Intra- and extraneuronal changes of immunofluorescence staining for TNF-alpha and TNFR1 in the dorsal root ganglia of rat peripheral neuropathic pain models

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-alpha and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury. 2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-alpha and TNFR1 proteins. The TNF-alpha and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-alpha and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-alpha and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury. 3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-alpha and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFalpha IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-alpha after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFalpha IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites. 4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF. 5. Our results suggest more sources of TNF-alpha protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-alpha molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-alpha effect on the primary sensory neurons can be mediated following nerve injury.     

Intra- and Extraneuronal Changes of Immunofluorescence Staining for TNF- and TNFR1 in the Dorsal Root Ganglia of Rat Peripheral Neuropathic Pain Models

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-α and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury.2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-α and TNFR1 proteins. The TNF-α and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-α and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-α and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury.3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-α and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFα IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-α after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFα IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites.4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF.5. Our results suggest more sources of TNF-α protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-α molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-α effect on the primary sensory neurons can be mediated following nerve injury.     

The Effects of Granulocyte-Colony Stimulating Factor on Regeneration in Nerve Crush Injuries in Rats

Granulocyte-colony stimulating factor (G-CSF) is widely known to have a neuroprotective effect, but its effects on function and morphology in mechanical nerve injury are not well understood. The aim of this study was to confirm the time course of the functional changes and morphological effects of G-CSF in a rat model of nerve crush injury. Twelve-eight rats were divided into three group: sham-operated control group, G-CSF-treated group, and saline treated group. 2 weeks after the nerve crush injury, G-CSF was injected for 5 days. After 4 weeks, functional tests such as motor nerve conduction velocity (MNCV), mechanical and cold allodynia tests, and morphological studies were performed. G-CSF-treated rats had significantly improved nerve function including MNCV and mechanical and cold allodynia. In addition, G-CSF-treated rats had significantly higher the density of myelinated fibers than saline-treated rats. In conclusion, we found that 100 μg/kg administration of G-CSF promoted long-term functional recovery in a rat model of nerve crush injury.     

Simvastatin对大鼠坐骨神经crush损伤修复作用研究

目的：探讨他汀类（statins）药物Simvastatin在大鼠坐骨神经损伤修复中的作用及可能的作用机制。方法：制作SD大鼠标准坐骨神经钳夹损伤（crush）模型后,分别予Simvastatin和溶媒对照干预2周。手术前后不同时间点进行趾展功能指数测定、神经电生理学、血脂水平、血清IL-6检测和组织学评价。结果：Simvastatin干预组与对照组比较,趾展功能指数在术后5d和8d显著增大（P〈0．05）,足趾展开速度快;2周肌肉复合动作电位幅度高,4周神经传导速度快;组织学显示有髓神经纤维数量多,髓鞘厚,排列相对整齐。各组手术前血脂水平无差异,手术后2周均有不同程度的降低,但Simvastatin干预组总胆固醇降低程度最轻,与对照组比较有显著差异（P〈0．05）;Simvastatin干预组手术后5d,血清IL-6水平明显低于对照组（P〈0．05）。结论：本研究发现,Simvastatin可能通过抑制免疫炎症反应,维持神经损伤后胆固醇的平衡,促进大鼠坐骨神经损伤的修复和再生。     

Rat sciatic nerve crush injury and recovery tracked by plantar test and immunohistochemistry analysis

An experimental crush injury to the sciatic nerve, with a crush force of 49.2 N (pressure p=1.98x10(8) Pa), was inflicted in 30 male rats (Wistar). A control group (sham), with the same number of rats, was also operated upon exactly as the experimental group but without the crush injury. We tested the sensory and motor recovery of the sciatic nerve with Hargreaves method, using an apparatus from Ugo Basile, Italy. Testing was continued for both legs of each rat, injured and uninjured, starting preoperatively (0 day), and then 1, 7, 14, 21, and 28 days postoperatively. The same experiment was run simultaneously with the sham group. The Plantar test showed recovery of the sensory and motor function of the sciatic nerve, though not complete recovery, by 28 days. An immunohistochemical experiment was run in parallel with the plantar test on L3-L6 segments of the spinal cord from where the sciatic nerve extends. We used antibodies for Myelin-associated glycoprotein (MAG), and gangliosides GD1a and GT1b on the aforesaid part of the spinal cord. The immunohistochemical methods showed changes in sensory and motor axons in the spinal cord segment L3-L6 which suggest correspondence with the results of the Plantar test, in terms of recovery of the sensory and motor function after injury of the sciatic nerve. The immunohistochemical results also show ipsilateral and contralateral changes following injury. Results of the plantar test are suggestive that the rat shows compensation for an injury in its contralateral leg.     

Neuronal CCL21 up-regulates microglia P2X4 expression and initiates neuropathic pain development

Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an untreatable pathological pain reaction occurring after peripheral nerve injury. How nerve injury in the periphery leads to this microglia reaction in the dorsal horn of the spinal cord is not yet understood. It is shown here that CCL21 was rapidly expressed in injured small-sized primary sensory neurons and transported to their central terminals in the dorsal horn. Intrathecal administration of a CCL21-blocking antibody diminished tactile allodynia development in wild-type animals. Mice deficient for CCL21 did not develop any signs of tactile allodynia and failed to up-regulate microglial P2X4 receptor expression. Microglia P2X4 expression was enhanced by CCL21 application in vitro and in vivo. A single intrathecal injection of CCL21 to nerve-injured CCL21-deficient mice induced long-lasting allodynia that was undistinguishable from the wild-type response. This effect of CCL21 injection was strictly dependent on P2X4 receptor function. Since neuronal CCL21 is the earliest yet identified factor in the cascade leading to tactile allodynia, these findings may lead to a preventive therapy in neuropathic pain.     

Decreased Resting Functional Connectivity after Traumatic Brain Injury in the Rat

Traumatic brain injury (TBI) contributes to about 10% of acquired epilepsy. Even though the mechanisms of post-traumatic epileptogenesis are poorly known, a disruption of neuronal networks predisposing to altered neuronal synchrony remains a viable candidate mechanism. We tested a hypothesis that resting state BOLD-fMRI functional connectivity can reveal network abnormalities in brain regions that are connected to the lesioned cortex, and that these changes associate with functional impairment, particularly epileptogenesis. TBI was induced using lateral fluid-percussion injury in seven adult male Sprague-Dawley rats followed by functional imaging at 9.4T 4 months later. As controls we used six sham-operated animals that underwent all surgical operations but were not injured. Electroencephalogram (EEG)-functional magnetic resonance imaging (fMRI) was performed to measure resting functional connectivity. A week after functional imaging, rats were implanted with bipolar skull electrodes. After recovery, rats underwent pentyleneterazol (PTZ) seizure-susceptibility test under EEG. For image analysis, four pairs of regions of interests were analyzed in each hemisphere: ipsilateral and contralateral frontal and parietal cortex, hippocampus, and thalamus. High-pass and low-pass filters were applied to functional imaging data. Group statistics comparing injured and sham-operated rats and correlations over time between each region were calculated. In the end, rats were perfused for histology. None of the rats had epileptiform discharges during functional imaging. PTZ-test, however revealed increased seizure susceptibility in injured rats as compared to controls. Group statistics revealed decreased connectivity between the ipsilateral and contralateral parietal cortex and between the parietal cortex and hippocampus on the side of injury as compared to sham-operated animals. Injured animals also had abnormal negative connectivity between the ipsilateral and contralateral parietal cortex and other regions. Our data provide the first evidence on abnormal functional connectivity after experimental TBI assessed with resting state BOLD-fMRI.     

Org.2766 improves functional and electrophysiological aspects of regenerating sciatic nerve in the rat

The beneficial effect of short-term (8 days) melanocortin therapy on regenerating peripheral nerves is demonstrated using functional and electrophysiological tests. Following a crush lesion of the rat sciatic nerve, recovery of sensory function is monitored by assessing the responsiveness of the rat to a small electric current applied to the footsole. Recovery of motor function is assessed by means of an analysis of walking patterns. Normalization of the walking pattern reflects reinnervation of different muscle groups. The motor and H-reflex related sensory nerve conduction velocity of the regenerated nerves are longitudinally investigated in the same rats in which the recovery of motor and sensory function had been assessed previously. Functional tests show an enhanced recovery under melanocortin therapy, but in the end both saline- and melanocortin-treated rats show 100% recovery. However, when compared to the contralateral sciatic nerve, in the peptide-treated animals motor nerve conduction in the regenerated nerves has fully recovered after about 90 days following the crush lesion and the sensory conduction after about 120 days, whereas in the saline-treated rats a deficit of 20-40% in both motor and sensory conduction remains. This difference is observed even 214 days following crush.     

骨髓间充质干细胞经BDNF基因修饰后移植治疗大鼠半横断脊髓损伤的电生理研究

目的采用电生理的研究方法,观察脑源性神经营养因子(BDNF)基因修饰的骨髓间充质干细胞对脊髓损伤的修复作用。方法随机将大鼠分成3组:空白组10只(只切除椎板,暴露脊髓硬脊膜);SCI组10只;SCI术后细胞移植组10只;从以上三组大鼠随机抽取8只于细胞移植后1 d、7 d、14 d、21 d、30 d、60 d进行SEP(皮层体感诱发电位)、MEP(运动诱发电位)等电生理检测技术,并观察大鼠的运动评分恢复程度。结果细胞移植4d后,大鼠饮食和活动开始增加;后肢变化过程如下:损伤后1~4 d损伤侧后肢迟缓性瘫痪,拖地行走,损伤对侧后肢由损伤初期的运动减弱逐渐恢复,损伤后5~9 d损伤侧后肢痉挛性瘫痪;10~14 d损伤侧下肢恢复少量活动,损伤对侧后肢恢复至较损伤前稍弱的状态;15~21 d损伤侧后肢活动能力较之前有明显改善,至30 d损伤侧后肢活动能力及肌张力恢复程度最明显,30 d以后无更明显改善。免疫组化发现损伤处诱导标记的骨髓间充质干细胞存活,行为学观察发现细胞移植改善了损伤大鼠运动能力。结论骨髓间充质干细胞经BDNF基因修饰后可以促进脊髓损伤大鼠的神经再生及部分传导功能恢复。     

对侧外周神经移位到损伤手臂引起的体感皮层功能动态重组

单侧肢体的外周神经损伤通常导致对侧体感皮层的功能重组. 然而,接受了对侧颈 7 (C7) 外周神经移位手术治疗单侧手臂臂丛全撕脱的病人,在术后早期当其患手被触摸时,只在其健手产生感觉. 在术后晚期,病人才逐渐恢复其患手和健手的正常、独立的功能. 我们在模拟对侧颈 7 (C7) 外周神经移位手术病例的大鼠模型上,用记录体感诱发电位的方法研究了患手和健手的体感代表区. 患手的体感和运动功能由于 C7 神经的再生而逐渐恢复. 术后第 5 个月始, 13 只大鼠患手的体感代表区只出现在其同侧的皮层,同时患手和健手的代表区在该皮层内是高度重叠的 (除掉一个例外),虽然刺激它们产生的体感诱发电位的潜伏期和反应幅度有很大的不同. 结果表明,移位到患手的对侧外周神经能够导致同侧体感皮层动态的功能重组,提示身体另侧感觉输入的介入激发了大脑显著的可塑性.     

Differential effects of post-natal development, animal strain and long term recovery on the restoration of neuromuscular function after neuromyotoxic injury in rat

We have analysed the effect of long term recovery, post-natal development and animal strain on the extent of restoration of neuromuscular function after neuromyotoxic injury in the rat (Rattus norvegicus). Muscle isometric contractile properties of soleus muscle in response to nerve stimulation were measured in situ in snake venom injured muscles and compared to contralateral uninjured muscles. We show here that neuromuscular function was not fully recovered until 24 weeks after injury in young adult (2-3 month old) Wistar rats. Moreover, the level of functional recovery 3 weeks after injury induced in juvenile rats (1 month old) was not globally different from that in younger adult, adult (10 month old) and older adult (24 month old) Wistar rats. Furthermore, the level of recovery of some contractile parameters differed between Wistar and Sprague-Dawley strains 3 weeks after injury. In conclusion, a very long time (>12 weeks) is required for full neuromuscular recovery following neuromyotoxic injury of young adult rats. Moreover, neuromuscular recovery during post-natal development is not markedly different from that during adult stage in the Wistar rat strain. Finally, some rat strain differences are observed in the recovery after injury of young adult rats.     

Nerve Growth Factor of Red Nucleus Involvement in Pain Induced by Spared Nerve Injury of the Rat Sciatic Nerve

Nerve growth factor (NGF), a member of the neurotrophin family, is essential for the development and maintenance of sensory neurons and for the formation of central pain circuitry. The current study was designed to evaluate the expression of NGF in the brain of rats with spared nerve injury (SNI), using immunohistochemical technique. The results showed that the level of NGF in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the effect of NGF in the development of neuropathic pain, different doses of anti-NGF antibody (20, 2.0 and 0.2 μg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The data suggested that the higher doses of anti-NGF antibody (20 and 2.0 μg/ml) significantly attenuated the mechanical allodynia of neuropathic rats, while the 0.2 μg/ml antibody showed no analgesic effect. These results suggest that the NGF of RN is involved in the development of neuropathic allodynia in SNI rats.     

Autotomy and decreased spinal substance P following peripheral cryogenic nerve lesion.

Cryotherapy has been clinically applied to relieve pain by blocking peripheral nerve function. Clinically, analgesia has been successfully achieved but there is suggestion that permanent pain relief may be accompanied by extended motor and sensory deficits. This study was undertaken to determine the effect of a peripheral cryogenic nerve lesion, i.e., of the sciatic nerve, on behavioral effects and substance P content in the dorsal horn of the spinal cord. In rats, the right sciatic nerve was exposed and cryolesioned using one freeze-thaw-refreeze cycle. In an alternate group, the right sciatic nerve was cut and a 3-mm region was excised. Animals were allowed to recover 7 or 21 days during which their behavior was assessed. Autotomy, an animal's tendency to attack the nerve-injured affected limb, occurred in both the cryolesioned and sectioned groups. They were killed by transcardiac perfusion of fixative and segments L4-S1 were processed for immunocytochemistry. The SP-like immunoreactivity (SPLI) in the right and left dorsal horns was compared and quantitated using a microcomputer imaging device. We utilized a fully automated program to digitize and quantitate the staining of the substantia gelatinosa. There was no significant difference in SPLI in the dorsal horns of the sham-operated controls at either time period. At 7 days the sectioned group demonstrated a 40% decrease in SPLI and 76% decrease at 21 days. In the cryolesioned group, there was a 34% decrease at 7 days and by 21 days there was a 68% decrease in immunoreactivity on the operated side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Effect of taste deafferentation on the preference for solutions of saccharose, saccharin and salt in rats

The preference of sucrose, saccharin and salt solutions to water was analyzed during 5 days in rats with bilateral section of the lingual nerve comprising the taste nerve--chorda tympani. In the process of the analysis of daily consumption and choice of solutions, different types of behavioural reactions were found: stable preference and change of preference. The number of rats preferring NaCl was greater among the animals with sectioned lingual nerve than among sham-operated control rats and rats with ligated ducts of submaxillary and sublingual salivary glands. The number of rats with the lingual nerve section preferring sucrose or saccharin solutions to water was equal to that among the sham-operated rats. At the same time the mean volume of sucrose solution taken in daily by rats with sectioned lingual nerve was twice as great as the volume of saccharin, drunk by the same animals. The role of taste in the process of choice and preference of NaCl to sweet solutions is discussed.     

Differential Regulation of Glucocorticoid Receptor Expression in Distinct Columns of Periaqueductal Grey in Rats with Behavioural Disability Following Nerve Injury

Neuropathic pain is diagnosed primarily by sensory dysfunction, which includes both spontaneous, and stimulus-evoked pain. Clinical evaluation highlights the disabilities which characterise this condition for most patients. Chronic constriction injury of the sciatic nerve (CCI) evokes sensory dysfunction characteristic of neuropathic pain. Approximately, 30 % of CCI rats show disabilities similar to those identified in clinical evaluation of neuropathic pain patients, these include: altered social behaviours; sleep disturbances; and endocrine dysfunction. The periaqueductal grey (PAG) is a nodal point in the brain circuits which regulate these functions, and undergoes a distinct set of neural and glial adaptations following CCI, in rats with disabilities. CCI increases corticosterone, which through its actions at the glucocorticoid receptor (GR), can trigger cellular adaptation. GR expression in PAG was quantified using qRT-PCR, Western blotting and immunohistochemical analyses and nerve-injured rats, with and without disabilities, were compared. Our data showed that the PAG of disabled rats has significantly increased expression of GR mRNA and protein. Further, this increased protein expression reflects contrasting patterns of change in GR expression in PAG subregions. The dorsolateral PAG had significant increases in the number of GR-immunoreactive (GR-IR) cells and the caudal lateral and ventrolateral PAG each had significant reductions in the number of GR-IR cells. These regional increases and decreases correlated with the degree of disability, as indicated by the degree of change in social behaviours. Our results suggest a role for altered PAG, GR–corticosterone interactions and their resultant cellular consequences in the expression of disabilities in a subpopulation of nerve-injured rats.     

5-Hydroxytryptophan-induced respiratory recovery after cervical spinal cord hemisection in rats.

The present study investigates the role of serotonin in respiratory recovery after spinal cord injury. Experiments were conducted on C(2) spinal cord hemisected, anesthetized, vagotomized, paralyzed, and artificially ventilated rats in which end-tidal CO(2) was monitored and maintained. Before drug administration, the phrenic nerve ipsilateral to hemisection showed no respiratory-related activity due to the disruption of the descending bulbospinal respiratory pathways by spinal cord hemisection. 5-Hydroxytryptophan (5-HTP), a serotonin precursor, was administrated intravenously. 5-HTP induced time- and dose-dependent increases in respiratory recovery in the phrenic nerve ipsilateral to hemisection. Although the 5-HTP-induced recovery was initially accompanied by an increase in activity in the contralateral phrenic nerve, suggesting an increase in descending respiratory drive, the recovery persisted well after activity in the contralateral nerve returned to predrug levels. 5-HTP-induced effects were reversed by a serotonin receptor antagonist, methysergide. Because experiments were conducted on animals subjected to C(2) spinal cord hemisection, the recovery was most likely mediated by the activation of a latent respiratory pathway spared by the spinal cord injury. The results suggest that serotonin is an important neuromodulator in the unmasking of the latent respiratory pathway after spinal cord injury. In addition, the results also suggest that the maintenance of 5-HTP-induced respiratory recovery may not require a continuous enhancement of central respiratory drive.     

Transplantation of bone marrow-derived mononuclear cells improves mechanical hyperalgesia, cold allodynia and nerve function in diabetic neuropathy

Relief from painful diabetic neuropathy is an important clinical issue. We have previously shown that the transplantation of cultured endothelial progenitor cells or mesenchymal stem cells ameliorated diabetic neuropathy in rats. In this study, we investigated whether transplantation of freshly isolated bone marrow-derived mononuclear cells (BM-MNCs) alleviates neuropathic pain in the early stage of streptozotocin-induced diabetic rats. Two weeks after STZ injection, BM-MNCs or vehicle saline were injected into the unilateral hind limb muscles. Mechanical hyperalgesia and cold allodynia in SD rats were measured as the number of foot withdrawals to von Frey hair stimulation and acetone application, respectively. Two weeks after the BM-MNC transplantation, sciatic motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV), sciatic nerve blood flow (SNBF), mRNA expressions and histology were assessed. The BM-MNC transplantation significantly ameliorated mechanical hyperalgesia and cold allodynia in the BM-MNC-injected side. Furthermore, the slowed MNCV/SNCV and decreased SNBF in diabetic rats were improved in the BM-MNC-injected side. BM-MNC transplantation improved the decreased mRNA expression of NT-3 and number of microvessels in the hind limb muscles. There was no distinct effect of BM-MNC transplantation on the intraepidermal nerve fiber density. These results suggest that autologous transplantation of BM-MNCs could be a novel strategy for the treatment of painful diabetic neuropathy.     

Nitric oxide-induced increase of excitatory amino acid levels in the trigeminal nucleus caudalis of the rat with tactile hypersensitivity evoked by the loose-ligation of the inferior alveolar nerves

To investigate whether or not N-methyl-D-aspartate (NMDA)/nitric oxide (NO) pathway in the trigeminal system is involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, we examined the alteration patterns of excitatory amino acid (EAA) level in the superficial layer of subnucleus caudalis of the brain-stem trigeminal sensory nuclear complex (SpVc-I,II) by in vivo microdialysis. A very high EAA release response was observed immediately after the start of the perfusion in ligated animals compared with sham-operated rats. The EAA level evoked by application of the 40-V tooth pulp-stimulation or 1% capsaicin cream was significantly higher in the ligated animals than those in the sham-operated animals. This increase of EAA level induced by capsaicin cream was inhibited by adding carboxy-PTIO (100 microM) to the perfusate. The applications of SNAP (2 mM) into the perfusate enhanced the level of EAAs in ligated animals and sham-operated animals. However, SNAP-evoked EAA levels in ligated animals were not significantly different compared with those of sham-operated animals. These results suggest that alterations in the stimulus-evoked raised EAA levels that occur in the site of the first synaptic relay of the dental pain pathway and which are expressed via endogenous NO, and which play an important role in development and/or maintenance of pathological pain states following dental peripheral nerve injury.