| Lung cancer is the cancer with the highest morbidity and mortality in China.Nearly 230,000 people in the United States are diagnosed with lung cancer each year,and surgical treatment is the preferred method in the early stages.Thoracotomy is usually accompanied with moderate to severe postoperative pain,which is not only the core factor affecting postoperative early rehabilitation,but also an independent risk factor for postoperative complications and chronic pain.The incidence of chronic pain after surgery has been reported to be as high as 30-50%.Multi-mode analgesia is recommended to control postoperative acute pain and prevent the transformation of acute pain into chronic pain in perioperative period.Recently,preoperative oral pregabalin has been recommended as a prophylactic analgesic measure,which has been reported to reduce postoperative pain scores and opioids consumption in thoracic,orthopedic,and breast surgery.In addition,a lot of basic studies have also shown that pregabalin can relieve neuropathic pain caused by different causes.Pregabalin is a Cav α 2-δ ligand antagonist,but the molecular mechanism of its prophylactic analgesia is unclear.Surgical trauma will immediately cause the inflammatory response of peripheral nerves to the spinal cord.The neurons in spinal dorsal horn will secrete a variety of pro-inflammatory factors,which result in glial cells overactivation and releasing a large number of inflammatory mediators and excitatory neurotransmitters and vice versa,named the neuron-glial pain regulation circuits.Furthermore,the innate immune system is considered as one of the important mechanisms for the regulation of postoperative acute and chronic pain.Peripheral and central nerve sensitization may be driven by the sensitization effect of inflammasome NLRP3(Nod-like receptor protein 3)and its products on peripheral and central nerves after injury.Therefore,preoperative use of pregabalin may inhibit excessive activation of the innate immune system,and reduce inflammation in spinal cord and then relieve acute pain after thoracotomy.In this study,a modified postoperative pain model of thoracotomy was established firstly in rats,and prophylactic oral pregabalin was used to observe the behavior change of postoperative pain and the effects on inflammasome NLRP3 and its products in spinal dorsal horn,which may provide a new theoretical basis for clinical medication and decision making.Part 1:To establish a model of postoperative pain after thoracotomy in rats[Objective]To establish an effective and stable acute and chronic pain model after thoracotomy in rats.[Methods]Forty-eight male Sprague-Dawley rats were divided into 4 groups(n=12),that were the blank control group,sham operation group A,sham operation group B and thoracotomy group,respectively.Rats in the thoracotomy group underwent endotracheal intubation after induction by inhalation of 2.0%to 3.0%isoflurane,connected to a small animal ventilator for mechanical ventilation and 1.5%to 2.0%isoflurane was used for maintenance of anesthesia.The operator selected the right T4～T5 intercostal incision from skin to intercostal muscle,rib retractor was used to separate 12 mm and maintain for 60 minutes.Finally,chest gas was extracted and then the muscle and skin were sewn layer by layer.The sham operation group A only incised the skin,fascia and the deep muscles of the intercostal muscle at right T4～T5.In the sham operation group B,skin to intercostal muscle incision was made at the same intercostal space,but the pleura was cut without rib distraction,and the muscle and skin were sutured after 60 minutes of anesthesia.Rats in the blank control group were only anesthetized and no invasive operations were performed.The operation was performed between 7:00 and 15:00 every day.The right thorax and back of the all three groups were shaved daily before and after surgery.The pain behavior tests of mechanical pain threshold and acetone cold stimulation were carried out within a specified period of time(9:00～12:00)at day 3 before modeling and day 1,2,3,5,7,10 and 14 after modeling.[Results]There were no significant difference in mechanical pain and cold pain thresholds between the four groups before modeling.Compared with the blank control group(n=12),sham operation group A(n=12)and sham operation group B(n=12),the mechanical pain threshold of the thoracotomy group(n=11)decreased to the lowest level on the first day after modeling(58.1±4.9g),which decreased significantly from day 1 and continued to day 14 after surgery(all P<0.05).Similarly,the cold pain threshold increased significantly from day 1 and continued to day 14 after surgery(all P<0.05).Compared with the blank control group,sham operation group A and sham operation group B only produced a short-term decrease in the mechanical and cold pain threshold,and gradually recovered within the day 3 after surgery,especially the difference in cold stimulus response was not significant.Compared with the other three groups,the rats in the thoracotomy group had significant hypersensitivity in terms of the predetermined threshold of mechanical and cold pain at the day 14 after surgery,with the incidence of 63.6%and 45.4%respectively.[Conclusions]Early neuropathic factors after thoracotomy increased the severity and duration of postoperative acute pain.The modified postoperative pain model after thoracotomy can provide effective and stable postoperative acute and chronic pain,meanwhile it provides an effective model for the study of postoperative acute pain and acute pain turns to chronic pain.Part 2:Effect of pregabalin on postoperative pain after thoracotomy in rats[Objective]To observe the effects of different doses of pregabalin on the behavior change of postoperative pain after thoracotomy in rats.[Methods]Sixty male Sprague-Dawley rats were divided into 5 groups(ne 12),that were the blank control+thoracotomy group,normal saline+thoracotomy group,pregabalin 10 mg/kg+thoracotomy group,pregabalin 30 mg/kg+thoracotomy group,pregabalin 90 mg/kg+thoracotomy group,respectively.The drug was given by gavage(normal saline or pregabalin of equal volume)once a day from 19:00 to 21:00 at day 3 before modeling.Thoracotomy was performed in the same way as the modeling described in the part one.The pain behavior tests of mechanical pain threshold and acetone cold stimulation were carried out within a specified period of time at day 3 before modeling and day 1,2,3,5,7 and 14 after modeling.[Results]There were no significant difference in mechanical pain and cold pain thresholds between the five groups before modeling.High dose of pregabalin significantly affected postoperative weight gain in rats.Compared with the control group(n=12)and the normal saline group(n=12),the pregabalin 30 mg/kg group(n=11)and the pregabalin 90 mg/kg group(n=10)significantly improved mechanical pain and cold pain hypersensitivity on day 1,2,3,5,7 and 14 after modeling(all P<0.05).Compared with the pregabalin 10 mg/kg group(n=12),the mechanical pain thresholds in the wound skin area significantly increased and cold pain threshold significantly decreased in the pregabalin 30 mg/kg and 90 mg/kg group from day 1 to 7 after thoracotomy.However,there were no statistical difference in mechanical pain and cold pain thresholds between each group at the day 14 after thoracotomy.There were no statistically significant difference in the mechanical pain and cold pain thresholds in the wound skin area between the pregabalin 30 mg/kg group and the pregabalin 90 mg/kg group from day 1 to day 14 after surgery.Compared with the other three groups,the incidence rate of postoperative chronic pain in the pregabalin 30 mg/kg and 90 mg/kg groups were significantly lower at day 14 after surgery.[Conclusions]Different doses of pregabalin had no significant effect on basal mechanical pain and cold pain thresholds in rats.Preoperative prophylactic use of pregabalin 30 mg/kg or 90 mg/kg can better inhibit the acute pain and reduce the incidence of chronic pain after thoracotomy.However,large doses of pregabalin(90 mg/kg)can significantly affect weight gain in rats.Part 3:Pregabalin exerts anti-inflammatory and analgesic effects by inhibiting the activation of inflammasome in spinal dorsal horn[Objective]To investigate the effect of prophylactic use of pregabalin on inflammaosome NLRP3 and its downstream products in the spinal dorsal horn after thoracotomy.[Methods]One hundred and eight male Sprague-Dawley rats were divided into 4 groups:blank control group(n=30),thoracotomy group(n=30),normal saline+thoracotomy group(n=18),and pregabalin 30 mg/kg+ thoracotomy group(n=30).The blank control group and the thoracotomy group were not treated with intervention before modeling,and the remaining two groups were given normal saline or pregabalin with the same volume,respectively.The dosing regimen,modeling methods,and behavioral tests were the same as those in part two.Six rats were randomly selected from each group and sacrificed at day 1,7 and 14 after thoracotomy and the spinal dorsal horn of the right thoracic segment(T3～T6)was taken.Western blot was used to analysis the expression of inflammasome NLRP3,caspase-1,IL-1β and IL-18,and immunofluorescence staining was used to determine the expressions of inflammasome NLRP3,IL-1β and activation of glial cells in the spinal dorsal horn cells at day 1 and 7 after surgery.[Results]The changes of mechanical pain threshold and cold pain thresholds before modeling and day 1,3,7 and 14 after modeling were the same as those in the part two,indicating the successfully establishment of the postoperative pain modeling after thoracotomy.The operation demonstrated significantly upregulation of inflammasome NLRP3,caspase-1,IL-1β and IL-18 in the ipsilateral dorsal horn.Pregabalin could significantly inhibited the expression of inflammasome NLRP3 and its downstream products in the ipsilateral dorsal horn at day 1 and 7 after modeling(all P<0.05).However,there were no significant statistical significance between the thoracotomy group,the normal saline group and the pregabalin group for the aforementioned proteins at day 14 after surgery.Immunofluorescence double staining technique showed that NLRP3 and IL-1β were co-stained with neurons in the ipsilateral dorsal horn,but they did not co-locate with microglia and satellite glial cells at day 1 after modeling.However,the notably activation of microglia and satellite glial cells in the ipsilateral dorsal horn were observed after thoracotomy,and pregabalin could significantly inhibited the excessive activation of microglia and satellite glial cells.[Conclusions]Prophylactic use of pregabalin can significantly inhibit the expression of inflammaosome NLRP3 and its downstream products in spinal dorsal horn neurons in the early stage after thoracotomy,and inhibit the excessive activation of microglia and satellite glial cells.Therefore,prophylactic use of pregabalin was involved in the regulation of inflammatory responses to postoperative pain after thoracotomy in rats. |
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