The Expression Of The NMDA Receptor NR2A And NR2B Subunits In The Spinal Cord, Hippocampus And Celebral Cortex Of Acute Opioid Tolerance Rats

Background:Opioids are widely used in the perioperative period owing to their profound analgesic effect.Acute administration of opioids results in analgesia and side effects,whereas tolerance and dependence only occur after chronic administration.However,it has become more recently recognized that tolerance can also develop rapidly after an acute opioid exposure in animals and humans.Tolerance to opioids is classically defined as a progressive reduction of their analgesic effect,thus explaining the need for a larger dose to achieve the same pharmacological effect.A growing body of evidence suggests that opioid analgesics can elicit hyperalgesia(exaggerated nociceptive response to noxious stimulation) in experimental models after repeated opioid administrations or continuous delivering.Such a pain sensitivity enhancement could explain a reduction of the opioid analgesic effect.It was reported that a single administration of an opioid also induces a longlasting increase of basal pain sensitivity, leading to delayed hyperalgesia,which,if not taken into account,gives the impression of less analgesia(i.e.,apparent tolerance) when a new opioid administration is performed.A series of clinical reports also indicate that opioids can produce abnormal pain including allodynia(nociceptive responses to innocuous stimulation) or hyperalgesia.If tolerance and pain facilitation share some common pathways, thenanimals(or humans) with acute tolerance to opioid analgesia should be hyperalgesic after the opioid administration.Fentanyl and morphine are widely used perioperative analgesics.It was reported that intraoperative administration of fentanyl could cause postoperative pain enhancement and morphine requirement increasement. There is now a substantial amount of evidence that glutamate via N-methyl-D-aspartate(NMDA) receptors play a pivotal role in the development and maintenance of central hyperactive states underlying the behavioral manifestations of pain facilitation such as hyperalgesia,allodynia,and spontaneous pain.Moreover, tolerance is prevented by NMDA-receptor antagonists.NMDA-receptor complex is comprised with NR1 subunit combined with subunits of NR2 family:NR2A,NR2B, NR2C,NR2D.The NMDA receptors containing different subunits may play different roles.In this study we examined the effects of acute Fentanyl administration on protein expression of two major NMDA receptors subunits(NR2B and NR2A) in the spinal cord,hippocampus and cerebral cortex of rats.PartⅠ.Establishment of Acute Opioid Tolerance ModelObjective:Fentanyl was injected subcutaneously to establishment the acute opioid tolerance model and morphine subcutaneous injection was used to test if the model is successful.Methods:24 male Sprague-Dawley rats,weighing 180-200g,were randomly divided into three groups with 8 rats each:Control,Saline and Fentanyl group.Fentanyl was injected subcutaneously four times at 15 min intervals(30μg/kg per injection) in Fentanyl group.Rats in Saline group received saline injections instead of Fentanyl. And no injections were given to the rats of Control group.The mechanical pain thresholds of hindpaw were measured every 30 min with a Von-Frey hair test.When the mechanical pain thresholds of Fentanyl group returned to the baseline which had been measured before injection,an injection of morphine(5mg/kg) was performed to every rat of these 3 groups.Subsequent changes of the mechanical pain thresholds were still measured every 30 min until the mechanical pain thresholds of Fentanyl group returned to the baseline again.The mechanical pain thresholds at each time point were compared with the baseline and the corresponding one of the other two groups.Results:Four fentanyl boluses(in group F) elicited an increase followed by an immediate decrease of the nociceptive threshold and reduction of the analgesic effect of a subsequent morphine administration compared with the other two groups(group S and C).Conclusions:Opioid tolerance could develop rapidly after an acute opioid exposure in rats.PartⅡ.The Expression of the NMDA Receptor NR2A and NR2B Subunits in the Spinal Cord,Hippocampus and Celebral Cortex of Acute Opioid Tolerance RatsObjective:To examine the effects of acute Fentanyl administration on protein expression of two major NMDA receptors subunits(NR2B and NR2A) in the spinal cord,hippocampus and cerebral cortex of rats.Methods:24 male Sprague-Dawley rats,weighing 180-200g,were randomly divided into three groups with 8 rats each:Control~*,Saline~* and Fentanyl~* group.Fentanyl was administrated as partⅠexcept no morphine administrated.When the mechanical pain thresholds of Fentanyl group retumed to the baseline,all of the rats were killed. The dorsal horns of the L4-L5 spinal cord were sampled and the protein level of NR2B and NR2A subunits were tested with the Western blot method. Results:According to the results of Western blot,in spinal cord and hippocampus,the protein levels of NR2B subunit of Fentanyl~* group were higher than that of the Control~* and Saline~* group,but there is no difference of the protein levels of NR2A subunit between Fentanyl~* group and the other two groups.In cerebral cortex,the protein levels of NR2B and NR2A subunit were similar in Fentanyl~* and the other two groups.Conclusion:The protein levels of NR2B subunit of spinal cord and hippoeampus increased in acute opioid tolerance rats,which might imply that NMDA receptors with NR2B subunits play an important role in acute opioid tolerance.