Preemptive analgesic eff ects of midazolam and diclofenac in rat model

Th e aim of the present study was to investigate the preemptive analgesic eff ects of intraperitoneally administrated midazolam and diclofenac, before acute and infl ammatory induced pain in rat model. One hundred twenty-eight (n= in each group) male Sprague Dawley rats were included in the study. Paw movements in response to thermal stimulation or paw fl inching in response to formalin injection were compared after midazolam (., ,  and  mg/kg) and diclofenac ( mg/ kg), intraperitoneal administration. Saline was used as a control. Preemptive analgesic eff ect was signifi cant in both tests when diclofenac and midazolam was administrated before the pain stimuli (p<. and p<.). Intraperitoneal injection of midazolam in doses  and  mg/kg, increase the response time in hot plate test and decrease the number of fl inches in formalin test (p<. vs. p<.). ED of midazolam (with diclofenac) in hot plate test was . mg/kg (CI =-.-. mg); and, . mg/kg (CI =-.-. mg) in phase I and . mg/kg (CI = .-. mg) in phase II, in formalin test. Intraperitoneally administered midazolam and diclofenac had preemptive analgesic eff ects on acute thermal, and infl ammatory induced pain in rats. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Preemptive analgesia is an antinociceptive treatment that prevents the establishment of altered processing of aff erent input that amplifi es pain [].Pain associated with tissue damage results in prolonged modulation of the somatosensory system, with increased responsiveness of both peripheral and central pain pathways [].Experimental evidence proposes that to 'prevent' or 'preempt' the noxious input to the CNS, may be more eff ective than treatment.Th e idea of preemptive analgesia was fi rst introduced into clinical practice by Crile in  [] and further developed by Wall [] and Woolf [].Th e defi nition of preemptive analgesia was formed by Kissin [].According to him, preemptive analgesia is "treatment that prevents establishment of central sensitization caused by incisional and infl ammatory injuries; it starts before incision and covers both the period of surgery and the initial postoperative period.Preemptive analgesia prevents pathologic pain that is diff erent from physiologic pain", which means: prevention or reversal of central and peripheral sensitization.
Midazolam, a benzodiazepine, eff ects mediated primarily via the central benzodiazepine receptors located in the central nervous system.Central benzodiazepine receptors are part of a macromolecular complex that also contains a γ-amino butyric acid (GABA) receptor site and a chloride ion channel.Midazolam potentiate the eff ects of GABA A receptors [].It is widely used during general anesthesia and decrease the requirement to other anesthetics.Analgesic eff ects of intrathecally given midazolam are well known [-].However, the antinociceptive eff ects of midazolam administered systematically are actually becoming well known in recent years.Nishiyama [] and Chiba et al. [] demonstrated the antinociceptive eff ect of systemically administrated midazolam in acute thermal and infl ammatory induced pain in animals.Diclofenac sodium, -[(,-dichlorophenyl) amino] benzene acetic acid, is a non-steroidal anti-infl ammatory drug (NSAID) with an approximate relative COX- ⁄ COX- specifi city ratio of one [].NSAIDs inhibit the cyclo-oxygenase enzymes (COX) and decrease peripheral and central prostaglandin production.To reduce the infl ammation that accompanies tissue injury, decreasing prostaglandin production attenuates the response of the peripheral and central components of the nervous system to noxious stimuli and reduce the pain occurred in response to following noxious stimuli [].Th ese properties would seem to make NSAIDs idea l Preemptive analgesic eff ect of diclofenac is discussed in many studies, but the results are still controversy [-].


In the present study the preemptive analgesic properties of systemically administered midazolam and diclofenac sodium were investigated in a rat model of acute and inflammatory pain.

MATERIALS AND METHODS
After Institutional Ethics Committee approval,  male ( for each group) Sprague Dawley rats, weighing - g, were included in the study.Th e animals were housed in a cage at -ºC under diurnal light condition and allowed to access food and water ad libitum.All experiment was done in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. -, revised ).Midazolam (F.Hoff mann-La Roche, Swiss) ., ,  and  mg/kg and diclofenac sodium (Proanalysis-Merck, Germany)  mg/kg, was dissolved in normal saline to achieve solutions for intraperitoneal (i.p.) administration.Th e total injected volume was adjusted to  ml/kg in each rat.Th ey were used for i.p. injection at -ml syringe with a -G needle.Th e rats are divided in three groups; Group I, midazolam ., ,  and  mg/kg (n= in each group) given i.p.  min before the nociceptive stimulus realized with hot plate test and formalin test; Group II, diclofenac at  mg/kg (n= in each group) given i.p.  min before the nociceptive stimulus realized with hot plate test and formalin test, and; Group III, midazolam ., ,  and mg/kg with diclofenac  mg/kg (n= in each group) given i.p.  min before the nociceptive stimulus, realized with hot plate test and formalin test.Saline group (n=), normal saline given i.p. was used as a control.Th e acute thermal pain was realized with hot plate test.Th e hot plate test was performed at  ºC on the paw of each rat.Animals were placed on the heated smooth surface and the latency of licking, shaking of the limbs, or jumping was measured.Hot plate tests were performed  min or  min after i.p. drug injection, and repeated every  min during  min.To prevent the tissue injury the rats were removed from the hot plate test after  sec.Pain inhibition percentage (PIP) was calculated with following formula []:

post drug latency; T  -pre drug latency
The formalin test, model of inflammatory pain, was performed  or  minutes after drug administration.Fifty microlitres of  formalin was injected subcutaneously into the dorsal surface of the right hind paw with a -G needle.Immediately after injection, the rat was placed in an open surface, and their paw response (fl inching or shaking) was observed at ten minutes intervals for a period of one hour.Th e number of movements was counted for one minute.Two phases were observed: phase  for the first six minutes after injection; and phase  beginning after about ten minutes.The paw movements were measured every  minutes during  minutes.A nociceptive score was determined by measuring the  behavioural categories: , the position and posture of the hind paw is indistinguishable from the contralateral paw; , the paw has little or no weight placed on it; , the paw is elevated and is not in contact with any surface; , the paw is licked, bitten or shaken.Behavioral side eff ects (agitation, allodinia, catatonic excitement and fl accidity) were observed in animals during the study.

Statistical analysis
Statistical analysis was performed using SPSS version .Data are expressed as the mean ± SD or  confi dence interval (CI).Th e experimental groups were compared by the nonparametric Kruskal-Wallis test.Multiple comparisons after the Kruskal-Wallis test were performed using the two-tailed Dunn test.A p value less than . were considered signifi cant.Preemptive antinociceptive effects of diclofenac sodium and midazolam in hot plate test were demonstrated by increase in response time compared with control group (saline).After i.p. administration of diclofenac  mg/kg and midazolam signifi cant increase in response time were observed in doses ., ,  and  mg/kg (p<.;p<., respectively).Dose depend antinociceptive eff ects were observed , , , , ,  and  min.after i.p. . and  mg/kg midazolam and diclofenac.Th e increase in response time, in doses  and  mg/kg midazolam and diclofenac, started in  min and continue to increase during  min (Figure ).ED  of midazolam (with diclofenac) in hot plate test was . mg/kg (CI  =-.-.mg).Preemptive antinociceptive effects of midazolam and diclofenac sodium in formalin test were demonstrated by observation of flinching or shaking of paw after formalin injection.The paw response observed at five minutes intervals during a period of  minutes were compared with control group (saline).The number of paw flinches decreased significantly, when diclofenac sodium was administrated i.p. in dose  mg/kg and midazolam in doses ., ,  and  mg/kg (p<.;p<. and p<., respectively).(Figure ).Dose depend antinociceptive eff ects were observed in both phases of formalin test; phase I and II.The  effective dose (ED  ) of the formalin test was calculated in both phases after the midazolam and diclofenac i.p. administration.The ED  of midazolam (with diclofenac) . mg/ kg (CI  =-.-.mg) in phase I and . mg/kg (CI  = .-.mg) in phase II.No behavioural side eff ects were observed in any animal.

DISCUSSION
In this recent study, we demonstrated that intraperitoneally administered diclofenac and midazolam had preemptive analgesic eff ects in the hot plate test and formalin test in rats.Preemptive analgesia can reduce both the acute and inflammatory pain and in this way can reduce peripheral and central sensitization.
In our study, we used before (pre) versus placebo (no) design; we applied diclofenac and midazolam before pain stimuli.Several studies have addressed midazolam as an analgesic during intrathecal or epidural administration.Naguib et al. [] examined the analgesic effi cacy of caudal administration of midazolam, bupivacaine, or a mixture of both drugs in  children, undergoing inguinal herniotomy.Th ey found that caudal midazolam in a dose of  μg kg − provides equivalent analgesia to bupivacaine ..Nishijama [] studied the antinociceptive properties of systemically vs. inthratecally administered midazolam in a rat model of thermal and infl ammatory pain.Th ey observed systemically administered midazolam induced antinociception for infl ammatory pain only, while intrathecal administration elicited antinociceptive effects on both acute thermal and infl ammatory-induced pain.In  Chiba et al. [] reported the other study which was performed to investigate antinociceptive effects of different types of nociception in mice.They concluded that systemically administered midazolam had antinociceptive eff ects on acute thermal, acute mechanical and acute infl ammatory-induced nociception in mice.Th e antinociceptive potency of midazol-  am was the same for both acute thermal-induced nociception and mechanical-induced nociception.Ong et al. [] found that IV midazolam treatment (. mg/kg) in humans has a pain-reducing eff ect after third molar surgery, thus improving postoperative pain management.However, it has been shown that midazolam by bolus and continuous infusion resulted in a reduction in morphine consumption and lower pain scores in  patients undergoing hysterectomy [].Anxyolitic, muscle relaxing, and sedative eff ects of midazolam may modify the responses in nociceptive tests.In our study we didn't use any test to evaluate sensorimotor impairment induced by midazolam.Chiba et al. [] used the running wheel test and the balance beam test in their experiment and showed that with midazolam in dose  mg/kg  and  min after i.p. drug administration, mice were still able to run on the wheel ().As well Orii et al. [] reported that midazolam (, , and  mg/kg) did not induce any detectable reduction in motor response in rats.Preemptive analgesic eff ect was obvious with NSAIDs due to their mode of action, competing with arachidonic acid for binding to cyclooxygenase and decreasing the formation of prostaglandins [].Treatment with NSAIDs should be started as early as possible and should be initiated before the input of nociceptive stimuli.However, the clinical value of this technique remains still uncertain.Instrumentation of the uterus and Fallopian tubes during laparoscopy or surgery leads to prostaglandin release and, the prostaglandins released play a role in pain following laparoscopy [].Inhibition of prostaglandin production by the NSAIDs both peripherally and centrally should, therefore, decrease postoperative discomfort and reduce opioid requirement [].Woolf et al. [] showed no difference with preoperative diclofenac from postoperative diclofenac in patients undergoing laparoscopic tubal ligation.However, Buggy et al. [] and Gillberg et al. [] demonstrate that preoperative administration of ketorolac, piroxicam and diclofenac did reduce postoperative pain in patients undergoing laparoscopy.Our fi ndings support these results as well.Bjorkman [] studied the site and nature of the antinociceptive eff ect of diclofenac and paracetamol in the central nervous system.He observed the antinociceptive eff ect of diclofenac engage with central nervous component in diff erent areas of central nervous system.Herrero et al. [] studied the central antinociceptive eff ect of NSAID ketoprofen in two experiment models in rats and conclude that ketoprofen has central while peripheral analgesic activity.The present results suggest that intraperitoneally administered diclofenac has few eff ects at the level of the spinal cord and antinociceptive effects in the periphery and the brain.However the available preoperative trials of preemptive NSAID use have modest or unclear results and it may be due to controversy associated with the defi nition of pre-emptive analgesia.Even though, NSAIDs may have an ability to induce a preemptive analgesic eff ect.Our study suggests how the preoperative use of diclofenac was more eff ective.It is expected that NSAIDs will play an increasing role in preemptive analgesia and pain relief in general.Th e hot plate test evaluates supraspinal antinociceptive eff ects, and it refl ects activity in thermally sensitive aff erent fi bers and activity of Aδ and C fi bers [].Responses in the formalin test are mediated by both the spinal and supraspinal sites.Th e phase  response of the formalin test is caused by the direct stimulation of nociceptors by formalin or tissue damage, and is thought to be an acute pain reaction.Th is refl ects activity that is prominent in Aβ, Aδ and high-threshold C nociceptor aff erent fi bers.Th e phase  response is caused by infl ammation after formalin injection and central sensitization related to C activity.It refl ects activity in mechanically insensitive aff erent fi bers and activity of Aδ and C fi bers [].Sensory fi bers respond to physical and chemical stimuli producing mediators with origin from tissue injury and infl ammation.Th ese infl ammatory mediators activate or sensitize aff erent fi bers, and the neural impulses originated from nociceptors are transmitted via peripheral nerves to the spinal cord and with cranial nerves to cranial nerve ganglia.Prostaglandins are among the most important mediators of infl ammatory pain.During inflammation prostaglandin formation is induced by COX enzymes.NSAIDs block COX enzymes production and produce analgesia [].Studies have highlight that NSAIDs do not increase the pain threshold in animal model such as tail-fl ick and hotplate tests, but they normalize the pain behavior, which is observed after tissue injury and infl ammation mechanism [, ].However, diclofenac cause dose dependant analgesia.Th e ED values for diclofenac are . mg/kg (. ± .) [].We assume that the inadequate administered doses of drug may decrease the concentration in peripheral nociceptive terminals and antinociceptive response may fail.Th e intraperitoneally administrated dose of diclofenac sodium, in our study, was  mg/ kg, the optimal dose to cause antinociceptive response in rats.

CONCLUSION
We concluded that i.p. administered diclofenac and midazolam has preemptive and antinociceptive effects in acute thermal and inflammatory induced pain.Furthermore diclofenac added to midazolam enhance the antinociceptive effect of systemically administered midazolam.

DECLARATION OF INTEREST
Th e authors state that there is no confl ict of interest.
BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2011; 11 (2): 114-118 ANTIGONA HASANI ET AL.: PREEMPTIVE ANALGESIC EFFECTS OF MIDAZOLAM AND DICLOFENAC IN RAT MODEL drugs to use in a pre-emptive approach.

FIGURE 3 .
FIGURE 3. Hot plate test.Response time after i.p. administration of diclofenac sodium 10 mg/kg in the hot plate test with control (saline) given during 120 min, as indicated.Data are presented as the mean ± SD (8 mice in each dose).*p<0.05;**p<0.01;***p<0.001.

FIGURE 2 .
FIGURE 2. Formalin test.Number of fl inches in the formalin test with control (saline) and different doses of midazolam (0.1, 1, 5 and 10 mg/kg) given during 120 min, as indicated.Data are presented as the mean ± SD (8 mice in each dose).

FIGURE 4 .
FIGURE 4. Formalin test.Number of fl inches in the formalin test with control (saline) and 10 mg/kg diclofenac sodium given during 60 min, as indicated.Data are presented as the mean ± SD (8 mice in each dose).

FIGURE 6 .
FIGURE 6. Formalin test.Number of fl inches in the formalin test with control (saline) and diclofenac sodium 10 mg/kg and diff erent doses of midazolam (0.1, 1, 5 and 10 mg/kg) given during 60 min, as indicated.Data are presented as the mean ± SD (8 mice in each dose).
ANTIGONA HASANI ET AL.: PREEMPTIVE ANALGESIC EFFECTS OF MIDAZOLAM AND DICLOFENAC IN RAT MODEL RESULTS ED  ) of the hot plate test was calculated  min after the midazolam i.p. administration.The ED  . mg/kg (CI  =-.-.mg).Dose depend antinociceptive eff ects were observed in both phases of formalin test; phase I and II.Th e  eff ective dose (ED  ) of the formalin test was calculated in both phases after the midazolam i.p. administration.The ED  . mg/kg (CI  =-.-.mg) in phase I and . mg/kg (CI  = .-.mg) in phase II.Preemptive antinociceptive effects of diclofenac sodium ( mg/kg) administered i.p., in hot plate test and formalin test were demonstrated by increase in response time and decrease in paw flinches, compared with control group (saline) (p <.; p <.; p <., respectively) (Figure , ).