| Background and Objective:Section 1:BACKGROUND Inguinal hernia repair is one of the common surgeries in clinical circumstance, a new rat model of persistent postoperative pain evoked by skin/muscle incision and retraction (SMIR), which is akin to a clinical procedure, was successfully set up. However, the mechanism of persistent pain evoked by this kind of minor surgeries is still obscure. OBJECTIVE With common anesthesia/analgesia method, our study will discuss the safety and efficacy of different anesthesia/analgesia formula to support the clinical anesthesia and analgesia consideration.Section 2:BACKGROUND Dexmedetomidine(DEX), is the novel α-2 receptor agonist, which has been proved as one strengthen local anesthetic adjuvant. However, no study showed that if the peripheral administration method is safe and effective enough for reducing acute postoperative pain. Pathology of the nervous system during the acute postoperative phase is not clear. At the same time preventive treatment is often ignored by clinical doctors. OBJECTIVE This study aimed to promote considerate understanding of the structural alteration of the related nervous system.Section 3:BACKGROUND Growing evidence has shown that unilateral nerve injury results in pain hypersensitivity in the ipsilateral and contralateral sides respective to the injury site. This phenomenon is known as mirror image pain (MIP). Glial cells have been indicated in the mechanism of MIP; however, it is not clear how glial cells are involved in MIP and changes after preemptive local injection of ropivocaine with dexmedetomidine. OBJECTIVE We aimed to observe how glial cells are involved in MIP and changes after preemptive local injection of ropivocaine with dexmedetomidine.Methods and materialsSection 1:Adult 32 SD(Sprague-Dawley) rats were selected and randomly allocated into 2 groups:Group S (SMIR group)and Group C(control group/skin incision and retraction). After procedures, behavior test were carried out at the day 0, day 1, day 3, day 5, day 7, day 14 and day 28.8 rats of each group were sacrificed at day 3 and day 28. Saphenous nerve, L3-4DRG and spinal cord were used to observed under optical microscope, electron-microscope and immunofluorescence(OX-42 and GFAP) test.Section 2:Rats (180-220g) were anaesthetised with i.p.10% chloral hydrate (100mg/ml), at doses of 400mg/kg, laid on their back and the medial thigh on one side was shaved. The shaved skin was then repeatedly swabbed with sterile alcohol wipes to sterilize the area and to enable visualisation of the saphenous vein. All rats were randomized allocated into 4 groups:S Group:SMIR(Skin/muscle incision and retraction) surgery group, R group(Ropivocaine group), RD1 group and RD5 group. All rats were sampled on days 3. The hind paw saphenous nerves, dorsal root ganglion and spinal cord were processed for light and electron microscopy. The differences among 4 groups were analyzed with one-way analysis of variance (ANOVA).Section 3:To observe phenomenon MIP and the following mechanism,48 adult male Sprague-Dawley rats (weighing 180-220g) were separated into 3 groups, Group S (n=16):before procedures and nerve extraction, Group S was injected 0.9% saline locally, then 8 rats were used for behavior test until 28 days,8 rats’ DRG were harvested at 3rd day after surgery; Group R (n=16):before procedures and nerve extraction, Group R was injected 0.5% ropivocaine locally, then 8 rats were used for behavior test until 28 days,8 rats’DRG were harvested at 3rd day after surgery; Group RD1 (n=16):before procedures and nerve extraction, Group RD1 was injected 0.5% ropivocaine combined lμg dexmedetomidine locally, then 8 rats were used for behavior test until 28 days,8 rats’ DRG were harvested at 3rd day after surgery. Immunofluorescent, and transmission electron microscopy using dorsal root ganglion (DRG) from both sides were then conducted.Results:Section 1:Group S mechanical pain of ipilateral side was evident from day 1, peaked at day 1 to day 5, duration of the first stage is 5 days and the second stage of pain is within 21 days. Group C mechanical pain was evident from day 1, peaked at day 3 and recovered within 5 days at the first stage and no second stage was observed. Group S thermal pain threshold of ipilateral side was expressed 2 stages as well, within 3 days threshold rise above the baseline at the first stage, peaked at day 1. The second stage from day 7 to day 21, thermal pain threshold of ipilateral side of Group C, rise above baseline from day 1, peaked at day 1 within day 3, and peaked at day 5 within day 3 to day 28. Except for day 14, thermal pain threshold of 2 groups express the same value from day 3 to day 5 (P>0.05). Nissl stain revealed that bilateral sides of saphenous nerve did not express degeneration. Grey level of 2 groups did not express significant differences with the value of (9110.45±135.21) and (8900.54±200.21) respectively at acute phase (p>0.05). (9210.45±155.56) and (9000.54±312.38) at chronic phase (p>0.05). Electron microscope observation (8000X) suggested that bilateral sides myelino-axon and C-fibers of saphenous nerve did not show great morphology changes after skin/muscle incision and retraction. Density of myelino-axon and C-fibers were the same between 2 groups(p> 0.05, t test). Density of myelino-axon and C-fibers microtubules (no./μm2), oblique microtubules of Group S at acute phase and chronic phase significantly increased comparing with Group C (p<0.05). Density of circular microtubules expressed no great difference between 2 groups (p> 0.05). Atypical mitochondria and lysosome in the neuron of DRG in Group S significantly increased significantly in Group C (p<0.05). Endoplasmic reticulum gaps enlarged (edema) after day 3, at the day 28, edema pathology changes did not change. Group C did not reveal enlarged endoplasmic reticulum gaps. Schwann and Satellite cells activity increased at the acute phase, and decreased at the chronic phase according to immunofluorescence(GFAP) test. According to immunofluorescence(GFAP) test, microglial cells expressed the same trend as GFAP(+) cells, and the activity of microglial cells showed much strong immunofluorescence than GFAP (+) cells.Section 2:We affirmed that peripheral adminstraion of DEX combined ropivocaine improved the SMIR model’s mechano-hyperalgesia in the acute phase and the peak time of pain was delayed. Combined dosage groups’thermal-hyperalgesia threshold is increased. Significant decrease of atypical mitochondria was obvious.Section 3:The results showed mechanical pain threshold in ipilateral hind-paws of the Group S, Group R, Group RD1 animals dropped to 4.52±0.67 g,5.827±0.73g,11.88±0.22g at 3d respectively, contralateral paws dropped to 7.10±0.91g,17.687±1.90g, 16.213±1.80g at 3d respectively. Immunofluorescent showed that glial cells actived in bilateral side DRG after surgery in 3 groups, but ipilateral paws expressed more active glial cells than contralateral paws. Transmission electron microscopy showed that mitochondria swelling/vacuolization and lysosomes were obvious in ipilateral paws than contralateral paws, but Group RD1 formula can reduce glial cells activity, mitochondria swelling/vacuolization and the amount of lysosomes.ConclusionSection 1:Postoperative persistent pain evoked by SMIR model, is the combined result of different effects:mitochondrial dysfunction, microtubule and endoplasmic reticulum stress, and serial activation of microglial cells, satellite cells and Schwann cells. With the alleviation of pain, the pathology changes were observed mitigated as well. Mitochondrial dysfunction has close relation with the damage of the axon, therefore Mitochondrial dysfunction might be the early symbol of nerve irritation.Section 2:Our research suggested a causal role of abnormalities in axonal mitochondria in SMIR acute pain, and the abnormal mitochondria could be connected to the severity of acute pain and persistent pain. The addition of low dose Dexmedetomidine to ropivacaine locally in SMIR surgery improved analgesia effect and mitigated postoperative pain.Section 3:Locally injection of ropivocaine and/or dexmedetomidine can inhibit the activation of glial cells in DRG, mitigated the pathological changes of neuron in DRG and reduce MIP. |
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