPC reduced CPP (-16.3, -28.5 and -47.5%), LVDP (-12.6, -19.6 and -21.3%) and +dP/dtmax (-8.7, -18.6 and -20.3%), respectively (p < 0.001). BPPC increased heart rate at 1 nM (+9.6%, p < 0.05 ) and at 10 nM (+14.2%, p < 0.01), however 100 nM decreased it (-15.3%, p < 0.001). CONCLUSIONS This study evidences that Des-Arg9-bradykinin and BPPC possess vasodilatory effect with modest negative inotropic action. Furthermore, high-dose of Des-Arg9-bradykinin and low-dose of BPPC may produce a tachycardic action, but high dose of BBPC may cause a bradycardic action.

Pregabalin Attenuates Carrageenan-Induced Acute Inflammation in Rats by Inhibiting Proinflammatory Cytokine Levels

Objective Pregabalin (PGB) is a compound used in the treatment of epilepsy, anxiety, and neuropathic pain. Experimental data also indicate that PGB can reduce inflammatory pain. We aimed to investigate the anti-inflammatory effects of PGB on carrageenan (CAR)-induced paw edema and its effects on tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) acting as acute phase cytokines in inflammation, and anti-inflammatory cytokine IL-10, in rats. Materials and Methods Single doses of PGB 30, 50, and 100 mg/kg and indomethacin (INDO) 5 mg/kg in the treatment groups and saline in the control group were injected once intraperitoneally prior to administration of 100 μl of 1% CAR into the right hind paw of the rats. The paw thickness was measured using gauge calipers (Vernier calipers) before (0 hour) and every hour afterwards for 6 hours following the inflammation induction. The cytokine levels in the blood serum obtained intracardiacally were determined after 6 hours using the enzyme-linked immunosorbent assay method. p<0.05 was considered statistically significant. Results There was no significant difference between the 0 and 6th hour considering the paw thickness in all groups, except in the CAR group. CAR significantly increased the paw thickness at 6 hours compared to the 0 hour. All doses of PGB and INDO significantly reduced the paw thickness after 6 hours compared to the CAR group. The TNF-α and IL-1β levels in the PGB and INDO groups were comparable to the control group, whereas in the CAR group, these levels were increased. The IL-10 level was enhanced in the PGB 50 mg/kg and INDO groups. Conclusion It was observed that all doses of PGB exerted anti-inflammatory-like effects comparable to INDO, supported by their effects on the levels of cytokines.

Agmatine co‐treatment attenuates allodynia and structural abnormalities in cisplatin‐induced neuropathy in rats

Cisplatin is a widely used antineoplastic agent in the treatment of various cancers. Peripheral neuropathy is a well‐known side effect of cisplatin and has potential to result in limiting and/or reducing the dose, decreasing the quality of life. Thus, effective treatments are needed. Agmatine is an endogenous neuromodulator that has been shown to exert antiallodynic effects in various animal studies. The first aim of this study was to investigate the in vitro effects of agmatine on cisplatin‐induced neurotoxicity. Primary cultures of dorsal root ganglia (DRG) which are the primary target of drug injury were prepared. DRG cells were incubated with cisplatin (100, 200, 500 μm). Then, agmatine (10, 100, 500 μm) was administered with the submaximal concentration of cisplatin. Cisplatin caused concentration‐dependent neurotoxicity, and agmatine did not alter this effect. The second aim was to investigate the effects of agmatine on cisplatin‐induced peripheral neuropathy in rats and the influence of nitric oxide synthase (NOS) inhibitor, L‐NAME, in this effect. Female Sprague Dawley rats received intraperitoneal saline (control), cisplatin (3 mg/kg), cisplatin+agmatine (100 mg/kg), or cisplatin+agmatine+L‐NAME (10 mg/kg) once a week for 5 weeks. The mechanical allodynia, hot plate, and tail clip tests were performed, and DRG cells and sciatic nerves were analyzed. Agmatine and agmatine+L‐NAME combination attenuated CIS‐induced mechanical allodynia and degeneration in DRG cells and sciatic nerves. However, L‐NAME did not potentiate the antiallodynic or neuroprotective effect of agmatine. These findings indicate that agmatine co‐administration ameliorates cisplatin‐induced neuropathy and may be a therapeutic alternative.

The Effect of Chemerin on Cardiac Parameters and Gene Expressions in Isolated Perfused Rat Heart

Background: Chemerin is a novel chemoattractant adipokine expressed in cardiovascular system, and its receptor has been detected in the epicardial adipose tissue. Aims: To determine the effects of chemerin on the cardiac parameters and gene expressions in the isolated perfused rat heart. Study Design: Animal experiment. Methods: The hearts were retrogradely perfused with Langendorff technique to measure the cardiac parameters. The experimental groups were acutely treated with 10, 100, and 1000 nM doses of chemerin. Another group was given 10 μM L-nitric oxide synthase inhibitor for 5 min before 1000 nM chemerin administration. The real-time polymerase chain reaction was performed for detecting the expression of target genes. Results: All doses of chemerin significantly decreased the left ventricular developed pressure (max 35.33 Δ%, p<0.001), and +dP/dtmax (max 31.3 Δ%, p<0.001), which are the indexes of cardiac contractile force. In addition, 1000 nM chemerin reduced the coronary flow (max 31 Δ%, p<0.001). N(W)-nitro-L-arginine methyl ester antagonized the negative inotropic effect of chemerin on contractility. Chemerin induced a 2.16-fold increase in endothelial nitric oxide synthase mRNA and increased the cyclic guanosine monophosphate levels (p<0.001) but decreased the PI3Kγ gene expression (1.8-fold, p<0.001). Furthermore, all doses of chemerin decreased the CaV1.2 gene expression (1.69-fold, p<0.001). Conclusion: Acute chemerin treatment induces a negative inotropic action with the involvement of nitric oxide pathway, CaV1.2, and PI3Kγ on isolated rat heart.

The Role of Cyclooxygenase Enzymes in the Effects of Losartan and Lisinopril on the Contractions of Rat Thoracic Aorta

OBJECTIVE It was suggested that prostaglandins which are synthesized by cyclooxygenase (COX) enzymes contribute to the actions of angiotensin-converting enzyme (ACE) inhibition and angiotensin AT1 receptor antagonism and there is an interaction between ACE signaling pathway and COX enzymes. We aim to investigate the role of COX enzymes in the effects of losartan, an angiotensin II (Ang II) receptor antagonist or lisinopril, an ACE inhibitor, on the contractions of rat thoracic aorta in isolated tissue bath. MATERIALS AND METHODS Responses of losartan (10-6, 10-5, 10-4 M), lisinopril (10-6, 10-5, 10-4 M), and non-selective COX inhibitor dipyrone (10-4, 7 × 10-4, 2 × 10-3 M) alone to the contractions induced by phenylephrine (Phe) (10-7 M), potassium chloride (KCl) (6 × 10-2 M), Ang II (10-8 M) and responses of losartan or lisinopril in combination with dipyrone to the contractions induced by Phe or KCl were recorded. RESULTS When used alone, dipyrone and losartan inhibited Phe, KCl, and Ang II-induced contractions, whereas lisinopril inhibited only Phe and Ang II-induced contractions. Inhibition of COX enzymes (COX-3, COX-3 + COX-1, COX-1+ COX-2 + COX-3 by dipyrone 10-4, 7 × 10-4, 2 × 10-3 M, respectively) augmented the relaxant effects of losartan or lisinopril. Also, dipyrone potentiated the effect of lisinopril on KCl-induced contractions. CONCLUSION We suggest that dipyrone increases the smooth-muscle relaxing effects of losartan or lisinopril and that COX enzyme inhibition may have a role in the enhancement of this relaxation.

PT670. Antiinflammatory and gastric side effect of gabapentin

Nicotinic acetylcholine (nACh) receptors evoke convulsive seizures both in nicotine intoxication and human epileptic disorders (e.g., autosomal dominant nocturnal frontal lobe epilepsy [ADNFLE]). Here, we performed behavioral and immunohistochemical studies to elucidate the mechanisms of nicotineinduced seizures. Nicotine at high doses (4 mg/kg, i.p.) evoked convulsive seizures, which was antagonized by non-selective (mecamylamine) and α7-selective (methyllycaconitine) nACh receptor antagonists. Nicotine-induced seizures were accompanied by significant and region-specific increments in Fos protein expression, a biological marker of neural excitation, in the piriform cortex (Pir), amygdala (AMG), medial habenular nucleus (MHb), thalamus (Th) and solitary tract, suggesting that these regions are potential causative sites for nicotine-induced seizures. Electrical lesioning (1 mA for 15 sec per side) of AMG significantly suppressed nicotine-induced seizures, whereas neither lesioning of the Pir, MHb nor Th affected the nicotine seizure induction. Furthermore, bilateral microinjection of nicotine (100 or 300 μg/μL/side) into the AMG effectively evoked convulsive seizure in a dose-dependent manner. The present results strongly suggest that acute nicotine treatment evokes convulsive seizures by activating amygdala neurons, mainly through α7nACh receptors. PT668 Development of behavioral dysfunction in mice cohabit with brain disordered cage-mate Seungmoon Jun1, Daejong Jeon2, Yong Jeong1, Hyunwoo Yang1 1Korea Advanced Institute of Science & Technology, Republic of Korea, 2Seoul National University, Republic of Korea Abstract People who live with patients with brain disorders are considered as a potential risk group leading to mental disorder. They suffer from a caregiving burden and distressing situation. Consequently, the longer caregiving, the worse their quality of life. In spite of their devoted effort, caregiver’s deteriorated state has been overlooked. Here, we attempted to set a longterm housing model reflecting the particular situation. Mice were housed with a conspecific temporal lobe epilepsy model mouse or inescapable foot-shock stress mouse models. After long-term housing, cage-mate was performed behavior testsPeople who live with patients with brain disorders are considered as a potential risk group leading to mental disorder. They suffer from a caregiving burden and distressing situation. Consequently, the longer caregiving, the worse their quality of life. In spite of their devoted effort, caregiver’s deteriorated state has been overlooked. Here, we attempted to set a longterm housing model reflecting the particular situation. Mice were housed with a conspecific temporal lobe epilepsy model mouse or inescapable foot-shock stress mouse models. After long-term housing, cage-mate was performed behavior tests and electrophysiological investigation. The conspecific cagemate showed increased anxiety and depression-like behavior. Furthermore, they showed significantly reduced social interaction with juvenile and anesthetized mouse. Behavioral dysfunction of cage-mate sustained four weeks after removing the mouse model. Interestingly, fluoxetine, serotonin selective reuptake inhibitor, hardly restored their behaviors. Our results suggest that a dweller whose cage-mate having brain disorder could develop abnormal behavior including reduced social and increased depression-like behavior. These findings may help to understand psychosocial or psychiatric symptoms frequently observed in at-risk nursing people or caregivers. PT669 Investigation of the antiinflammatory and gastric side effects of pregabalin Fatma Sultan Kilic1, Bilgin Kaygisiz1, Sule Aydin1, Hadi Karimkhani2, Cafer Yildirim1, Setenay Oner3 1 Eskisehir Osmangazi University Medical School, Department of Pharmacology 2 Eskisehir Osmangazi University Medical School, Department of Biochemistry 3 Eskisehir Osmangazi University Medical School, Department of Biostatistics Abstract Objectives: Nonsteroidal antiinflammatory drugs are used for the relief of inflammation, however gastrointestinal side effects restrict their clinical use. We aimed to investigate the antiinflammatory effects of pregabalin, a drug used in epilepsy, anxiety, neuropathic pain treatment, on carrageenaninduced paw edema and to evaluate its gastric side effects in Wistar rats. Methods: Pregabalin 30,50,100mg/kg; indomethacin 5mg/ kg(reference drug), vehicle(saline) were injected intraperitoneally before 100μl of 1% carrageenan administration into the right hind paws of the rats. Paw thickness was measured by a gauge calipers(Vernier Calipers) before (0th hour) and in every hour during 6 hours after induction of inflammation. Paw thickness of treated groups were compared with control group with One-way ANOVA. Also paw thickness in 0th and 6th hours were compared within each group with twoway ANOVA. Pregabalin was administered orally for 10 days to evaluate gastric side effect. At the end of 10 day treatment, rats were sacrificed, gastric tissues were removed out, mucus secretion was determined spectrophotometrically, ulcer index was scored from score 0:(no petechia) to score 3:(petechia>5mm). Results: There was no significant difference between 0th and 6th hours in paw thickness of all groups, except carrageenan group. Carrageenan significantly increased paw thickness in 6th hour compared to 0th hour. All doses of pregabalin and indomethacin significantly reduced paw thickness in 6th hour compared to carrageenan group. Pregabalin 50 and 100mg/kg similar to indomethacin significantly reduced mucus secretion and increased ulcer index compared to control while pregabalin 30mg/kg did not. Conclusion: All doses of pregabalin exerted antiinflammatory effects comparable to indomethacin, 50 and 100mg/kg pregabalin showed gastric side effects as reduced mucus secretion and ulcer formation similar to indomethacin and 30mg/kg pregabalin may be reasonable dose for antiinflammatory effect without showing gastric side effects. This study was supported by ‘Scientific Researchs Projects’ of Eskisehir Osmangazi University.

PT669. Investigation of the antiinflammatory and gastric side effects of pregabalin

Nicotinic acetylcholine (nACh) receptors evoke convulsive seizures both in nicotine intoxication and human epileptic disorders (e.g., autosomal dominant nocturnal frontal lobe epilepsy [ADNFLE]). Here, we performed behavioral and immunohistochemical studies to elucidate the mechanisms of nicotineinduced seizures. Nicotine at high doses (4 mg/kg, i.p.) evoked convulsive seizures, which was antagonized by non-selective (mecamylamine) and α7-selective (methyllycaconitine) nACh receptor antagonists. Nicotine-induced seizures were accompanied by significant and region-specific increments in Fos protein expression, a biological marker of neural excitation, in the piriform cortex (Pir), amygdala (AMG), medial habenular nucleus (MHb), thalamus (Th) and solitary tract, suggesting that these regions are potential causative sites for nicotine-induced seizures. Electrical lesioning (1 mA for 15 sec per side) of AMG significantly suppressed nicotine-induced seizures, whereas neither lesioning of the Pir, MHb nor Th affected the nicotine seizure induction. Furthermore, bilateral microinjection of nicotine (100 or 300 μg/μL/side) into the AMG effectively evoked convulsive seizure in a dose-dependent manner. The present results strongly suggest that acute nicotine treatment evokes convulsive seizures by activating amygdala neurons, mainly through α7nACh receptors. PT668 Development of behavioral dysfunction in mice cohabit with brain disordered cage-mate Seungmoon Jun1, Daejong Jeon2, Yong Jeong1, Hyunwoo Yang1 1Korea Advanced Institute of Science & Technology, Republic of Korea, 2Seoul National University, Republic of Korea Abstract People who live with patients with brain disorders are considered as a potential risk group leading to mental disorder. They suffer from a caregiving burden and distressing situation. Consequently, the longer caregiving, the worse their quality of life. In spite of their devoted effort, caregiver’s deteriorated state has been overlooked. Here, we attempted to set a longterm housing model reflecting the particular situation. Mice were housed with a conspecific temporal lobe epilepsy model mouse or inescapable foot-shock stress mouse models. After long-term housing, cage-mate was performed behavior testsPeople who live with patients with brain disorders are considered as a potential risk group leading to mental disorder. They suffer from a caregiving burden and distressing situation. Consequently, the longer caregiving, the worse their quality of life. In spite of their devoted effort, caregiver’s deteriorated state has been overlooked. Here, we attempted to set a longterm housing model reflecting the particular situation. Mice were housed with a conspecific temporal lobe epilepsy model mouse or inescapable foot-shock stress mouse models. After long-term housing, cage-mate was performed behavior tests and electrophysiological investigation. The conspecific cagemate showed increased anxiety and depression-like behavior. Furthermore, they showed significantly reduced social interaction with juvenile and anesthetized mouse. Behavioral dysfunction of cage-mate sustained four weeks after removing the mouse model. Interestingly, fluoxetine, serotonin selective reuptake inhibitor, hardly restored their behaviors. Our results suggest that a dweller whose cage-mate having brain disorder could develop abnormal behavior including reduced social and increased depression-like behavior. These findings may help to understand psychosocial or psychiatric symptoms frequently observed in at-risk nursing people or caregivers. PT669 Investigation of the antiinflammatory and gastric side effects of pregabalin Fatma Sultan Kilic1, Bilgin Kaygisiz1, Sule Aydin1, Hadi Karimkhani2, Cafer Yildirim1, Setenay Oner3 1 Eskisehir Osmangazi University Medical School, Department of Pharmacology 2 Eskisehir Osmangazi University Medical School, Department of Biochemistry 3 Eskisehir Osmangazi University Medical School, Department of Biostatistics Abstract Objectives: Nonsteroidal antiinflammatory drugs are used for the relief of inflammation, however gastrointestinal side effects restrict their clinical use. We aimed to investigate the antiinflammatory effects of pregabalin, a drug used in epilepsy, anxiety, neuropathic pain treatment, on carrageenaninduced paw edema and to evaluate its gastric side effects in Wistar rats. Methods: Pregabalin 30,50,100mg/kg; indomethacin 5mg/ kg(reference drug), vehicle(saline) were injected intraperitoneally before 100μl of 1% carrageenan administration into the right hind paws of the rats. Paw thickness was measured by a gauge calipers(Vernier Calipers) before (0th hour) and in every hour during 6 hours after induction of inflammation. Paw thickness of treated groups were compared with control group with One-way ANOVA. Also paw thickness in 0th and 6th hours were compared within each group with twoway ANOVA. Pregabalin was administered orally for 10 days to evaluate gastric side effect. At the end of 10 day treatment, rats were sacrificed, gastric tissues were removed out, mucus secretion was determined spectrophotometrically, ulcer index was scored from score 0:(no petechia) to score 3:(petechia>5mm). Results: There was no significant difference between 0th and 6th hours in paw thickness of all groups, except carrageenan group. Carrageenan significantly increased paw thickness in 6th hour compared to 0th hour. All doses of pregabalin and indomethacin significantly reduced paw thickness in 6th hour compared to carrageenan group. Pregabalin 50 and 100mg/kg similar to indomethacin significantly reduced mucus secretion and increased ulcer index compared to control while pregabalin 30mg/kg did not. Conclusion: All doses of pregabalin exerted antiinflammatory effects comparable to indomethacin, 50 and 100mg/kg pregabalin showed gastric side effects as reduced mucus secretion and ulcer formation similar to indomethacin and 30mg/kg pregabalin may be reasonable dose for antiinflammatory effect without showing gastric side effects. This study was supported by ‘Scientific Researchs Projects’ of Eskisehir Osmangazi University.