Differential effects of endogenous brain-derived neurotrophic factor on the survival of axotomized sensory neurons in dorsal root ganglia: a possible role for the p75 neurotrophin receptor

After peripheral nerve injury, axotomized sensory neurons in dorsal root ganglia (DRG) undergo apoptosis and up-regulate brain-derived neurotrophic factor (BDNF). We tested whether endogenous BDNF plays any role in the survival of axotomized sensory neurons using in vitro and in vivo models. In the in vitro model, treatment with BDNF antibody significantly reduced apoptosis of sensory neurons in DRG explants from both adult and neonate rats and adult mice cultured for 48 h. Consistently, exogenous BDNF increased the percentage of apoptotic neurons in the DRGs from mice. The effects of the BDNF antibody and BDNF were not seen in DRGs from p75NTR(-/-) mice. In the in vivo model, sciatic nerve transection in neonatal rats decreased the total number of neurons in the injured DRG and treatment with antiserum to BDNF significantly exaggerated the loss of DRG neurons. Numbers of sensory neurons expressing BDNF and p75NTR in cultured DRGs increased but that expressing TrkB decreased. In contrast, sciatic nerve transection in vivo reduced the numbers of neurons expressing both p75NTR and TrkB but increased the numbers of cells expressing BDNF, 1 and 7 days after the surgery. These results suggest that BDNF may have differential effects on the survival of sensory neurons depending on the expression of p75NTR. While endogenous BDNF induced apoptosis of axotomized sensory neurons through p75NTR in vitro where more neurons expressed p75NTR, it prevented apoptosis in vivo where fewer neurons expressed p75NTR after sciatic nerve transection.     

Hyperexcitability of axotomized and neighboring unaxotomized sensory neurons is reduced days after perineural clonidine at the site of injury

Hyperexcitability after peripheral nerve injury occurs in axotomized and neighboring unaxotomized dorsal root ganglion (DRG) neurons and contributes to hypersensitivity. Previous studies have focused on proximal nerve injury and have not examined unaxotomized neurons innervating the site of sensory testing. The current study used a distal nerve injury (partial sciatic nerve ligation [PSNL]), and identified, using fluorescent tracers, axotomized and unaxotomized neurons innervating the site of hypersensitivity. We hypothesized that reduced hypersensitivity after perineural clonidine was associated with reduced hyperexcitability of DRG neurons. Rats underwent sham or PSNL surgery, followed 2 wk later by a single injection at the injury site of clonidine or saline. PSNL, but not sham surgery, reduced hindpaw mechanical withdrawal threshold, and clonidine, but not saline, partially reversed this effect 3 days after injection. Intracellular recording of neurons in whole DRG demonstrated similar changes in membrane properties and excitability in unaxotomized and axotomized neurons after PSNL compared with sham surgery, primarily depolarized resting membrane potential, reduced rheobase, presence of oscillations, and capability to fire repetitively. Most of these changes were present in small-, medium-, and large-diameter neurons. Perineural clonidine 3 days later significantly reversed many of these effects, whereas saline was without effect. We speculate that perineural clonidine reduces signals, likely proinflammatory cytokines and prostaglandins produced during Wallerian degeneration after nerve injury, which drive changes in ion channel expression in DRG somata leading to hyperexcitability and hypersensitivity.     

Expression of c-JUN, JUN B and JUN D proteins in rat nervous system following transection of vagus nerve and cervical sympathetic trunk.

Expression of c-JUN, JUN B and JUN D proteins was investigated in axotomized neurons following transection of the vagus nerve and the cervical sympathetic trunk in the rat. Vagotomy induced the expression of c-JUN and JUN D in the nodose ganglion, dorsal motor nucleus of the vagus nerve and nucleus ambiguus, whereas JUN B was not expressed in these areas, c-JUN and JUN D appeared after 10 h in the nodose ganglion and after 24 h in the dorsal motor nucleus of the vagus nerve with a maximum of immunoreactivity after 48 h. The c-JUN protein remained expressed at an increased level up to 100 days, whereas the immunoreactivity of JUN D declined after five days. Crush of the vagus nerve initially evoked an intense expression of c-JUN and JUN D, but in the course of regeneration the expression of c-JUN and JUN D had returned to more basal levels after 100 days. Similar to vagotomy, application of colchicine and vinblastine on to the intact vagus nerve induced expression of c-JUN and JUN D. On the other hand, application of lidocaine prior to vagotomy did not prevent the expression of these proteins. Transection of the cervical sympathetic trunk induced expression of c-JUN and JUN D, but not of JUN B, in the preganglionic sympathetic neurons of thoracic spinal cord. In these neurons, expression of c-JUN was still enhanced after 60 days whereas JUN D had returned to basal level. One hour after vagotomy, c-JUN and JUN B were transynaptically expressed in the area of central termination of sensory vagal neurons and declined within 10 h to basal levels. JUN D showed a late onset of expression, it appeared after 5 h and persisted for 60 days in this area. We postulate that the expression of c-JUN and JUN D in axotomized neurons is induced by deprivation of a target-derived suppressor.     

Increased body sway at 3.5-8 Hz in patients with phobic postural vertigo

Gicerin is an integral membrane glycoprotein which mediates cell-cell and cell-extracellular matrix (ECM) interactions in the nervous system. We studied gicerin expression in the hypoglossal nucleus post transection using in situ hybridization and immunocytochemistry. We found that hypoglossal nerve injury resulted in a significant increase in gicerin expression. Its expression levels reached peak values in reactive astrocytes surrounding axotomized motoneurons of the ipsilateral hypoglossal nucleus 14 days after hypoglossal nerve injury. The results indicate that gicerin is up-regulated during nerve regeneration, suggesting that gicerin expressed in the reactive astrocytes might be involved in the processes of nerve regeneration.     

Role of decreased sensory neuron membrane calcium currents in the genesis of neuropathic pain

The pathogenesis of neuropathic pain is incompletely understood and treatments are often inadequate. Cytoplasmic Ca(2+) regulates numerous cellular processes in neurons. This review therefore examines the pathogenic contribution of altered inward Ca(2+) flux (I(Ca)) through voltage-gated Ca(2+) channels in sensory neurons after peripheral nerve injury. We reviewed studies that recorded membrane currents through intracellular and patch-clamp techniques, as well as intracellular Ca(2+) levels using fluorimetric indicators, and performed behavioral analysis of rodent nerve injury models. Following nerve injury by partial ligation, a response characterized by sustained lifting, shaking, and licking of the paw after sharp mechanical stimulation is a reliable indicator or neuropathic pain. Primary sensory neurons isolated from animals with this behavior show a decrease in high-voltage activated I(Ca) by approximately one third. Low voltage-activated I(Ca) is nearly eliminated by peripheral nerve injury. Loss of I(Ca) leads to decreased activation of Ca(2+)-activated K(+) currents, which are also directly reduced in traumatized neurons. As a result of these changes in membrane currents, membrane voltage recordings show increased action potential duration and diminished afterhyperpolarization. Excitability is elevated, as indicated by resting membrane potential depolarization and a decreased current threshold for action potential initiation. Traumatized nociceptive neurons develop increased repetitive firing during sustained depolarization after axotomy. Concurrently, cytoplasmic Ca(2+) transients are diminished. In conclusions, axotomized neurons, especially pain-conducting ones, develop instability and elevated excitability after peripheral injury. Treatment of neuronal I(Ca) loss at the level of injury of the dorsal root ganglion may provide a novel therapeutic pathway.     

Long-term IL-1β exposure causes subpopulation-dependent alterations in rat dorsal root ganglion neuron excitability

The effect of interleukin-1β (IL-1β) on the electrical properties of sensory neurons was assessed at levels and exposure times comparable to those found in animal models of neuropathic pain. Experiments involved whole cell current-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined-medium, neuron-enriched cultures. Five- to six-day exposure to 100 pM IL-1β produced subpopulation-dependent effects on DRG neurons. These included an increase in the excitability of medium-diameter and small-diameter isolectin B(4) (IB(4))-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large-diameter neurons, while no effect was found in small-diameter IB(4)-negative neurons. Further characterization of changes in medium and small IB(4)-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Although the acute actions of IL-1β on sensory neurons have been well studied and related to acute and/or inflammatory pain, the present study shows how sensory neurons respond to long-term cytokine exposure. Such effects are relevant to understanding processes that contribute to the onset of neuropathic pain.     

Calcium dependence of axotomized sensory neurons excitability

Hyperexcitability of axotomized dorsal root ganglion neurons is thought to play a role in neuropathic pain. Numerous changes in ionic channels expression or current amplitude are reported after an axotomy, but to date no direct correlation between excitability of axotomized sensory neurons and ionic channels alteration has been provided. Following sciatic nerve injury, we examined, under whole-cell patch clamp recording, the effects of calcium homeostasis on the electrical activity of axotomized medium-sized sensory neurons isolated from lumbar dorsal root ganglia of adult mice. Axotomy induced an increase in excitability of medium sensory neurons among which 25% develop a propensity to fire repetitively. The condition necessary to get burst discharge in axotomized neurons was the presence of a high intracellular Ca²⁺ buffer concentration. The main effect was to amplify the increase in threshold current and apparent input resistance induced by axotomy. These data supply evidence for a role of Ca²⁺-dependent mechanisms in the control of excitability of axotomized sensory neurons.     

Upregulation of retinal transglutaminase during the axonal elongation stage of goldfish optic nerve regeneration

Fish CNS neurons can repair their axons following nerve injury, whereas mammalian CNS neurons cannot regenerate, and become apoptotic within 1-2 weeks after the nerve lesion. One explanation for these differences is that one, or several molecules are upregulated in fish CNS neurons during nerve regeneration, and this same molecule is downregulated in mammalian CNS neurons before the development of apoptosis caused by nerve injury. A molecule satisfying these criteria might successfully rescue and repair the mammalian CNS neurons. In this study, we looked for such a candidate molecule from goldfish retinas. Transglutaminase derived from goldfish retina (TG(R)) was characterized as a regenerating molecule after optic nerve injury. A full-length cDNA for TG(R) was isolated from the goldfish retinal cDNA library prepared from axotomized retinas. Levels of TG(R) mRNA and protein increased only in the retinal ganglion cells (RGCs) between 10 and 40 days after optic nerve transection. Recombinant TG(R) protein enhanced neurite outgrowth from adult fish RGCs in culture. Specific interference RNA and antibodies for TG(R) inhibited neurite outgrowth both in vitro and in vivo. In contrast, the level of TG(R) protein decreased in rat RGCs within 1-3 days after nerve injury. Furthermore, the addition of recombinant TG(R) to retinal cultures induced striking neurite outgrowth from adult rat RGCs. These molecular and cellular data strongly suggest that TG(R) promotes axonal elongation at the surface of injured RGCs after optic nerve injury.     

mRNA expression changes of slit proteins following peripheral nerve injury in the rat model

Slit family of proteins is one of the repulsive axonal guidance cues, and it also plays an important role in neuronal migration and branching through the interaction with roundabout receptors. The function and role of Slit family proteins during peripheral nerve regeneration are still unknown. We examined the expressions of Slits 1–3 mRNAs in the facial nerve nuclei after facial nerve transection by in situ hybridization, using Sprague–Dawley rats. Slit 1 mRNA was weakly expressed in the facial motoneurons, and its expression increased from day 5 to day 28 after transection, with the peak on day 14 after axotomy. Slits 2 and 3 mRNAs were expressed in the motoneurons of the facial nerve before injury, but the expression of Slit 2 mRNA was down-regulated from day 1 to day 7 after axotomy, with the peak on the first day after injury. Slit 3 mRNA expression in the axotomized side remained unchanged throughout the examination period. Slits 1 and 2 mRNA expression returned to the normal level on day 56 postoperatively. The difference in expression pattern of Slit family mRNA in the neurons during peripheral nerve regeneration suggests that it plays a different role in axonal regeneration after axotomy of peripheral nerves.     

mRNA expression changes of slit proteins following peripheral nerve injury in the rat model

Slit family of proteins is one of the repulsive axonal guidance cues, and it also plays an important role in neuronal migration and branching through the interaction with roundabout receptors. The function and role of Slit family proteins during peripheral nerve regeneration are still unknown. We examined the expressions of Slits 1-3 mRNAs in the facial nerve nuclei after facial nerve transection by in situ hybridization, using Sprague-Dawley rats. Slit 1 mRNA was weakly expressed in the facial motoneurons, and its expression increased from day 5 to day 28 after transection, with the peak on day 14 after axotomy. Slits 2 and 3 mRNAs were expressed in the motoneurons of the facial nerve before injury, but the expression of Slit 2 mRNA was down-regulated from day 1 to day 7 after axotomy, with the peak on the first day after injury. Slit 3 mRNA expression in the axotomized side remained unchanged throughout the examination period. Slits 1 and 2 mRNA expression returned to the normal level on day 56 postoperatively. The difference in expression pattern of Slit family mRNA in the neurons during peripheral nerve regeneration suggests that it plays a different role in axonal regeneration after axotomy of peripheral nerves.     

Reduction of oligodendrocyte myelin glycoprotein expression following facial nerve transection

Oligodendrocyte myelin glycoprotein (OMgp) has been thought to be expressed in the oligodendrocytes and inhibit the regeneration of the nerves by binding to the Nogo receptor expressed in neurons in the central nervous system (CNS). However, OMgp is expressed in the CNS in the neurons as well as oligodendrocytes. In order to help understanding the physiological role of neuronal OMgp, we examined the change of OMgp expression in the facial nucleus after the facial nerve transection. Real-time RT-PCR and Western blot analysis showed a down-regulation of OMgp expression in the facial nucleus 5-7 (mRNA) or 5-14 (protein) days after transection. Thereafter, expression of OMgp returned to the control level at 28 days after axotomy. Subsequent analysis using in situ hybridization histochemistry and immunohistochemistry established that the decrease of OMgp expression was attributable to the expression in facial motoneurons, but not in oligodendrocytes. These findings suggest a possibility that the change of neuronal OMgp expression might be involved in reconnection of neural circuit between axotomized facial neuron and upper motor neuron after transection.     

Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury

Robos are transmembrane receptors that mediate Slit signaling to repel growth cone outgrowth and neural migration in the developing central nervous system. Their distribution and function in the peripheral nervous system remains unclear. In the present study, we examined expression of Slit1 and Robo2 in adult rat dorsal root ganglion (DRG), spinal cord and sciatic nerve after peripheral nerve injury (axotomy). In control rats, Slit1 and Robo2 mRNA and protein were expressed at basic levels in the L5 and L6 DRGs. Sciatic transection resulted in a significant up-regulation of both Robo2 and Slit1 mRNA and protein (p<0.05 versus control). The peak of Slit1 and Robo2 expression occurred at days 7 and 14, respectively, and returned to control levels at days 28 and 21 post-axotomy, respectively. By contrast, injury to the central axons of the DRG by dorsal rhizotomy did not up-regulate Slit1 and Robo2 expression. Robo2 staining was stronger in small diameter neurons than in large diameter neurons in control DRG. Interestingly, post-axotomy, Robo2 immunostaining increased in the large diameter neurons and the number of Robo2 positive large diameter neurons increased significantly relative to controls. Non-neuronal cells surrounding the primary sensory neurons, including the satellite cells, were Slit1-positive, and Slit1 protein was expressed in the myelin sheath and non-neural cells in both intact and degenerating sciatic nerve axons. Sciatic nerve transection also led to an accumulation of Slit1 protein in peripheral region of the traumatic neuroma. In conclusion, we report an altered expression and redistribution of Robo2 and Slit1 in the DRG and sciatic nerve trunk after peripheral axotomy. Our results indicate that Slit1 and Robo2 likely play an important role in regeneration after peripheral nerve injury.     

Expression of beta-preprotachykinin mRNA and tachykinins in rat dorsal root ganglion cells following peripheral or central axotomy

The changes in gene expression and protein synthesis induced in neurons by axotomy usually lead to increased production of axon constituents and decreased production of molecules related to neurotransmission. Exceptions to this generalization occur, however, and it is unclear whether the injury itself changes the pattern of synthesis or whether individual mechanisms regulate the synthesis of the various axonal components. We used in situ hybridization histochemistry and immunocytochemistry to compare the changes in L4 and L5 rat dorsal root ganglion neuron levels of preprotachykinin mRNA and tachykinin peptides caused by sciatic nerve injury with those caused by dorsal root injury. Both lesions elicit regeneration, although only the axotomized peripheral processes re-establish functional contact with their targets. In the contralateral, intact dorsal root ganglia approximately 17% of neurons contained detectable levels of both mRNAs and peptides. Sciatic nerve section decreased by 70% the number of neurons labeled for preprotachykinin mRNA at three days post-operatively. Not all cells in the ganglion are axotomized by the sciatic nerve lesion; grain counts over the cells spared by the lesion showed an increased level of labeling, possibly a result of collateral sprouting by these spared cells. By two weeks, the number of cells labeled for preprotachykinin mRNA had decreased to 80% of control levels. The numbers of neurons labeled for tachykinin peptides decreased more slowly and reached approximately 50% of control numbers at two weeks. By six months post-operatively, when regeneration is largely complete, the number of neurons containing both mRNAs and peptides returned to normal. In contrast, dorsal root section did not elicit a decrease in the number of neurons labeled either for the mRNAs or the peptides at any of the post-operative intervals examined. These results indicate that axotomy is not the stimulus that elicits changes in the expression of genes coding for tachykinins. Evidence is considered indicating that interruption of the supply of peripherally derived nerve growth factor may be responsible for the changes in gene expression for tachykinins after axotomy.     

Expression of Semaphorin3C in axotomized rodent facial and rubrospinal neurons

Semaphorins are a family of axonal guidance molecules that, by virtue of their chemorepulsive or chemoattractive actions, may be the important factors in determining the success or failure of axonal regeneration in the mature nervous system after injury. Here, we have used two adult mouse models of nervous system injury to evaluate the neuronal expression of Semaphorin3C (Sema3C) in regenerating (facial motoneurons) and non-regenerating (rubrospinal) neurons following axonal injury. Using in situ hybridization (ISH), we observed that uninjured facial motoneurons express Sema3C mRNA and, following axonal injury, there is a transient up-regulation in Sema3C mRNA expression in injured motoneurons. In contrast, Sema3C mRNA was not detected in uninjured rubrospinal neurons; however, following axotomy, injured rubrospinal neurons significantly up-regulate Sema3C mRNA expression. The increase in Sema3C mRNA expression in axotomized rubrospinal neurons was not limited to the mouse nervous system: serial dilution RT-PCR analysis revealed a similar increase in Sema3C mRNA expression in the axotomized rat rubrospinal nucleus, 3 days following a rubrospinal tract lesion. This demonstrates that increased Sema3C mRNA levels in axotomized rubrospinal neurons is common to both mouse and rat injury models.     

Reduction of oligodendrocyte myelin glycoprotein expression following facial nerve transection

Oligodendrocyte myelin glycoprotein (OMgp) has been thought to be expressed in the oligodendrocytes and inhibit the regeneration of the nerves by binding to the Nogo receptor expressed in neurons in the central nervous system (CNS). However, OMgp is expressed in the CNS in the neurons as well as oligodendrocytes. In order to help understanding the physiological role of neuronal OMgp, we examined the change of OMgp expression in the facial nucleus after the facial nerve transection. Real-time RT-PCR and Western blot analysis showed a down-regulation of OMgp expression in the facial nucleus 5–7 (mRNA) or 5–14 (protein) days after transection. Thereafter, expression of OMgp returned to the control level at 28 days after axotomy. Subsequent analysis using in situ hybridization histochemistry and immunohistochemistry established that the decrease of OMgp expression was attributable to the expression in facial motoneurons, but not in oligodendrocytes. These findings suggest a possibility that the change of neuronal OMgp expression might be involved in reconnection of neural circuit between axotomized facial neuron and upper motor neuron after transection.     

Injury-induced CRMP4 expression in adult sensory neurons; a possible target gene for ciliary neurotrophic factor

Neurotrophic cytokines, such as ciliary neurotrophic factor (CNTF) play an important role in the development and regeneration of the nervous system. In the present study, we screened gene expression induced by CNTF in adult dorsal root ganglion (DRG) neurons using the Illumina microarray. We found that the expression of both short and long forms of collapsin response-mediator protein 4 (CRMP4) was increased in cultured primary sensory neurons by CNTF. In addition, sciatic nerve injury induced the expression of CRMP4 mRNA and protein in DRG neurons. Finally, the increased CRMP4 protein was transported into peripheral axons following nerve injury. These findings indicate that CRMP4 may be a target gene for CNTF in the regenerative axon growth of DRG neurons after injury.