The Effects Of Minocycline Exerted On Microglial Activation In Spinal Cord Of The Rats' Neuropathic Pain Model

Objectives:1. By observing the expression of spinal microglial OX-42 (clone nameof monoclonal antibody of complement receptor type 3) to detect themicroglial responses toward peripheral nerve injury and its activative statesduring the time course after peripheral nerve injury through the CCI (chronicconstriction of sciatic nerve) model;2. To observe the effects toward the expression of spinal microglialOX-42 exerted by minocycline so as to investigate the role microglia cellsplaying in neuropathic pain and the potential mechanisms.3. It had been proved minocycline had a neuroprotective function. Byobserving the effects it exerted during the neuropathic pain, we could givesome scientific evidence for its further clinical using.Methods:(1) Building of CCI model: The unilateral peripheral mononeuropathyof groupIII, group IVand group Vwas produced according to the methoddescribed earlier by Bennett-Xie(1988). Allodynia was determined bybehavior test in sham and nerve-injured rats. Seventy-two SD rats wereallocated into five groups.Briefly, rats were anesthetized with10％chloraldurat (400mg/kg). A small lengthways incision about 1.5-2.0cmto the skin at midpiece of the left thigh was made followed by retraction ofthe biceps femoris. The left sciatic nerve was isolated under a surgicalmicroscope for about 5-7mm, and the nerve were tightly ligated with 4-0chromicized catgut suture near the trigeminal proximal end for a interval of1.0mm between every two nodes. The ligation intensity was identified liftedslightly chatter to shank musculature. The wound was irrigated with salineand closed in two layers with 4-0 silk suture and surgical skin staples. TheSham control rats were surgically prepared as described above except for thenerve ligation.All the surgical procedure was completed in an biocleanenvironment.(2) Division of groups:70 Sprague-Dawley rats were randomlydivided into 5 groups. GroupⅠ, normal controlling group(n=10); groupⅡ, sham-operated group(n=15); GroupⅢ, preemptive treated group(In asystemic preventive paradigm 40 mg/kg minocycline was administered bythe i. p route. Treatment was initiated 1 h before surgery and continued dailyto day 15 post-operation, n=15); GroupⅣ, post-operation treated group(Thesame dose of minocycline was administered after establishment ofneuropathic pain and continued daily to day 15 post-operation, n=15); GroupⅤ, saline administered group(saline vehicle was administered by the i. proute after establishment of neuropathic pain). All injections were completed15 h before behavioral testing.(3) Praxiology test: Mechanical nociceptive thresholds were evaluatedby an paw tactile sensation Analgesy-Meter (biomedical engineeringresearch institute of Chinese Academy of Medical Sciences) to calculate the50％paw withdrawal threshold. The tactile stimulus producing a50％likelihood of withdrawal response was calculated by using the updownmethod. Rats were placed in individual plastic boxes on a mesh floor andallowed to acclimate for at least 30 min. A series of calibrated von Freyfilaments ranged from 0.45g to 15.10g were applied perpendicularly to theplantar surface of the left hindpaw with sufficient forces to bend thefilaments for 6 s. Brisk withdrawal or paw flinching was considered apositive response. In the absenceof a response, the filament of next greaterforce was applied. In the presence of a response, the next lower force wasapplied. Unitil we found the two consecutive filaments could or could notelicit a paw withdrawal response respectively, then continued to do 4 timesthe same as above, finding out 6 values finally. Each trial was repeated twoor three times at approximately 2-min intervals, and the mean value was usedas the force to produce withdrawal responses. If it needed a strength beyondthe given range,we would record it as 15.00g or 0.25g directly. The formulafor calculating the 50％paw withdrawal threshold is: 101g(x)+kδ(X is thescale of the last filament; k is the coefficient of the different stimulatemethods,δis the mean value of the intervals between every consecutivetwo stimulate strength(g),δ=0.224). All the behavior was recorded beforesurgery, and on everyday after surgery.(4) Method of measuring OX-42: Picking out 3 rats randomly fromevery experimental group at timepoint of 7, 11, 13, 14, 15day after surgery. Rats were anesthetized with 10%chloraldurat by the i.p route,then theirbodies were perfused and fixed by 0.01 mol/ L phosphate bufferedsolution containning 40g/L paraformaldehyde,and then get the spinalsegment (lumbosacral enlargement) which connecting with sciaticnerve. Making the spinal segment to be imbeded by paraffin and coronallysliced into microtome section.Every section was incubated with mouseanti-OX-42 and visualized with 3,3'-diaminobenzidine. Using lightmicroscope to observe OX-42 positive microglia cells and then measured themean gray values of them by an image analysis software.Results:(1) Rats ofgroupV shown us that the 50% paw withdrawal thresholdsbefore surgery and 7,11,13,14,15day after operation were as follow: 13.30±1.71g, 2.66±0.46g, 2.89±0.34g, 2.83±0.48g, 2.41±0.228g, 2.75±0.36g. From the data we could draw a conclusion that neuropathic painemerged at the seventh day post-operation and exposed most obviously atfourteenth day and kept the values less than 4.0g to the end of theexperiment in this group.(2) There was no apparent difference between groupⅡand groupⅠatevery timepoint(P＞0.05).(3) There was no apparent difference between groupⅢand groupⅠatevery timepoint(P＞0.05).(4) Compared the 50％paw withdrawal thresholds among groupⅢ,groupⅣand groupⅤ,the resulrts tured out to be that: groupⅢ＜groupⅣ(P＜0.05), groupⅣ＜groupⅤ(P＜0.05). we were given a result shown iamong the there groups in degree of neuropathic pain: groupⅤ＜groupⅣ＜groupⅢ.(5) Immunohistochemistry results of groupⅤshown us that: theresponses of microglia cells turned from quiescent condition to earlyreactive state 7 days post surgery, OX-42 anachromasised, cell morphousseemed clearly but small; OX-42 manifestly anachromasised, OX-42positive cells got the peak not only in quantity, but also in volume of cells,cell morphous were clear and presented in a chubby state 11 days postsurgery; the responses of microglia cells were getting weaker across 13,14,15 and 17 days after operation, OX-42 understained, OX-42 positive cells decreased gradually, cell morphous was becoming vague. It is thus evidentthat, microgial activation appeared early after nerve injury, and then to bemost manifest at the eleventh day after surgery, keeping active statethroughout the experiment,but getting gradually weaker.(6) The mean gray values of OX-42 positive cells shown us that: themean gray value resulted from the corresponding part of groupI was 157.41±5.40; in groupV, the mean gray value of the seventh day post-operationwas 121.62±6.67, and the value of the eleventh day post-operation was115.47±6.12(it was the lowest one), and the value of the fourteenth daypost-operation was 119.34±4.71, and then the mean gray values presented anascendant tendency. There was obvious variance between saline administeredgroup and normal controlling group (P＜0.05).Conclusions:(1) Microglia cells in spinal cord were actived early post-operstion, andreached the peak at the eleventh day after surgery in the CCI rats'model.(2)Preemptive administered minocycline could inhibit neuropathicpain by blocking microglial activiation. But after establishment ofneuropathic pain, administer minocycline could only partially reverse it.