Effects Of Brain-Derived Neurotrophic Factor On Modulating Nerve Regeneration And Neuropathic Pain After Peripheral Nerve Injury

BackgroundChronic pain has become a worldwide public health problem because of the great economic and social costs associated with patient treatment. Recently, an epidemiological study in Europe showed that19%of the adult European population suffers from moderate to severe chronic pain and that40%of these patients receive inadequate analgesic treatment, which affects their social and working life. These observations not only reveal the high human and social economics cost (one trillion dollars per year in developed countries) but also highlight the current limitations of the available analgesic treatments. Neuropathic pain and inflammatory pain are the two principal types of chronic pain. Inflammatory pain arises as a consequence of tissue damage/inflammation and neuropathic pain from nervous system lesions. In contrast to inflammatory pain hypersensitivity, which usually returns to normal if the disease process is controlled, neuropathic pain persists long after the initiating event has healed. Major surgeries such as breast and thoracic surgery, leg amputation, and coronary artery bypass surgery also lead to chronic pain in10-50%of individuals after acute postoperative pain, in part due to surgery induced nerve injury.Regeneration and neuropathic pain usually coexist after peripheral nerve injury. Owing to the similar pathophysiological basis of communication of neurons, glia cells and immunocytes, the process of nerve regeneration and neuropathic pain might be modulated by some factors affecting both of them. Brain-derived neurotrophic factor (BDNF) is one of such a candidate, which is up-regulated after peripheral nerve injury to promote axonal growth and neuronal survival, and on the other hand is sufficient to trigger the onset of neuropathic pain and central sensitization by itself. Local administration of BDNF promotes axonal regeneration after axonotomy; and anti-BDNF antibody produces a dramatic reduction in the number of myelinated axons distal to the neuropathy and a decrease in the elongation of regenerating axons. In terms of excitatory synaptic transmission, both BDNF and CCI promote a similar "injury footprint"; a similar analysis of actions on delay neurons show that the actions of CCI and BDNF on mEPSC properties are similar; multiple evidences implicate microglia-derived BDNF in attenuation of Cl" mediated, GABA/glycine inhibition in the dorsal horn; inhibition of BDNF attenuate allodynia in different neuropathic pain models. Basing on the multiple effects of BDNF after peripheral nerve injury, we postulate that BDNF would be a key point to connect nerve regeneration and neuropathic pain.In the present research, we developed a rat model of graded neuropathic pain by graded sciatic constriction and assess the behavioral and pathologic changes of the sciatic nerve. We concluded that allodynia postoperatively and central activations of neurons and astrocytes in the ipsilateral lumbar spinal cord were well correlated with the degrees of peripheral nerve injury. In addition, ATF-3and BDNF expression in L3-L6DRG neurons were also detected to reveal the regenerative profile of the graded neuropathic pain model. In subsequent experiments, we evaluated the effects of rhBDNF, TrkB-Fc and anti-BDNF antibody on the development of neuropathic pain and the process of nerve regeneration, and explored the relationship between them.Methods and results1. Development and assessment of the graded neuropathic pain model in ratsMethods:45male SD rats were randomly allocated to5groups (group No, N1, N2, N4and C, n=9for each group) and received graded right sciatic constriction. Briefly, rats in group No, Ni, N2and N4received0,1,2and4loose ligations on the trunk of the right sciatic nerve with4-0chromic gut respectively; meanwhile another4,3,2and0segments of5mm-long chromic gut were put subcutaneously in the correspondent rats. No surgical treatment was taken in rats of group C. Mechanical allodynia was assessed with von Frey hairs before operation and on the3rd,7th,10th and14th day postoperatively, and the50%withdrawal threshold was calculated with up-and-down method. On the15th day postoperatively,30of the rats (n=6for each group) were sacrificed and the right sciatic nerve were dissected for transverse slicing and myelin staining with Luxol fast blue; the lumbar enlargement of the spinal cord were snap frozen at-80℃and sliced for immuno-histochemical staining of c-Fos and GFAP. The left15rats (n=3for each group) were sacrificed on the same day for quantitative analysis of c-Fos and GFAP in the ipsilateral lumbar spinal cord with Western blot.Results:The50%withdrawal thresholds of the right hind foot dropped dramatically in group N1, N2and N4from the3rd day and maintained at the lowest level from the7th10th day to2weeks postoperatively compared to those in group No and C (P<0.001). On the14th day, the50%withdrawal thresholds presented a graded manifestation of No> N1> N2> N4(P<0.05). Graded demyelination (No<Ni and N2<N4, P<0.001) was also observed in the distal area of the ligated sciatic nerve, represented digitally by the myelin sheath counts and the DPR value (the ratios of the myelin sheath counts in the distal areas to those in the proximal areas). The Spearman correlation coefficient between DPR values and the50%withdrawal thresholds on the14th day was0.901(P<0.001). Both immuno-histochemistry and Western blot analysis revealed the graded increases of c-Fos and GFAP in the lumbar enlargement of the spinal cord on the15th day (No> N1> N2> N4). The Spearman correlation coefficients between the counts of c-Fos positive neurons, the integrated optical densities of GFAP in the superficial laminae of spinal cord and the50%withdrawal thresholds on the14th day were-0.910and-0.859respectively (P<0.001). 2. ATF-3and BDNF expressions in the lumbar DRG neurons in the graded neuropathic pain modelMethods:Animals in experiment1were reused in this procedure. On the15th day postoperatively, the ipsilateral L3-L6DRGs of rats in group No, Ni, N2and N4(n=6for each group) were fixed in4%paraformaldehyde and prepared for immuno-histochemical staining of ATF-3and BDNF. The ipsilateral L3-L6DRGs in another12rats (n=3for each group) were obtained on the same day for quantitative analysis of BDNF with Western blot.Results:Sciatic constriction obviously activated the ipsilateral lumbar DRG neurons represented by elevated ATF-3expression. The ratios of ATF-3positive neurons in the ipsilateral L3～L6DRGs showed a graded increase as No<N1<N2<N4(P<0.001) as correlated with the demyelination of the sciatic nerve. BDNF was also up-regulated in the lumbar DRG neurons in a roughly graded manner in this model (No、 N1<N2<N4, P<0.05). The majority of BDNF positive cells were small to medium primary sensory neurons.3. Effects of BDNF intervention on development of neuropathic pain and nerve regeneration after sciatic constrictionMethods:Adult male SD rats weighing200～220g were prepared for intrathecal cannulation through L3/4intervertebral space.5days later,42rats which received cannulation successfully without sensory or motor deficiency were selected for subsequent experiments.18of them received single intrathecal injection of rhBDNF in different dosages of0,0.01,0.1,1,10or100ng respectively (n=3for each group). Allodynia was recorded2h,6h,1d,2d,4d and7d after injection with von Frey hairs, and on the7th day the rats were sacrificed for detection of central sensitization in the lumbar spinal cord. The other24rats were randomized into8groups (n=3for each group):group C, group C+TrkB-Fc, group C+anti-BDNF, group N2, group N2+TrkB-Fc, group N2+anti-BDNF, group N2D7+TrkB-Fc, group N2D7+anti-BDNF. We detected the effects of TrkB-Fc and anti-BDNF antibody on allodynia after sciatic constriction, the central sensitization in spinal cord, the activation of neurons in the lumbar DRGs and the demyelination of sciatic nerve in two different timing of treatment.Results:A single intrathecal injection of different doses of rhBDNF evoked significant allodynia in healthy rats, and caused over-expressions of c-Fos and GFAP in the lumbar spinal cord. Early sequestering BDNF with TrkB-Fc or anti-BDNF antibody since the day of surgery alleviated allodynia after sciatic constriction and prevented central sensitization in the spinal cord; the ATF-3expression in DRG neurons was also inhibited by early intervention with TrkB-Fc and anti-BDNF antibody (P<0.001), while demyelination in sciatic nerve was not influenced (P>0.05). No detectable effects in the above aspects could be seen when delayed TrkB-Fc or anti-BDNF antibody was given to rats undergoing neuropathic pain (P>0.05).4. Statistical analysisAll data were expressed as mean±standard deviation (x±SD). Statistical analysis was performed with SPSS13.0. Results of50%withdrawal threshold were compared by repeated measures of multiple variances, and the other data were analyzed by one-way analysis of variance. P<0.05with two-tailed test was considered statistically significant.Summary1. Main results:(1) We successfully developed a graded neuropathic pain model in rats through graded constriction of the sciatic nerve. Demyelination of the injured nerve and central sensitization in the spinal cord were assessed and proved to correlate with allodynia postoperatively. (2) ATF-3and BDNF expressions in the ipsilateral L3～L6DRG neurons increased significantly in the graded neuropathic pain model in a corresponding graded manner.(3) Intrathecal BDNF injection evoked obvious allodynia in healthy rats. Early sequestering BDNF with TrkB-Fc and anti-BDNF antibody could inhibit central sensitization in the spinal cord and activation of primary sensory neurons in DRGs, alleviating neuropathic pain without deterioration of the injured nerve.2. Conclusions:The graded model of neuropathic pain can be developed by graded constriction of the sciatic nerve on a pathologic basis of different degrees of demyelination, accompanied by a graded activation of neurons and astrocytes in the lumbar enlargement of the spinal cord. At the same time, the corresponding DRG cells can be activated with over-expressions of ATF-3and BDNF to promote nerve regeneration. Early sequestering BDNF with TrkB-Fc and anti-BDNF antibody could inhibit central sensitization in the spinal cord and the activation of DRG neurons, alleviating neuropathic pain without deterioration of the injured nerve. The intrinsic mechanism underlying BDNF and activation of primary sensory neurons and the follow-up reactivities need further investigations.