Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation. Ye Huang and Yonghua Liu contributed equally to this work.     

Moderately Different NADPH-Diaphorase Positivity in the Selected Peripheral Nerves after Ischemia/ Reperfusion Injury of the Spinal Cord in Rabbit

1. To vicariously investigate the nitric oxide synthase (NOS) production after spinal cord injury, NADPH-d histochemistry was performed on the selected peripheral nerves of adult rabbits 7 days after ischemia. The effect of transient spinal cord ischemia (15 min) on possible degenerative changes in the motor and mixed peripheral nerves of Chinchilla rabbits was evaluated.2. The NADPH-diaphorase histochemistry was used to determine NADPH-diaphorase activity after ischemia/reperfusion injury in radial nerve and mediane nerve isolated from the fore-limb and femoral nerve, saphenous nerve and sciatic nerve separated from the hind-limb of rabbits. The qualitative analysis of the optical density of NADPH-diaphorase in selected peripheral nerves demonstrated different frequency of staining intensity (attained by UTHSCSA Image Tool 2 analysis for each determined nerve).3. On the seventh postsurgery day, the ischemic spinal cord injury resulted in an extensive increase of NADPH-d positivity in isolated nerves. The transient ischemia caused neurological disorders related to the neurological injury—a partial paraplegia. The sciatic, femoral, and saphenous nerves of paraplegic animals presented the noticeable increase of NADPH-d activity. The mean of NADPH-diaphorase intensity staining per unit area ranged from 134.87 (±32.81) pixels to 141.65 (±35.06) pixels (using a 256-unit gray scale where 0 denotes black, 256 denotes white) depending on the determined nerve as the consequence of spinal cord ischemia. The obtained data were compared to the mean values of staining intensity in the same nerves in the limbs of control animals (163.69 (±25.66) pixels/unit area in the femoral nerve, 173.00 (±32.93) pixels/unit area in saphenous nerve, 186.01 (±29.65) pixels/unit area in sciatic nerve). Based on the statistical analysis of the data (two-way unpaired Mann–Whitney test), a significant increase (p≤0.05) of NADPH-d activity in femoral and saphenous nerve, and also in sciatic nerve (p≤0.001) has been found. On the other hand, there was no significant difference between the histochemically stained nerves of fore-limbs after ischemia/reperfusion injury and the same histochemically stained nerves of fore-limbs in control animals.4. The neurodegenerative changes of the hind-limbs, characterized by damage of their motor function exhibiting a partial paraplegia after 15 min spinal cord ischemia and subsequent 7 days of reperfusions resulted in the different sensitivity of peripheral nerves to transient ischemia. Finally, we suppose that activation of NOS indirectly demonstrable through the NADPH-d study may contribute to the explanation of neurodegenerative processes and the production of nitric oxide could be involved in the pathophysiology of spinal cord injury by transient ischemia.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

Peripheral Nerve Injury Is Associated with Chronic,Reversible Changes in Global DNA Methylation in the Mouse Prefrontal Cortex

Changes in brain structure and cortical function are associated with many chronic pain conditions including low back pain and fibromyalgia. The magnitude of these changes correlates with the duration and/or the intensity of chronic pain. Most studies report changes in common areas involved in pain modulation, including the prefrontal cortex (PFC), and pain-related pathological changes in the PFC can be reversed with effective treatment. While the mechanisms underlying these changes are unknown, they must be dynamically regulated. Epigenetic modulation of gene expression in response to experience and environment is reversible and dynamic. Epigenetic modulation by DNA methylation is associated with abnormal behavior and pathological gene expression in the central nervous system. DNA methylation might also be involved in mediating the pathologies associated with chronic pain in the brain. We therefore tested a) whether alterations in DNA methylation are found in the brain long after chronic neuropathic pain is induced in the periphery using the spared nerve injury modal and b) whether these injury-associated changes are reversible by interventions that reverse the pathologies associated with chronic pain. Six months following peripheral nerve injury, abnormal sensory thresholds and increased anxiety were accompanied by decreased global methylation in the PFC and the amygdala but not in the visual cortex or the thalamus. Environmental enrichment attenuated nerve injury-induced hypersensitivity and reversed the changes in global PFC methylation. Furthermore, global PFC methylation correlated with mechanical and thermal sensitivity in neuropathic mice. In summary, induction of chronic pain by peripheral nerve injury is associated with epigenetic changes in the brain. These changes are detected long after the original injury, at a long distance from the site of injury and are reversible with environmental manipulation. Changes in brain structure and cortical function that are associated with chronic pain conditions may therefore be mediated by epigenetic mechanisms.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

Proteome of synaptosome‐associated proteins in spinal cord dorsal horn after peripheral nerve injury

Peripheral nerve injury may lead to neuroadaptive changes of cellular signals in spinal cord that are thought to contribute to central mechanisms underlying neuropathic pain. Here we used a 2‐DE‐based proteomic technique to determine the global expression changes of synaptosome‐associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury (SNI). The fifth lumbar dorsal horns ipsilateral to SNI or sham surgery were harvested on day 14 post‐surgery, and the total soluble and synaptosomal fractions were isolated. The proteins derived from the synaptosomal fraction were resolved by 2‐DE. We identified 27 proteins that displayed different expression levels after SNI, including proteins involved in transmission and modulation of noxious information, cellular metabolism, membrane receptor trafficking, oxidative stress, apoptosis, and degeneration. Six of the 27 proteins were chosen randomly and further validated in the synaptosomal fraction by Western blot analysis. Unexpectedly, Western blot analysis showed that only one protein in the total soluble fraction exhibited a significant expression change after SNI. The data indicate that peripheral nerve injury changes not only protein expression but also protein subcellular distribution in dorsal horn cells. These changes might participate in the central mechanism that underlies the maintenance of neuropathic pain.     

Effect of Treadmill Exercise on Serotonin Immunoreactivity in Medullary Raphe Nuclei and Spinal Cord Following Sciatic Nerve Transection in Rats

The serotoninergic system modulates nociceptive and locomotor spinal cord circuits. Exercise improves motor function and changes dopaminergic, noradrenergic, and serotonergic central systems. However, the direct relationship between serotonin, peripheral nerve lesion and aerobic treadmill exercise has not been studied. Using immunohistochemistry and optic densitometry, this study showed that the sciatic nerve transection increased the serotoninergic immunoreactivity in neuronal cytoplasm of the magnus raphe nuclei of trained and sedentary rats. In the dorsal raphe nucleus the increase only occurred in sedentary-sham-operated rats. In the spinal cord of trained, transected rats, the ventral horn showed significant changes, while the change in dorsal horn was insignificant. Von Frey’s test indicated analgesia in all exercise-trained rats. The sciatic nerve functional index indicated recovery in the trained group. Thus, both the aerobic treadmill exercise training and the nervous lesion appear to contribute to changes in serotonin immunoreactivity.     

c-fos Expression in Gracilothalamic Tract Neurons after Electrical Stimulation of the Injured Sciatic Nerve in the Adult Rat

The number of c-fos protein-like immunoreactive (Fos-LI) cells in the gracile nucleus was determined after electrical stimulation at Aα/Aβ-fiber strength of the normal and of the previously injured sciatic nerve in adult rats. No Fos-LI cells were seen after electrical stimulation of the noninjured sciatic nerve, or after sciatic nerve injury without electrical stimulation. However, stimulation 21 days after sciatic nerve transection resulted in numerous Fos-LI cells in the ipsilateral gracile nucleus. Combined Fos immunocytochemistry and retrograde labeling from the thalamus showed that the majority (76%; range = 70–80%) of the cells in the gracile nucleus that expressed Fos-LI after nerve injury projected to the thalamus. The results indicate that morphological, biochemical, and physiological alterations in primary sensory central endings and second-order neurons, which have earlier been demonstrated in the dorsal column nuclei after peripheral nerve injury, are accompanied by changes in the c-fos gene activation pattern after stimulation of the injured sciatic nerve. A substantial number of the c-fos-expressing neurons project to the thalamus.     

Permanent alterations of spinal cord reflexes following nerve lesion in newborn rats

Sciatic nerve lesion in newborn rats is known to cause degeneration of a large number of axotomized motoneurones and spinal ganglion cells. Some of the surviving motoneurones exhibit abnormal firing properties and the projection pattern of central terminals of sensory neurones is altered. We report here on long-term changes in spinal cord reflexes in adult rats following neonatal nerve crush. In acutely spinalized and anaesthetized adult rats 4-6 months old in which the sciatic nerve had been crushed on one side at birth, the tibial nerve, common peroneal nerve or sural nerve were stimulated on the reinnervated and control side and reflex responses were recorded from the L5 ventral spinal roots. Ventral root responses (VRRs) to tibial and peroneal nerve stimulation on the side of the nerve lesion were significantly smaller in amplitude representing only about 15% of the mean amplitude of VRRs on the control side. The calculated central delay of the first, presumably monosynaptic component of the VRR potential was 1.6 ms on the control side while the earliest VRR wave on the side of the nerve lesion appeared after a mean central latency of 4.0 ms that seems too long to be of monosynaptic origin. These results suggest that neonatal sciatic nerve injury markedly alters the physiological properties and synaptic connectivity in spinal cord neurones and causes a marked depression of spinal cord responses to peripheral nerve stimulation.     

FK506 Protects Neurons Following Peripheral Nerve Injury Via Immunosuppression

In this study, we have evaluated neuroprotective effect of an immunosuppressant immunophilin ligand, FK506, in the sciatic nerve injury model in rats. FK506 was injected to the sciatic nerve transected 3-month-old female Wistar rats (2 mg/kg/day starting 1 day prior to sciatic nerve injury up to 7 day post operation). Equal number of sciatic nerve transected animals served as injured untreated controls. The contralateral side served as respective control. L4-L5 region of the spinal cord was removed on day 1, 3, 7, 14, 21, and 28, post operation and then processed for cryo-sectioning and paraffin sectioning. The cryocut sections were used for immunohistochemistry for localizing all microglia (using anti-Iba-1) and MHC-II expressing microglia (with OX-6). The physical dissector method was applied on Nissl stained paraffin sections for absolute motor neuron counting in the L4-L5 region of spinal cord. FK506 treated animals presented 88.7% neuronal survival while the injured alone had 79.12%, which is significantly less than the treated animals. FK506 caused early proliferation of microglia at 1 and 3 days post operation. FK506 also significantly restricted transformation of these cells in to phagocytes. Colocalization of activated microglia by anti-Iba-1 and OX-6 antibodies, confirms that the MHC-II expressing cells in injured spinal cord are none other than microglial cells and MHC-II expressing cells are significantly less in treated as compared to untreated injured animals. We propose that immunosuppression is one of the main mechanisms by which FK506 protects the central neurons following peripheral injury.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Botulinum toxin's axonal transport from periphery to the spinal cord

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.     

大鼠坐骨神经切断后外源性bcl-2对脊髓运动神经元的保护作用

采用大鼠坐骨神经切断损伤模型,行神经外膜端端对线缝合,术中依不同组别,动物于神经缝合处远端0．5cm处分别注射人的正义和反义bcl-2重组腺病毒(Ad／s-bcl-2、Ad／as-bcl-2),报道基因重组腺病毒(Ad／lacZ)和生理盐水。术后48h,7d,15d和30d常规灌注固定大鼠,取L4-L6脊髓节段,应用X-gal染色、bel-2原位杂交和免疫组化染色、TUNEL染色以及乙酰胆碱酯酶(AChE)组织化学染色方法,观察到外源基因能在脊髓中表达,同时外源性Ad／s-bcl-2能显著减少L4到L6节段脊髓前角运动神经元凋亡的数目,减少脊髓前角运动神经元中因坐骨神经切断导致的AChE活性的降低幅度,并加快其恢复。而Ad／as-bcl-2可显著增加坐骨神经切断诱导的脊髓前角运动神经元凋亡数目以及AChE活性降低幅度,并延缓其恢复。这些观察结果表明,外源性bcl-2能保护周围神经切断后引起的脊髓运动神经元损伤。     

Early Changes of β-Catenins and Menins in Spinal Cord Dorsal Horn after Peripheral Nerve Injury

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Aβ-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. β-Catenin and menin as synaptogenic factors are critically involved in synapse formation. However, the roles of β-catenin and menin in neuropathic pain are still unclear. Using Western blot analysis we investigated the changes of β-catenin and menin in the spinal dorsal horn after unilateral spared nerve injury (SNI). We demonstrated an increase in both β-catenin and menin protein levels in the ipsilateral spinal dorsal horn at days 1 and 3 following spared nerve injury (P < 0.05). These increases were associated with changes in paw withdrawal threshold to mechanical stimuli and weight bearing deficit suggestive of pain behavior and spontaneous ongoing pain respectively. However, the injury-associated increases in β-catenins and menins levels returned to control levels at day 14. In conclusion, these results indicate that peripheral nerve injury induces upregulation of β-catenins and menins in the dorsal horn of the spinal cord, which may contribute to the development of chronic neuropathic pain. Antagonists of these molecules may serve as new therapeutic agents.