Stereological Study Of The Structural Plasticity In The Rats Spinal Dorsal Horn Associated With Postdenervation Pain And Early Postoperative Analgesia

Neuropathic pain is defined as"pain initiated or caused by a primary and (or) secondary lesion, dysfunction or transitory perturbation in the peripheral and (or) central nervous system". The menifestations of neuropathic pain are dysesthesia, paraesthesia, hyperalgesia and allodynia. Postdenervation pain syndrome (e.g. phantom limb pain), a form of neuropathic pain, is commonly seen in patients after limb amputation or limb denervation, and was seriously disrupted the quality of patients'life. Treatment of the pain is difficult and our knowledge on the underlying mechanism is limited. The prevalently accepted theory considers that phantom limb pain is caused by a maladaptive plasticity i.e. central sensitization, in the central nervous system. Central sensitization is characterized as an enduring increase in the neuronal circuit in the spinal dorsal horn, thus the neuronal circuit reacts excessively to the external stimulus.Central sensitization is associated with a synaptic plasticity in the spinal dorsal horn. In terms of nerve pulse transmission, central sensitization is characterized by the long term potentiation (LTP) of the spinal synaptic transmission, which was reported to be associated with increase of synthesis of synaptophysin. And with respect to the structural changes associated with central sensitization, sprouting of low threshold Aβ-fibers from laminaⅢto the laminaⅡcould be found in which might result in more synaptic contacts. All of the above synaptic plasticity strongly indicates that the number of synapses in the spinal dorsal horn might increase after peripheral nerve injury. However, there was no quantification study about the plasticity of synaptic number in the spinal dorsal horn after unilateral sciatic nerve transection by now.The activated microglial cells and astrocytes in the spinal dorsal play a key role in the development and maintenance of neuropathic pain, they form"conversation"between synapses or neurons through the secretion of inflammatory mediators after peripheral nerve injury thus inprove the sensitization of secondary sensory neurons. Peripheral nerve injury only activates microglia in the spinal dorsal horn and the activation of astrocytes relies on the activation of microglia. So, activation of glial cells mainly occurs in the spinal dorsal horn. It is believed that the inflammation of the injured nerve fiber induces this activation, thus promote hypertrophy of cell body and increase of cell number. However, the inflammation in the nerve trunk after transection is rather mild, whether it can promote the activation of the above glial cells and the plasticity change of their numbers remains still unknown.As above mentioned, synapses, microglial cells and astrocytes play important roles in the development of central sensitization after peripheral nerve injury, the plasticity change of their numbers might necessarily influence their functions and resulted in the development and maintenance of chronic pain. At present, numbers of the above particles had been rarely reported. Furthermore, the type of nerve injury associated with phantom limb pain is rather different from other types of neuropathic pain, whether transection of a peripheral nerve will result in the same plasticity change in the spinal dorsal horn with other types of nerve injury need to be clarified. In this study, we applied desige-based stereological methods and a new stereological technique– the optical disector with special staining methods for the above particles to quantification the plasticity changes about numbers of synapses, microglial cells and astrocytes in the rat spinal dorsal horn after unilateral sciatic nerve transection. The aim of this study is to provide freshnew anatomic data and baseline indexes for understanding the mechanism of phantom limb pain.Alagesia for the acute pain after amputation may prevent or alleviate phantom limb pain. It was reported that parecoxib, a selective cyclooxygenase (COX)-2 inhibitor used for postoperative analgesia, might inhibit the central sensitization, so we applied parecoxib as a positive control in this study to determine whether parecoxib could inhibit, if any, the changes of the particles mentioned above.The major object of stereological morphometry is pathological sections. The processes of embedding and staining associate with obvious crimple of tissues. This crimple must be taken into account when the morphometry study is carried out. Therefore, we estimated the constriction coefficient and crimple coefficient by stereology and Cavalieri's principle firstly, in order to revise the morphometric results and to acquire more precise conclusions.PartⅠObjectiveTo estimate changes of spinal cord volume and length after paraffin embedding and staining to revise the following morphometric results. Methods12 rats were fixed with transcardiacally perfusing and the lumber intumescentia (the L4-6 segments of the spinal cord) was separated through laminectomy. Afterwards, the length, weight and density were measured, so as to estimate its volume. Then the lumbar intumescentia were transected into 6 blocks equally, each approximately 2mm thick, with one block being randomly sampled for paraffin embedding, serially sectioning at the thickness of 14μm. According to the systematic random manner, one section was sampled per 15 sections. These sampled sections were used for hematoxylin and eosin staining. The cross-section area and the thickness of each section were measured by Visiopharm stereological system. Thus, the volumes of spinal cord after embedding and staining could be calculated. Compared to their initial volumes and lengths after fixation, the compression coefficient or crimple coefficient could thus be calculated.ResultsAfter fixation, the volume of the spinal cord was 96.80±13.85 mm2; the length of it was 1.53±0.25 cm. After been embedding or staining, its volume compressed to 60.19±19.15 mm2 or 42.97±16.02 mm2 respectively; the length of grey matter or white matter crimpled to 0.85±0.17 cm or 1.00±0.18cm respectively. The compression coefficient after embedding or staining was 62.9%±20.5% or 44.8%±16.6% respectively. After staining, the crimple coefficient of the grey matter or white matter was 56.9%±17.1% or 66.7%±19.5% respectively.PartⅡObjectiveTo determine whether the postdenervation pain syndrome caused by unilateral sciatic nerve transection was associated with the plasticity changes of the numbers of neurons, synapses, microglia or astrocytes. Methods11 normal adult Sprague-Dawley rats were randomly divided into 2 groups undergoing (1) unilateral sham operation, and (2) unilateral sciatic nerve transection. 28 days post operation, rats were sacrificed, their lumber enlargements (L4-6) were removed and embedded with paraffin and were sectioned at the thickness of 14μm. Sections were sampled with a systemic random manner and were stained with Nissl's methods (for the displaying and counting of neurons), synaptophysin immunohistochemistry (for the displaying and counting of synapses), iba-1 immunohistochemistry (for the displaying and counting of microglial cells) or GFAP immunohistochemistry (for the displaying and counting of astrocytes) respectively. A sophisticated stereology set was used to measure all sections. The optical disector (a contemporary stereological technique) was used to estimate the numbers of particles mentioned above in the spinal dorsal horn.Results(1) After unilateral sciatic nerve transection, majority of rats developed autotomy. (2) Compared to the non-operated side, the axotomy induced a 74.3% increase in the number of synapses, a 44.9% or 35.8% decrease in the number of microglial cells or astrocytes per unit length of spinal cord in the middle tissue block from the L4-6 segment on the operated side but not in either the rostral or caudal tissue block.PartⅢObjectiveTo determine effects of early postoperative use of parecoxib on the structural plasticity in the rat spinal dorsal horn associated with unilateral sciatic nerve transection.Methods13 normal adult SD rats were randomly divided into 2 groups undergoing (1) unilateral sciatic nerve transection, and (2) unilateral sciatic nerve transection with postoperative use of parecoxib for 3 days. Autotomy, tissuse processing and numbers of synapses, microglia or astrocytes were also processed as PartⅡ.Results(1) Parecoxib had anti-autotomy effects to some degree; (2) Parecoxib had no effect on the plasticity change of synaptic number, while it had anti-decrease effects on microglia and astrocytes to some degree.Conclusions1. The dehydration process during paraffin embedding and staining causes obvious compression of the spinal cord. This compression cannot be ignored when the morphometric study is done.2. Axotomy, a model for postdenervation pain, was associated with serials of plasticity changes in the neuronal circuits in the spinal dorsal horn. The plasticity may play a key role in the central sensitization thus may lead to the refractory of treatment on the postdenervation pain (e.g.: phantom limb pain).3. Early postoperative analgesia with parecoxib had anti-autotomy effect to some degree, this effect may relate to the inhibition of plasticity changes in the number of microglia and astrocytes in the spinal dorsal horn.