Effects of antipsychotic drugs on pain threshold and motor behavior in a rat model of schizophrenia

Gang LI，Meng Meng JI，Shuai YANG，Dong Hong CUI，Huan Jun CAO，Jian Feng YU*

Effects of antipsychotic drugs on pain threshold and motor behavior in a rat model of schizophrenia

Gang LI1，Meng Meng JI1，Shuai YANG1，Dong Hong CUI2，Huan Jun CAO1，Jian Feng YU1*

Background：Previous investigations have suggested that patients with schizophrenia have decreased pain sensitivity that is partially reversed with antipsychotic treatment.One way to assess this hypothesis is to test it in animal models of schizophrenia.

Objective：Determine whether or not rats that manifest the expected behavioral changes of a ketamine-induced rat model of schizophrenia have an increased pain threshold，and test whether or not pretreatment with antipsychotic medication reverses this increase in the pain threshold.

Methods：30 male Wistar rats were randomly assigned to five groups：three groups received intraperitoneal antipscyhotics［risperidone(0.3mg/kg)，risperidone(0.9mg/kg)or haloperidol(1mg/kg)］30 minutes prior to receiving intraperitoneal ketamine(100mg/kg);one group received normal saline followed by intraperitoneal ketamine;and a control group received two injections of normal saline.The threshold values for pressure pain and thermal pain were assessed at baseline and at 5，15，30 and 45 minutes after the second injection.The behaviors of another 30 rats treated in the same manner were assessed using the open field test for 120 minutes after receiving the second injection.

Results：Compared to the control group，rats in the ketamine group(without pretreatment with antipsychotics)had decreased thresholds for pressure pain and increased thresholds for thermal pain at all time periods after administration of ketamine.Pretreatment with haloperidol significantly diminished the ketamine-induced decreased pressure pain threshold at all time periods but low-or high-dose risperidone had no effect on pressure pain thresholds. Pretreatment with low-dose risperidone reduced the ketamine-induced increase in thermal pain threshold 5 minutes after the ketamine injection but not at 15-45 minutes after the ketamine injection;high-dose risperidone and haloperidol had no significant effect on thermal pain thresholds.In the open field test the groups pretreated with antipsychotics had fewer standing upright behaviors(goal-directed behavior)，fewer crossed grids(less hyperactivity)，and fewer head shakes and circuling body movements(less stereotyped behavior)than the rats treated with ketamine that did not receive pretreatment with antipsychotics.Falls(i.e.，ataxia)were significantly more common in the haloperidol pretreatment group than in the ketamine group(without pretreatment)and than in the two risperidone pretreatment groups.

Conclusion：We did not confirm previous findings about the inhibition of ketamine-induced increased pain thresholds by antipsychotics in a rat model of schizophrenia.The surprising decrease in pressure-pain thresholds with ketamine may be related to the increased overall activity following ketamine injection(which made it difficult to reliably conduct the pressure pain threshold test).Risperidone and haloperidol effectively reduce many of the psychomimetic effects of ketamine，but haloperidol increased ketamine-induced ataxia while risperidone decreased ataxia，a finding that could be clinically relevant for elderly patients who are prone to falls.

Risperidone;Haloperidol;Ketamine;Schizophrenia;Pain threshold;Motor behavior;Animal models

1 Introduction

Ketamine is a glutamate N-Methyl-D-aspartate (NMDA)receptor antagonist and an important intravenous anesthetic.It has satisfactory analgesic effects and mildly inhibits respiratory and circu

latory functions，but the adverse reactions of this drug have limited its clinical applications［1］.Among these adverse reactions are ketamine's psychomimetic properties，which can be used to induce an animal model of schizophrenia［2-4］that is better than the animal model induced by dopamine agonists［5］.

Recent studies report reduced responsiveness to various kinds of pain stimuli in patients with schizophrenia［6］which may be related to a functional disorder of NMDA receptors［7］.Though not a core symptom of schizophrenia，changed sensitivity to pain may be clinically important because it has been associated with functional recovery，reintegration into the community and the occurrence of violent behavior［8］.

Antipsychotic drugs can alleviate some symptoms of schizophrenia in both living patients and in animal models［6，9］，but findings from studies on the relationship of antipsychotic treatment to the pain threshold in schizophrenia have been contradictory.In some studies haloperidol and risperidone have no effect on pain thresholds［10］.But Becker's group［8］—which used an indirect method of assessing pain thresholds and low doses of ketamine (30mg/kg)—reports that pretreatment with antipsychotic drugs can affect pain sensitivity in ketamine-induced rat models for schizophrenia.The present study aims to replicate Becker's findings using an acute dose of ketamine(100mg/kg)to induce a rat model of schizophrenia and direct measures of pain thresholds.

2 Materials and methods

2.1 Experimental animals

Sixty clean-grade male Wistar rats weighing 150-200g purchased from the Shandong Lukang Pharmaceutical Company were housed in a quiet and warm animal room with proper humidity，dimmed lighting and free access to food and water for at least 48 hours before the experiment.The study was carried out from April 1，2010 to May 1，2010.None of the rats died during the experiment.

2.2 Medications

Medications used were as follows：ketamine hydrochloride injection(50mg/ml)，produced by the Jiangsu Hengrui Medicine Company;risperidone powder，purchased from the Sigma Company in the USA，dissolved in acetic acid and diluted to 0.5mg/ml;and haloperidol injection(5mg/ml) produced by the Shanghai Xudong Haipu Pharmaceutical Company.

2.3 Medication administration

Two separate sets of 30 male Wistar rats were used in the study，one for the pain threshold assessment and one for the open field test(Figure 1). Each set of 30 rats was divided into five groups using a random numbers table，with six rats in each group.The first three groups were administered intraperitoneal injections with antipsychotic drugs before receiving ketamine;physiological saline was added to each injection to result in a final volume of 0.4ml.After 30 minutes，these three groups were administered 100mg/kg ketamine diluted to result in a final intraperitoneal injection of 0.4 ml.The antipsychotic doses for the three groups were 1)a low-dose risperidone pretreatment group received 0.3mg/kg risperidone;2)a high-dose risperidone pretreatment group received 0.9 mg/kg risperidone.And 3)a haloperidol pretreatment group received 1mg/kg of haloperidol.(These dosages for assessing pain thresholds were based on the study by Alimohamad and colleagues［11］.)The last two groups were administered an intraperitoneal injection of 0.4ml physiological saline as a pretreatment control;the fourth group was subsequently administereda0.4mlintraperitonealinjectionwith 100mg/kg ketamine and the fifth group was subsequently administered a second 0.4ml intraperitoneal injection of physiological saline(the control condition for the ketamine administration).

2.4 Behavioral experiments

Pain threshold testing and the open field test were independently conducted by two researchers.

2.4.1 Determination of pressure pain and thermal pain thresholds

The pressure pain and thermal pain thresholds were determined using the Electronic Von Frey 2390-5 tenderness meter(IITC Life Science Corporation，USA)and the SW-200 tail light and heat pain test instrument(Chengdu TME Technology Company).Baseline(pretreatment)values of the pressure and thermal pain thresholds for each rat were the average of six measurements assessed with a 15-minute interval between each measurement.

The specific method of determining the pain thresholds was based on the protocol developed by Johnson and colleagues［12］.When assessing the pressure pain threshold a researcher gently holds the rat and after the rat was in a quiet state presses their posterior instep until the rat lifts its leg in avoidance.The pressure pain meter records the force on the rat's instep in grams.When assessing the thermal pain threshold the tail of a quiet rat is exposed to a light source(thus generating heat);the time until the rat flicks its tail(the flick reflex)is recorded as the thermal pain threshold.To prevent burning of the tail，exposure is terminated if the rat fails to flick its tail for 20 seconds，and 20 seconds is recorded as the threshold.The tenderness and thermal pain thresholds were assessed 5，15，30 and 45 minutes after the second intraperitoneal injection(which was ketamine in 5 groups and normal saline in one group).

2.4.2 Open field test

An open field test was conducted to verify that the ketamine dosages employed were inducing pschyomimetic(i.e.，schizophreniform)activity in the rats.Based on the standard method［13］，the open field test was conducted with a 100cm×100cm× 40cm box with black walls and a bottom divided into a 5×5 grid with 25 squares of equal size.A digital camera attached to the top of the box recorded the test，which was conducted in a soundproof room.The rats had 30 minutes of environmental acclimation at 10：00 to 12：00 every morning for seven continuous days before the actual test.On the day of the test，the rats were kept in the open box for acclimation for 10 minutes，and then they were administered medications following the same procedure as that described for the pain threshold tests(above).After the second injection the rats were placed in the central square of the grid and their behavior was recorded for 120 minutes.Each of the 30 rats was tested once.The feces and urine in the box were removed after each test.

The following variables were recorded：the number of grids crossed(assessing motor status)，the frequency of falling(assessing ataxia)，the frequency of upright standing behavior(assessing conscious activities)，the frequency of circling body movements(assessing stereotyped behavior)，and the frequency of head shaking(assessing stereotyped behavior).This is the standard method of assessing results for the open field test and has been employed in previous research on rat behavior after administration of ketamine［14，15］.

2.5 Statistical Methods

To show percent change in pain thresholds from baseline in the different groups，we used the standard method employed by Becker and colleagues［8］：percent change=［(Experimental pain threshold-baseline pain threshold)/baseline pain threshold］×100%.

The SPSS 11.5 software package was used for statistical analysis.All results were expressed as means and standard deviations.One-way ANOVA was used to compare the five groups，if significantly different，post-hoc tests were used to make subsequent pairwise comparisons between groups(LSD if the groups had equal variances and Dunnett's T3if the groups had unequal variances).Statistical significance was defined at the 0.05 level.

3 Results

3.1 Effects of ketamine on pain threshold and open field motor behavior

In comparison to the control group，the pressure pain thresholds of rats that received ketamine without antipsychotic pretreatment were significantly lower at all four time points(Table 1)，while their thermal pain thresholds were significantly higher at all four time points(Table 2).

In the open field trial compared to the control group，rats that received ketamine without antipsychotic pretreatment had significant increases in grid crossings，falls，head shaking behaviors and circling body movements;but the number of upright standing behaviors(which are considered goal-oriented behaviors)were significantly decreased(Table 3).

3.2 Effects of antipsychotics on pain threshold

3.2.1 Pressure pain threshold

At all four time points the pressure pain thresholds for rats treated with ketamine were significantly higher in rats pretreated with high-dose risperidone than in those with no antipsychotic pretreatment and than in those pretreated with haloperidol or low-dose risperidone.The differences between the pain threshold in the latter three groups were not statistically significant(Table 1).Thus pretreatment with high-dose risperidone reduced，but did not eliminate，the decrease in the pressure pain threshold induced by ketamine.

3.2.2 Thermal pain threshold

At the fifth minute following ketamine injection，the thermal pain threshold in rats pretreated with low-dose risperidone was significantly lower than in the other three groups.At other time points，however，therewerenostatisticallysignificant differences in the thermal pain thresholds between the four groups(Table 2).Thus pre-treatment with low-dose risperidone had a transient effect onreducing the increase in the thermal pain threshold induced by ketamine.

3.3 Effects of antipsychotic pretreatment on ketamine-induced motor behaviors

3.3.1 Total numbers of passed grids

The total number grid crossing of rats in the three antipsychotic pretreatment groups were significantly less than in ketamine-treated rats that did not have pretreatment with antipsychotic medications.The number of grid crossings in the high-dose risperidone group and in the haloperidol group were not significantly different from the control group，but the number of grid crossings in the lowdose risperidone group remained significantly higher than in the control group.Thus high-dose risperidone and haloperidol eliminated the effect of ketamine on grid crossings while low-dose resperidone reduced，but did not eliminate，this effect.

3.3.2 Total numbers of upright standing behaviors

None of the rats in the three pretreatment groups exhibited any upright standing behaviors during the 120-minute observation period;this was significantly less than in ketamine-treated rats who did not receive pretreatment with antipsychotics and significantly less than in the control group.

3.3.3 Total numbers of falls

3.3.4 Total numbers head shakes circling bodymovements

The total number of head shakes and circling body movements in the three antipsychotic pretreatment groups were significantly less than in rats that received ketamine without pretreatment with antipsychotics.The high-dose risperidone group had fewer head shaking and circling body movements than rats in the other two antipsychotic pretreatment groups but the differences between the low-dose risperidone group and the haloperidol group were not statistically significant(Table 3).

4 Discussion

4.1 Main findings

Similar to previous studies we found that ketamine induces hyperactivity，stereotyped behavior and ataxia and decreases goal-directed behavior［16］.The reduction in goal directed behavior (i.e.，standing on hind legs)induced by ketamine was magnified，not ameliorated，by pretreatment with both risperidone and haloperidol.Risperidone had a significant inhibitory effect on the ketamineinduced hyperactivity，stereotyped behavior and ataxia.Haloperidolalso alleviated hyperactivity and stereotyped behaviors，but appeared to aggravate ataxia.Thus the therapeutic efficacy of risperidone on the psychomimetic effects induced by high-dose ketamine was better than that of haloperidol，a finding which parallels conclusions from two clinical investigations［11，17］.Falls are a major cause for morbidity and can be fatal in the elderly，particularly in elderly with mental disorders［18］，so the finding of decreased ataxia with risperidone has important clinical implications.

As expected the thermal pain threshold was increased with ketamine but，against expectations，we found that the pressure pain threshold was significantly decreased after the intraperitoneal injection of ketamine.We hypothesize that this inconsistency between the decrease in the pressure pain threshold and the analgesic effects of ketamine may be related to the side effect of increased movements induced by ketamine.The test method for the pressure pain threshold in the present study was based on the lifting of lower limbs after manually applying pressure.Unfortunately，as we saw in the open box test，ketamine induces hyperactivity(i.e，increased movement).This made any results obtained from this measure of the pressure pain threshold suspect.Our results could be interpreted as increased movements with ketamine(thus appearing to have a decreased pressure pain threshold)that were reduced by high-dose risperidone. The opposite effects of ketamine on the pressure pain and thermal pain thresholds could also be explained by differential effects of ketamine on the different afferent nerves which relay pressure and thermal pain，though we consider this a less plausible hypothesis.

Low-dose risperidone had some effect in reversing the ketamine-induced increase in the thermal pain threshold at 5 minutes after the ketamine injection but not at 15，30 or 45 minutes after the injection.High-dose risperidone and haloperidol had no statistically significant effect on the ketamine-induced increased thermal pain threshold at any of the time points.The transient effect of lowdose risperidone in inhibiting the increased ketamine-induced thermal pain threshold suggests that risperidone's 5-serotonin(5-HTA)and dopamine (DA)antagonistic effects［19，20］play a role in the pain threshold abnormalities seen in schizophrenia，but this hypothesis would need to be tested further. The lack of inhibition of the increased pain threshold over longer periods with low-dose risperidone and with high-dose risperidone and haloperidol—which contradicts the results of Becker and colleagues［8］—remains unexplained，but it may be related to the complex relationship between the analgesic and psychomimetic effects of ketamine or to the higher doses of ketamine(100mg/kg)employed in this study compared to previons studies (30mg/kg)［8］.

4.2 Limitations

The biggest limitation of the study was that the pressure pain threshold test might have been contaminated by the hyperkinetic side-effects of ketamine.There do not appear to be many remedies to address this problem.One cannot ethically test pain thresholds in humans with schizophrenia.And most measures of pain rely on when a pain stimulus causes movement so other factors that affect movement(such as ketamine administration)can confound the results.Future studies need to find creative ways to control for this issue.The indicators that are frequently used to assess the analgesic effect of ketamine include thermal pain，pressure pain，and visceral pain thresholds［21］.This study did not assess the visceral pain threshold，so different results might have been obtained had this measure been included.And the small number of experimental animals in each group(six)may have made it difficult to identify real differences between the groups.

4.3 Implications

The present study has supported the notion that that risperidone and haloperidol can effectively reduce some，but not all，of the psychomimetic effects of ketamine.One of the most interesting findings was that risperidone was better at alleviating ketamine-induced ataxia than haloperidol，which could have important implications for the treatment of schizophrenia，particularly elderly individuals with schizophrenia(and，perhaps，dementia).

No clear conclusions can be drawn about the effect of antipsychotic treatment on the pain thresholds of persons with schizophrenia.Pressure pain thresholds are typically assessed in terms of voluntary movement，so the assessment can be confounded when the subject is treated with a medication that increases movement(such as ketamine). The test of thermal pain was less affected by this problem because it was based on involuntary movement(the rat's flick reflex)but in this case we only found a significant inhibition of the ketamine-induced decreased thermal pain threshold for one of the three medication regimens at one of the four time periods.

Funding

This study was funded by the‘Innovation of Technology for College Students'fund of Weifang Medical University(KX2009012).The study did not receive any financial support from pharmaceutical companies.

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(received date：2010-12-22;accepted date：2011-05-04)

抗精神病藥物對精神分裂癥大鼠模型痛閾和運動行為的影響

李 剛1紀蒙蒙1楊 帥1崔東紅2曹煥軍1于劍鋒1

基金項目：濰坊醫(yī)學院大學生科技創(chuàng)新基金（KX2009012)

作者單位：1濰坊醫(yī)學院261053；2上海交通大學醫(yī)學院附屬精神衛(wèi)生中心200030。

通信作者：于劍鋒，電子信箱yujf@wfmc.edu.cn

背景既往研究提示精神分裂癥患者的疼痛敏感性降低并且可以被抗精神病藥物部分逆轉。對這一假說的評價方法之一是檢測精神分裂癥模型。

目的在顯示出預期的行為學改變的氯胺酮誘導的精神分裂癥大鼠模型中，檢測是否出現(xiàn)痛閾升高，并且檢測抗精神病藥物預處理是否逆轉這種痛閾升高。

方法將30只雄性Wistar大鼠隨機分為5組，其中3組先腹腔注射抗精神病藥物預處理［利培酮（0.3 mg/kg）、利培酮（0.9 mg/kg）或氟哌啶醇（1 mg/kg）］，30 min后再腹腔注射氯胺酮（100 mg/kg）；1組先腹腔注射生理鹽水，再腹腔注射氯胺酮；1個對照組接受2次生理鹽水注射。測定大鼠在基線以及第二次注射后第5、15、30和45 min的壓痛和熱痛閾值。另外的30只大鼠做同樣處理，用曠場實驗觀測大鼠在第二次注射后120 min內的行為改變。

結果 與對照組相比，在所有時間段內，氯胺酮組大鼠（未用抗精神病藥物）出現(xiàn)壓痛閾降低、熱痛閾升高。在所有時間段內，氟哌啶醇預處理明顯減輕了氯胺酮誘導的壓痛降低，但高或低劑量的利培酮對壓痛閾無明顯影響。用低劑量利培酮預處理減輕了氯胺酮誘導的第5 min時段的熱痛閾升高，但不包括第15～45 min時段。高劑量利培酮和氟哌啶醇對熱痛閾無明顯影響。在曠場實驗中，抗精神病藥物預處理的各組比僅用氯胺酮處理的一組有較少的直立行為（目標指向的行為），較少的穿越格子行為（較低的高運動性），較少的搖頭和轉圈運動。氟哌啶醇預處理組比氯胺酮組（無預處理）和2個利培酮預處理組相比，摔倒（即共濟失調）更為常見。

結論我們不能肯定先前所見的在精神分裂癥大鼠模型中抗精神病藥物對氯胺酮誘導的痛閾升高有抑制作用。氯胺酮引起的壓痛閾明顯降低可能與氯胺酮注射后增強的全身活動性有關（這使壓痛閾檢測難以可靠進行）。利培酮和氟哌啶醇有效減輕多方面的氯胺酮引起的擬精神病癥狀，而氟哌啶醇增強了氯胺酮引起的共濟失調，利培酮降低了共濟失調。這一資料與易于摔倒的老年患者有臨床相關性。

利培酮 氟哌啶醇 氯胺酮 精神分裂癥 痛閾 運動行為 動物模型

number of falls in rats that

pretreatmentwithhaloperidolwassignificantly higher than in the other four groups.Rats that received ketamine without pretreatment with antipsychotics had significantly more falls than rats pretreated with risperidone，and rats pretreated with low-dose risperidone had significantly more falls than rats pretreated with high-dose risperidone.

10.3969/j.issn.1002-0829.2011.04.005

1Weifang Medical University，Weifang 261053;

2Shanghai Mental Health Center，Shanghai Jiao Tong University School of Medicine，Shanghai 200030

*Correspondence：yujf@wfmc.edu.cn