Effect Of Ovariectomy On Nociceptive Responses And Its Possible Mechanisms In Adult Female Rats

Pain is one of the common complications of many diseases. It is an unpleasant sense in all the human being, with high individual differences. Usually, acute pain is a symptom, but chronic pain is a type of disease. Compare to male, females usually encounter more periodical, severe and persistent pain. But, the occurrence rate of low back pain is significantly increasing over time after menopause (Adera et al.1994).However, most of the current research on pain and analgesia are carried out in male animals and there are relatively few studies to compare sex differences in pain. The male centered research strategy resulted in women receiving treatment therapy derived from researches which are not based on studies for female, so it is very urgent to study pain and analgesia in female.To a large extent, the sex difference of pain happens due to the action of sex hormones, among which, estrogen (especially Estradiol) plays an important role. Mechanisms of estrogen involvement in pain are very complicated in terms of the role of pro-or anti-pain, which is still controversial at present.Ovariectomy (OVX) is an ideal model to study the response to pain by estrogen substitute therapy. Presently, the model was used to evaluate the effect of estrogen on pain and analgesia (Fillingim and Ness 2000). Is there any change of pain threshold of rats after OVX? What kind of change will happen? What is the mechanism leading to the change, and is there any involvement of spinal cord in the process? As we known, estrogen acts on its specific receptor to produce effect, so we also aimed to ask if there is any alternation of estrogen receptor (ER) in spinal cord in this model. To answer the questions mentioned above, in this study, we carried a systematic research for the first time on the pain behavior in basal pain response of OVX rats, pain response of OVX rats after inflammatory pain and neuropathic pain. By measuring the change of ER in spinal cord, the effect of ER on pain and the possible mechanism was also explored. The main findings will be described bellow.1. Pain threshold change after OVX surgery.Mechanical allodynia:using von-Frey testing, OVX and OVX+vehicle group showed decreased mechanical pain threshold to the tactile stimuli (allodynia) in both hindpaws at 4 week after surgery. Significant allodynia appeared at 5 week post surgery and lasted at least until 7 weeks after OVX surgery. Compare to the naive group and sham OVX surgery (sham OVX) group, hormone substitution (OVX+E2 group) can reverse the allodynia after OVX surgery.Thermal hyperalgesia:OVX did not affect the paw withdrawal response, even though hormone substitution treatment (OVX+E2 group) produced a trend of increased paw withdrawal latency.Tail-flick reflex response:OVX surgery caused super sensitization of the tail-flick reflex. All the OVX rats displayed decreased tail-flick response latency at 2 week after surgery, and this lasted at least 5-7 weeks. After estrogen substitution, the pain super sensitization was reversed completely and hypoalgesia was even observed as the tail-flick response latency in OVX+E2 group is significantly higher than naive group and sham OVX control group.Inflammatory pain:Similarly, rats under OVX were tested by formalin test at 5 week post-surgery and found the scores significantly increased, and then estrogen was administrated for 1 week. We found that estrogen may inhibit rat formalin pain behavior, almost the same as, even stronger than, the group which using estrogen one week ealier. Hence, OVX may enhance the nociceptive action of formalin.Neuropathic pain:Three days after single side chronic constriction injury (CCI) surgery, Sham OVX group showed obvious allodynia, which indicated the CCI model was successfully established. Interestingly, for those rats which already developed allodynia, CCI did not produce further decrease of mechanical pain threshold. In contrast, OVX rats received sham surgery displayed severer allodynia. This indicated the relatively lower level of estrogen might inhibit the allodynia accompanied by neuropathic pain.2. Spinal cord estrogen receptor is involved in pain super sensitization after OVX surgery.Formalin pain response was significantly reduced by intrathecal injection of estrogen in OVX rats, especially the late phase response. This suggested that spinal cord is involved in the pain modulation effect of ER. Immunofluorescence labeling detected the existence of ER in spinal cord. Western blot results suggested that although long term OVX resulted in the upregulation of ERa and down regulation of ERβ accompanied with pain super sensitization. Considering the estrogen substitution can reverse both the pain behavior and ERa expression level, but has no effect on ERβ expression, we suppose that the long term OVX induced pain is mainly mediated by spinal cord ERa.3. PI3K/Akt signal pathway is involved in modulation of pain by upregulating spinal cord ERaLong term of low estrogen level resulted in the increase of spinal cord tAKt level and estrogen substitution can down regulate tAkt level. The change of pAkt level is in same direction. Intrathecal pretreatment with Wortmannin, an Akt upstream inhibitor-PI3K inhibitor, dose dependently inhibited formalin pain response.In summary, lower estrogen level after long term OVX caused pain super sensitization and upregulation of the ERa and tAkt in the spinal cord dorsal horn. The pain super sensitization can be reversed by substitute treatment of estrogen and PI3K inhibitor. It was indicated that ERa in the spinal cord might play an important role in rat pain super sensitization after OVX through PI3K/Akt signal pathway.