Horn Of Rats Research Articles

Relationship between abnormal complement activation in the spinal dorsal horn and neuropathologic pain in rats

Objective To observe the change of complement 3 in dorsal horn of spinal cord and to investigate the role of ab-normal activation of complement protein in pathogenesis of NPP. Methods This study comprises two parts. In part 1, 84 healthy male Sprague-Dawley rats were divided randomly into seven groups:normal control group, sham I d group, sham 3 d group, sham 7 d group, CCI 1 d group, CCI 3 d group, CCI 7 d group (n=12). The mechanical pain threshold were measured in different time ac-cording to the group, while the mRNA and protein of Complement 3 were determined by RT-PCR, immunoturbidimetry and immuno-histochemistry. In part 2, 48 healthy male Sprague-Dawley rats were divided randomly into sham operation + saline group, CCI + saline group, CCI + CVF group(continual injection CVF group) and CCI + saline + CVF group(single injection CVF group)(n=12). Pain thresholds by mechanical stimulation were recorded at preoperation and 1, 3, 7, 14 d after operation in each group. Comple-ment 3 expression in the spinal dorsal horn was determined immunohistochemically to confirm the efficacy of CVF. The SOD activity and MDA content in the spinal cord homogenate were determined, and intracellular changes of spinal dorsal horn neurons were ob-served under electronmicroscopy. Results The expression of mRNA and protein of complement 3 in spinal dorsal horn increased at 1, 3, 7 d after CCI, but those were not shown in rats in sham operation group and normal control group. The pearson analysis demonstrated that there was an initate relation between complement 3 content and pain threshold. Hyperalgesia was obviously alleviat-ed in CCI + saline + CVF group after administration of CVF, and the threshold revived following attenuation of the role of CVF. Hy-peralgesia was not evoked after sciatic nerve ligation in CCI + CVF group. Compared with CCI + saline group, the SOl) activity in-creased sinificianfly and MDA content decreased obviously in CCI + CVF group (P<0.05). Electronmicroscopy demonstrated mito-chondrial swelling and cytomembrane breakdown of spinal dorsal horn neurons in CCI + saline group while mitochondrial swelling was mitigated in spinal dorsal horn neurons in CCI + CVF group. Conclusion Abnormal complement activation occurred in the spinal dorsal horn of rats with peripheral nerve injury induced NPP and participated in the development of hyperalgesia. Key words: Neuropathic pain; Chronic constriction injury; Hyperalgesia; Complement 3 ; Cobra venom factor

Activation of NMDA Receptor is Associated with Up-regulation of COX-2 Expression in the Spinal Dorsal Horn during Nociceptive Inputs in Rats

Cyclooxygenases-2 (COX-2) in the spinal dorsal horn is up-regulated and plays an important role in pain and hyperalgesia induced by nociceptive stimulation. The mechanisms involved in the up-regulation of spinal COX-2 during nociceptive stimulation are yet not well understood. Because the important role of NMDA and its receptor in transmission of nociceptive information in the spinal cord, activation of the spinal NMDA receptor might contribute to the up-regulation of spinal COX-2 expression. The present study was undertaken to demonstrate the above hypothesis by observing changes of COX-2 expression in the spinal dorsal horn in rats subjected to formalin test and intrathecal administration of NMDA, a selective NMDA receptor agonist, in conditions with or without presence of MK-801, an antagonist of NMDA receptor, using methods of Western blotting, reverse transcription polymerase chain reaction and immunohistochemistry. The results showed that intrathecal injection of MK-801, a noncompetitive antagonist of NMDA receptor, significantly suppressed the up-regulation of the COX-2 expression and characteristic pain behavior responses evoked in formalin test. Whereas, intrathecal injection of NMDA significantly up-regulated the expression of COX-2 in the spinal dorsal horn in a time course corresponding to that of nociceptive behavioral responses elicited by the intrathecal NMDA administration. In addition, the up-regulation of the COX-2 expression induced by the intrathecal NMDA was dose-dependent and blocked by prior administration of MK-801. These findings proved that activation of NMDA receptor is associated with the up-regulation of COX-2 expression in the spinal dorsal horn during nociceptive stimulation in rats.

Interleukin-1β Induces Long-Term Potentiation of C-Fiber Evoked Field Potentials in Spinal dorsal Horn in Rats with Neuropathic Pain

Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), impairs long-term potentiation (LTP) in hippocampus, a synaptic model of memory, while in spinal dorsal horn TNF-α is found to induce LTP of C-fiber evoked field potentials in rats with nerve injury. In the present work the role of IL-1β in the spinal LTP of C-fiber evoked field potentials, which is important for pathological pain, was investigated in both physiological and pathological conditions. We found that spinal application of IL-1β at the concentrations up to 500 ng/ml affected neither basal synaptic transmission mediated by C-fiber nor spinal LTP induced by tetanic stimulation in intact rats. In rats with neuropathic pain produced by spared nerve injury (SNI) or lumbar 5 ventral root transection (L5 VRT), however, IL- 1β at a low concentration (5 ng/ml) induced LTP of C-fiber evoked field potentials. Pretreatment with either p38 MAPK inhibitor (SB203580) or NF-κB inhibitor (PDTC) but not with JNK inhibitor (SP600125) completely blocked LTP induced by IL-1β in SNI rats. Taken together, the results indicated that exogenous IL-1β might induce spinal LTP only in rats with nerve injury but not in intact rats. The differential effects of proinflammatory cytokines on synaptic plasticity in spinal dorsal horn and in hippocampus may be clinically significant.

Spinal substance P and neurokinin-1 increase with high repetition reaching

Musculoskeletal injury and inflammation is associated with performance of repetitive and forceful tasks. In this study, we examined the effects of performing a voluntary, highly repetitive, negligible force (HRNF) reaching task on spinal cord neurochemicals involved in nociception. To our knowledge, no other laboratories are examining spinal cord nociceptive neurochemicals in response to repetitive motion-induced injury and inflammation. The purpose of this study was to extend our earlier findings related to central neurotransmitters from a low demand task to a higher demand task. Specifically, this study determined immunoreactivity of a peptidergic pro-nociceptive transmitter (substance P) and one of its receptors, neurokinin-1 (NK-1) receptor, in spinal cord dorsal horns in rats performing a HRNF reaching task for 6–10 weeks. The relationship of these spinal cord changes with the number of TNFα immunopositive cells in flexor forelimb muscles and with previously observed forearm grip strength changes from these same rats were examined. Performance of the HRNF task resulted in significantly increased substance P and NK-1 receptor immunoreactivity in the superficial lamina of spinal cord dorsal horns at 6 and 10 weeks compared to trained controls ( p < 0.01). The increased substance P and NK-1 receptor immmunoreactivity were positively correlated with declines in forearm grip strength, an assay of movement-related hyperalgesia ( r = 0.70, p < 0.01 and r = 0.64, p < 0.05, respectively). The increased substance P and NK-1 receptor immmunoreactivity were also positively correlated with increased TNF immunopositive cells in forelimb flexor muscles ( r = 0.85, p < 0.001 and r = 0.88, p < 0.001, respectively). Thus, our highly repetitive task leads to increased spinal cord pro-nociceptive neurochemicals that are most likely directed by forelimb muscle inflammation and pain.

Effect of acupuncture with varied intensity on NMDA and SP expression in spinal dorsal horn in rats with gastric distension-induced pain

Effect of acupuncture with varied intensity on NMDA and SP expression in spinal dorsal horn in rats with gastric distension-induced pain

Spinal cord stimulation induces c-Fos expression in the dorsal horn in rats with neuropathic pain after partial sciatic nerve injury

Spinal cord stimulation (SCS) is an established treatment for intractable neuropathic pain, especially CRPS-1. The mechanisms of action of SCS have only been partly elucidated and include suppression of the hyper-excitability of the Wide Dynamic Range neurons and a GABA increase in the dorsal horn. In the present study we demonstrate an increase of c-Fos immunoreactive cells in the dorsal horn after SCS, suggesting early cellular activation that may preclude earlier described electrophysiological and biochemical changes in the dorsal horn after SCS. In a rat model of neuropathic pain, allodynia was induced and quantified using the von Frey test. In 11 rats a SCS device was implanted and spinal cord stimulation performed. Withdrawal threshold were measured every 15 min up to 90 min. A sham group ( n = 6) also had a SCS device implanted, but did not receive SCS. After SCS the animals were perfused and histology was performed for quantification of c-Fos immunoreactivity in the dorsal horns. We found a significant increase in c-Fos in the SCS group compared to our sham group and control tissue, indicating late cellular activity in the dorsal horn after SCS.

Cholinergic mechanisms involved in the pain relieving effect of spinal cord stimulation in a model of neuropathy

The mechanisms underlying the pain relieving effect of spinal cord stimulation (SCS) on neuropathic pain remain unclear. We have previously demonstrated that suppression of tactile hypersensitivity produced by SCS may be potentiated by i.t. clonidine in a rat model of mononeuropathy. Since the analgesic effect of this drug is mediated mainly via cholinergic mechanisms, a study exploring the possible involvement of the spinal cholinergic system in SCS was undertaken. The effect of SCS was assessed with von Frey filaments in rats displaying tactile hypersensitivity after partial ligation of the sciatic nerve and both SCS-responding and non-responding as well as normal rats were subjected to microdialysis in the dorsal horn. Acetylcholine (ACh) was analyzed with HPLC before, during and after SCS. SCS produced significantly increased release of ACh in the dorsal horn in rats responding to SCS whereas the release was unaffected in the non-responding animals. Furthermore, the basal release of ACh was significantly lower in nerve lesioned than in normal rats. In another group of rats it was found that the response to SCS was completely eliminated by i.t. atropine and a muscarinic M 4 receptor antagonist while a partial attenuation was produced by M 1 and M 2 antagonists. Blocking of nicotinic receptors did not influence the SCS effect. In conclusion, the attenuating effect of SCS on pain related behavior is associated with the activation of the cholinergic system in the dorsal horn and mediated via muscarinic receptors, particularly M 4, while nicotinic receptors appear not to be involved.

The proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100–300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.

Central glucocorticoid receptors regulate the upregulation of spinal cannabinoid-1 receptors after peripheral nerve injury in rats

Previous studies have shown that peripheral nerve injury upregulated both glucocorticoid receptors (GR) and cannabinoid-1 receptors (CB1R) within the spinal cord dorsal horn in rats. However, the relationship between the expression of spinal GR and CB1R after nerve injury remains unclear. Here, we examined the hypothesis that the upregulation of spinal CB1R induced by chronic constriction nerve injury (CCI) in rats would be regulated by spinal GR. CCI induced the upregulation of spinal CB1R primarily within the ipsilateral spinal cord dorsal horn as revealed by Western blot and immunohistochemistry. The expression of CB1R in CCI rats was substantially attenuated by intrathecal treatment with either the GR antagonist RU38486 or a GR antisense oligonucleotide given twice daily for postoperative day 1–6, whereas the expression of spinal CB1R was enhanced following intrathecal administration of a GR sense oligonucleotide twice daily for postoperative day 1–6. Furthermore, the upregulation of spinal CB1R after nerve injury was prevented in adrenalectomized rats, which was at least partially restored with the intrathecal administration of an exogenous GR agonist dexamethasone, indicating that corticosteroids (endogenous GR agonists) were critical to spinal GR actions. Since the development of neuropathic pain behaviors in CCI rats was attenuated by either RU38486 or a GR antisense oligonucleotide, these results suggest that CB1R is a downstream target for spinal GR actions contributory to the mechanisms of neuropathic pain.

Iteration of high-frequency stimulation enhances long-lasting excitatory responses in the spinal dorsal horn of rats: Characterization by optical imaging of signal propagation

To investigate plastic changes in nociceptive sensitivity of the dorsal horn, slow excitatory responses elicited by iteration of high-frequency stimulation were spatiotemporally observed in spinal cord slices of young-adult rats using membrane excitation imaging techniques. Single-pulse stimulation to the dorsal root elicited membrane excitation in lamina II, and high-frequency pulse-train stimulation evoked long-lasting excitation that expanded widely in the dorsal horn. Iteration of high-frequency stimulation enhanced the strength and extent of the excitatory responses, but such augmentation of the excitatory responses disappeared in the presence of an NMDA receptor antagonist (CPP) and was hindered by an NK1 receptor antagonist (L-703.606). The results suggest that activation of both NMDA and NK1 receptors is involved in the enhancement of slow excitatory responses evoked by iteration of high-frequency stimulation.

Coronary artery occlusion alters expression of substance P and its mRNA in spinal dorsal horn in rats

The painful sensation during acute myocardial ischemia or infarction is a common symptom and results from neural activity in humans. Little is known about the role of neuropeptides in this effect of myocardial ischemia. The aim of the current study was to investigate the role of substance P in mediating the noxious neural signals in spinal cord in acute myocardial ischemia by exploring the change in substance P and its mRNA in thoracic dorsal root ganglia and spinal dorsal horn (T1–T5) after coronary artery occlusion. The experiment was performed with immunohistochemistry, enzyme immunoassay and real time reverse transcription–polymerase chain reaction techniques on rats’ hearts. In acute myocardial ischemia (<6 h), substance P and preprotachykinin mRNA were up-regulated in the neurons of the dorsal root ganglia and spinal dorsal horn. The increase in the density of immunoreactive material was mainly observed in small-diameter neurons of the dorsal root ganglia and the superficial laminae (I and II) of the spinal cord. The increase in the expressions was statistically significant compared with the control and the sham surgery groups ( P<0.05). The results suggest that substance P is involved in the mediation of the noxious neural signals of acute myocardial ischemia in spinal cord. The pathophysiological role and significance need to be investigated.

Reduced postoperative intra-abdominal adhesions using Carbylan-SX, a semisynthetic glycosaminoglycan hydrogel

To compare the efficacy of crosslinked Carbylan-SX (Carbylan BioSurgery, Inc., Palo Alto, CA) hydrogel films and sprayable gels as physical barriers in reducing postoperative intra-abdominal adhesions in the rat cecum-abdominal wall and rat uterine horn models. Pre-formed crosslinked Carbylan-SX films and sprayable in situ crosslinkable Carbylan-SX gels were evaluated in rat cecum-abdominal wall and rat uterine horn models and compared with commercially available and clinically used Seprafilm. University animal research facility. Female Wistar rats. Abrasions were made with the foot-pedal-operated Flex-shaft (Dremel, Racine, WI) on both the cecum and abdominal wall (each area 10 mm in diameter) in female rats as one model and on both uterine horns (3 x 10 mm) in female rats as the other model. In each of the two adhesion models, four groups were assigned with eight rats in each group: (1) untreated control, (2) treated with Seprafilm (Genzyme Corporation, Cambridge, MA), (3) treated with preformed Carbylan-SX hydrogel films, and (4) treated with sprayable Carbylan-SX gel. Extent and severity of postoperative adhesions between the cecum and the abdominal wall in rat cecum-abdominal wall model and between the uterine horns in rat uterine horn model. The Carbylan-SX film and the Carbylan-SX sprayable gel led to fewer adhesions than Seprafilm in both rat adhesion models. Interestingly, a single physical form was not optimal for both models: the Carbylan film was more efficacious in the rat uterine horn model, whereas Carbylan gel gave the best results in the rat cecum-abdominal wall model. Both Carbylan-SX film and gel were efficacious in reducing postoperative intra-abdominal adhesion formation in rat cecum-abdominal wall and uterine horn models.

Segmental somatotopic organization of cutaneous afferent fibers in the lumbar spinal cord dorsal horn in rats

In the present study, we investigated the central representation of segmental cutaneous afferent fiber projection fields in the horizontal plane of the spinal cord dorsal horn in adult rats. The neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) was applied to spinal nerves T12-S2 and cutaneous ventrodorsal axial lines T13-S1. The Dil fluorescent zones in transverse sections of the dorsal horn were observed microscopically. Mediolateral locations of Dil fluorescent zones were measured, followed by reorganization on the horizontal plane through lamina I-I111. Rostral and caudal boundary lines of the central projection fields of spinal nerves T12-S2 formed 'waves' in the horizontal plane of the dorsal horn, pitching rostrocaudally about one spinal cord segment. The rostral and caudal apexes of the waves could be linked with those of adjacent segments, suggesting that the wave pattern is continuous rostrocaudally in the dorsal horn. The waves were markedly transformed in the central projection fields of the hindlimb and genital regions, in the L5 and L6 spinal cord segments.

Tumor necrosis factor-α induces long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn in rats with nerve injury: The role of NF-kappa B, JNK and p38 MAPK

Compelling evidence has shown that in hippocampus tumor necrosis factor α (TNF-α) at pathological concentration inhibits long-term potentiation (LTP), a synaptic model of learning and memory. In the present work we investigated the role of TNF-α in LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathological pain. We showed that spinal application of TNF-α affected neither basal synaptic transmission mediated by C-fibers nor spinal LTP of C-fiber evoked field potentials induced by tetanic stimulation in intact rats. However, in rats with neuropathic pain, produced by either lumbar 5 ventral root transection (L5 VRT) or spared nerve injury (SNI), spinal application of TNF-α induced LTP of C-fiber evoked field potentials. Spinal application of JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) did not affect the spinal LTP induced by tetanic stimulation in intact rats, but completely blocked LTP induced by TNF-α in L5 VRT rats. NF-kappa B (NF-κB) inhibitor (PDTC) also blocked LTP induced by TNF-α. These results suggest that TNF-α and its downstream molecules may have no acute effect on spinal synaptic transmission in intact animals and induce LTP in rats with neuropathic pain produced by nerve injury.

Prostaglandin E2 potentiates the excitability of small diameter trigeminal root ganglion neurons projecting onto the superficial layer of the cervical dorsal horn in rats

The aim of the present study was to investigate how prostaglandin E2 (PGE2) affects the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, using fluorescence retrograde tracing and perforated patch-clamp techniques. TRG neurons were retrogradely labeled with fluorogold (FG). The cell diameter of FG-labeled neurons was small (< 30 microm). Under the voltage-clamp mode, application of PGE2 (0.01-10 microM) concentration-dependently increased the magnitude of the peak tetrodotoxin-resistant sodium current (TTX-R I(Na)) and this current was maximal at a concentration of 1 microM. One micromolar PGE2 application caused a hyperpolarizing shift of 8.3 mV in the activation curve for TTX-R I(Na). In the current-clamp mode, the PGE2 (1 microM) application significantly increased the number of action potentials during the depolarizing step pulses as well as the level of overshoot but had no significant effect on the resting membrane potential. These results suggest that the excitability of small diameter TRG neurons seen after 1 microM PGE2 application is involved in an increase in the

Dermatomes and the central organization of dermatomes and body surface regions in the spinal cord dorsal horn in rats.

Dermatomes and the associated central projection fields were studied with the application of fluorescent neurotracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), to 21 reference points on rat trunk and hindlimb skin. Segmental distribution and rostrocaudal central level of dorsal root ganglion (DRG) neurons innervating reference points were examined and DiI-induced fluorescent areas were mapped in the horizontal plane through lamina II of the dorsal horn. Segmental levels of DRG neurons innervating reference points were generally identical to the level determined using dye-extravasation methods. However, innervation of the first digit was situated in the L4 dermatome, not the L3 reported previously using those methods. Generally, afferents from a reference point projected to a single field in the ipsilateral dorsal horn. Reference points on ventral and dorsal median lines of the trunk were represented bilaterally. Afferents from reference points located on the ventral median line of the hindlimb projected to two separate fields: one on the medial margin of spinal cord segments L2-L5 and the other on the medial half of spinal cord segment L5. From the distribution of central projection fields of reference points, central projection fields of dermatomes were revealed as even in shape and located within corresponding spinal cord segments. The arrangement of peripheral and central fields of dermatomes and body surface regions suggests that peripheral and central projection fields of cutaneous afferent fibers are reshaped from the common prototypical pattern that exhibits an orderly and evenly sequenced arrangement.

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord

The inhibitory neurotransmitter GABA is synthesised by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

Inflammation induced increase of fluoride resistant acid phosphatase (FRAP) activity in the spinal dorsal horn in rats

Fluoride-resistant acid phosphatase (FRAP) has been suggested as an enzymatic marker for nociceptive primary afferent terminals in the spinal dorsal horn, however there has not been demonstrated a direct functional relation between FRAP activity and an increased nociceptive transmission. For this purpose, we quantitated FRAP activity in the spinal dorsal horn of the rat in a heat-induced cutaneous inflammatory model. Male Sprague–Dawley rats anaesthetised with thiopental were separated in two groups where the left hindpaw was submerged during 60 s either in water at room temperature (control group) or in water at 60°C (inflammation group) which induce in this group a progressive hindpaw inflammation. After 8 h, the lumbar enlargement of the spinal cord was extracted, cut in slices and 1 mm micropunch fragments were obtained from the right and left dorsal horn. The activity of FRAP was determined using the Gomori colorimetric method and corrected by the protein concentrations. FRAP activity in the left dorsal horn was statistically higher than right dorsal horn in the inflammation group (3.05±0.54 versus 1.91±0.23 u/g per l; P<0.05). Also, FRAP activity from the left dorsal horn of the control and inflammation groups show a significant increase in the last group (3.05±0.54 versus 2.17±0.23 u/g per l; P<0.05). This results demonstrate that FRAP is not only an enzymatic marker for neuronal and fibre integrity of nociceptive primary afferents but also it is associated to the nociceptive afferent activation.

Clinical and electrophysiological expression of deafferentation pain alleviated by dorsal root entry zone lesions in rats.

The aims of this study were to construct an animal model of deafferentation of the spinal cord by brachial plexus avulsion and to analyze the effects of subsequent dorsal root entry zone (DREZ) lesions in this model. To this end, the authors measured the clinical and electrophysiological effects of total deafferentation of the cervical dorsal horn in rats and evaluated the clinical efficacy of cervical DREZ lesioning. Forty-three Sprague-Dawley rats were subjected to total deafferentation of the right cervical dorsal horn by performing a posterior rhizotomy from C-5 to T-1. The clinical effects of this deafferentation, namely self-directed mutilations consisting of scraping and/or ulceration of the forelimb skin or even autotomy of some forelimb digits, were then evaluated. As soon as some of these clinical signs of pain appeared, the authors performed a microsurgical DREZ rhizotomy ([MDR], microincision along the deafferented DREZ and dorsal horn). Before and after MDR, single-unit recordings were obtained in the deafferented dorsal horn and in the contralateral (healthy) side. The mean frequency of spontaneous discharge from the deafferented dorsal horn neurons was significantly higher than that from the healthy side (36.4 Hz compared with 17.9 Hz, p = 0.03). After deafferentation, 81.4% of the rats developed clinical signs corresponding to pain following posterior rhizotomy. Among these animals, scraping was observed in 85.7% of cases, ulceration (associated with edema) in 37.1%, and autotomy in 8.5%. These signs appeared a mean 5.7 weeks (range 1-12 weeks) after deafferentation. Thirteen rats benefited from an MDR; nine (69%) experienced a complete cure, that is, a total resolution of scraping or ulceration (a mean 4.6 weeks after MDR). In contrast, only one of 11 sham-operated animals showed signs of spontaneous recovery (p = 0.01). These results emphasize the role of the spinal dorsal horn in the genesis of deafferentation pain and suggest that dorsal horn deafferentation by cervical posterior rhizotomy in the rat provides a reliable model of chronic pain due to brachial plexus avulsion and its suppression by MDR.

EFFECTS OF MIDAZOLAM IN THE SPINAL NERVE LIGATION MODEL OF NEUROPATHIC PAIN IN RATS

Potential changes in the spinal GABAergic activity after nerve injury were studied by comparing the effects of systemic administration of the benzodiazepine midazolam on the noxious evoked responses of dorsal horn in rats with spinal nerve ligation of neuropathy and control animals. The tight ligation of the L5 and L6 spinal nerves was performed in adult male Sprague‐Dawley rats and resulting mechanical and cold allodynia were assessed with von Frey hairs and the acetone drop test. Single unit extracellular recordings of dorsal horn neurones were performed 15‐18 days after the surgery under halothane anaesthesia using transcutaneous electrical stimulation of the receptive field at three times the C‐fibre threshold. The rats in the spinal nerve ligation group, but not in the sham‐operated control group developed mechanical and cold allodynia. Subcutaneous administration of midazolam 0.1–3.0 mg/kg reduced the A delta‐fibre evoked activity in a dose‐related manner in all study groups, but the C‐fibre evoked activity was significantly reduced only in the spinal nerve ligation group. The inhibitory effects of s.c. midazolam were significantly reversed by i.t. administration of flumazenil, suggesting a spinal site of action. Midazolam reduced C‐fibre evoked firing significantly more in the spinal nerve ligation model than in the non‐operated or sham controls. These results indicate changes in the spinal GABAergic system in the neuropathic animals and could be of importance in the development of new treatments for neuropathic pain.