Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

Effect of injury on nerve growth factor uptake by sensory ganglia

The level of the nerve growth factor protein, NGF, in vivo has a profound influence on axonal sprouting by sensory neurons of vertebrate dorsal root ganglia. There is evidence also that NGF may play similar roles in cholinergic central structures in brain. In both instances, retrograde transport of NGF has been demonstrated. Here we examined uptake of NGF by DRG neurons in response to contusion injury of the spinal cord. Under these conditions there was uptake and transport of NGF into large DRG neurons via central processes but no uptake by non-DRG central neurons. Thus, any effects of NGF on spinal neurons or their processes would be secondary to the direct effects of NGF on DRG neurons.     

Contribution of degeneration of motor and sensory fibers to pain behavior and the changes in neurotrophic factors in rat dorsal root ganglion

To elucidate the role of the degeneration of motor and sensory fibers in neuropathic pain, we examined the pain-related behaviors and the changes of brain-derived neurotrophic factor (BDNF) in the L4/5 dorsal root ganglion (DRG) and the spinal cord after L5 ventral rhizotomy. L5 ventral rhizotomy, producing a selective lesion of motor fibers, produced thermal hyperalgesia and increased BDNF expression in tyrosine kinase A-containing small- and medium-sized neurons in the L5 DRG and their central terminations within the spinal cord, but not in the L4 DRG. Furthermore, L5 ventral rhizotomy up-regulated nerve growth factor (NGF) protein in small to medium diameter neurons in the L5 DRG and also in ED-1-positive cells in the L5 spinal nerve, suggesting that NGF synthesized in the degenerative fibers is transported to the L5 DRG and increases BDNF synthesis. On the other hand, L5 ganglionectomy, producing a selective lesion of sensory fibers, produced heat hypersensitivity and an increase in BDNF and NGF in the L4 DRG. These data indicate that degeneration of L5 sensory fibers distal to the DRG, but not motor fibers, might influence the neighboring L4 nerve fibers and induce neurotrophin changes in the L4 DRG. We suggest that these changes of neurotrophins in the intact primary afferents of neighboring nerves may be one of many complex mechanisms, which can explain the abnormal pain behaviors after nerve injury. The ventral rhizotomy and ganglionectomy models may be useful to investigate the pathophysiological mechanisms of neuropathic pain after Wallerian degeneration in motor or sensory or mixed nerve.     

Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat

Injury to a peripheral nerve induces in the dorsal root ganglia (DRG) sprouting of sympathetic and peptidergic terminals around large-diameter sensory neurons that project in the damaged nerve. This pathological change may be implicated in the chronic pain syndromes seen in some patients with peripheral nerve injury. The mechanisms underlying the sprouting are not known. Using in situ hybridization and immunohistochemical techniques, we have now found that nerve growth factor (NGF) and neurotrophin-3 (NT3) synthesis is upregulated in satellite cells surrounding neurons in lesioned DRG as early as 48 h after nerve injury. This response lasts for at least 2 months. Quantitative analysis showed that the levels of mRNAs for NT3 and NGF increased in ipsilateral but not contralateral DRG after nerve injury. Noradrenergic sprouting around the axotomized neurons was associated with p75-immunoreactive satellite cells. Further, antibodies specific to NGF or NT3, delivered by an osmotic mini-pump to the DRG via the lesioned L5 spinal nerve, significantly reduced noradrenergic sprouting. These results implicate satellite cell-derived neurotrophins in the induction of sympathetic sprouting following peripheral nerve injury.     

Embryonic sensory development: Local expression of neurotrophin-3 and target expression of nerve growth factor

Development and maintenance of peripheral sensory and sympathetic neurons are regulated by target-derived neurotrophins, including nerve growth factor (NGF). To determine whether trophins are potentially critical prior to and during target innervation, for neuronal survival or axon guidance, in situ hybridization was performed in the rat embryo. We examined the expression of genes encoding NGF, neurotrophin-3 (NT-3), and their putative high-affinity receptors, trk A and trk C, respectively. Trks A and C were detected in dorsal root sensory ganglia (DRG) on embryonic day 12.5 (E12.5), implying early responsiveness to NGF and NT-3. NGF mRNA was expressed in the central spinal cord target and by the peripheral somite, at this early time, which thereby may function as a transient “guidepost” target for sensory fibers. Somitic expression was transient and was undetectable by E17.5. NT-3 was expressed in the DRG itself from E13.5 to 17.5, suggesting local transient actions on sensory neurons. NT-3 was also expressed in the ventral spinal cord at low levels on E13.5. We examined the trigeminal ganglion to determine whether cranial sensory neurons are similarly regulated. Trk A was detected in the trigeminal ganglion, while NGF was expressed in the central myelencephalon target, paralleling observations in the DRG and spinal cord. However, NT-3 and trk C were undetectable, in contrast to DRG, suggesting that the environment or different neural crest lineages govern expression of different trophins and trks. Apparently, multiple trophins regulate sensory neuron development through local as well as transient target mechanisms prior to innervation of definitive targets.     

Nerve growth factor stimulates development of substance P in the embryonic spinal cord

Development of the putative neurotransmitter, substance P (SP), in the embryonic rat dorsal root ganglion (DRG) and spinal cord was defined in vivo. SP was not detectable by radioimmunoassay before day 17 of gestation (E17). On E17, cervical sensory ganglia contained 4 pg SP/ganglion, rising to 49 pg/ganglion at birth. The dorsal cervical spinal cord contained 0.75 ng SP/mg protein on E17, rising to 6 ng SP/mg protein on postnatal day 3. The ventral spinal cord contained approximately 20% of the SP content in the dorsal cord at each gestational age. Intrauterine forelimb amputation partially prevented the normal development increase of SP in sensory ganglia destined to innervate that limb, suggesting that target structures regulate the development of peptidergic neruons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF peptidergic neruons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF peptidergic neruons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF action on peripheral ganglia, but a direct effect on the spinal cord has not been excluded. However, treatment with antiserum to NGF failed to significantly inhibit development of ganglion SP. The system of SP-containing neurons in the DRG may provide a convenient model for defining events regulating peptidergic maturation.     

NGF message and protein distribution in the injured rat spinal cord

Nerve growth factor (NGF) content of the spinal cord is increased after cord injury. NGF can cause central sprouting of sensory fibers after spinal cord injury (SCI), leading to autonomic dysfunction and pain. NGF also can promote the death of oligodendroglia after SCI. Knowing the source of intraspinal NGF would benefit strategies for minimizing abnormal plasticity and cell death after SCI. We identified these sources, using RNA in situ hybridization to detect NGF mRNA and double-labeling immunocytochemistry for NGF and cell-marking antigens. In uninjured and sham-injured rats, we identified NGF mRNA in leptomeningeal cells and in neurons in the intermediate grey matter, whereas NGF protein was observed only in leptomeningeal cells. At 3-7 days after transection or clip-compression SCI, NGF mRNA and protein were expressed in the lesion and throughout the intermediate grey matter and white matter rostral and caudal to the injury site. Transection-SCI was used to permit comparisons to previous studies; clip-compression injury was used as a more clinically relevant model. mRNA and protein in adjacent sections were expressed in ramified microglia, astrocytes, intermediate grey neurons, pial cells, and leptomeningeal and Schwann cells in the lateral white matter and the lesion site. Rounded macrophages in the lesion were immunoreactive (Ir) for NGF, but the cells expressing NGF mRNA were not in the same areas of the lesion and were not stained by a macrophage marker. Our data demonstrate that glia, neurons, meningeal cells and Schwann cells but not macrophages contribute to the increased intraspinal NGF after SCI.     

NGF levels decrease in the spinal cord and dorsal root ganglion after spinal hemisection

To examine changes in nerve growth factor (NGF) levels in spinal cord and dorsal root ganglia (DRG) after spinal injury, male Sprague-Dawley rats weighing 150-175 g were given spinal hemisections. NGF content was measured at various post-surgical times and compared with naive controls (n = 4 per time point) in the spinal cord, DRG and blood serum by ELISA techniques (Promega). Levels of NGF in the blood serum were significantly increased 8-fold at 48h but were significantly decreased in the spinal cord and DRG by 2- to 4-fold until 7 days postsurgery (ANOVA, p < 0.05). Contrary to accepted dogma, spinal injury results in decreased levels of NGF in the spinal cord and DRG following spinal injury.     

Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation

Expression of pituitary adenylate cyclase-activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT-3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4-L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT-3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT-3 infusion, suggesting a role for NT-3 in returning the 'injured phenotype' back towards an 'intact phenotype'. NGF dramatically up-regulated PACAP expression in trkA-positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT-3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT-3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small-medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons.     

Activation and circuitry of uterine-cervix-related neurons in the lumbosacral dorsal root ganglia and spinal cord at parturition

Stimulation of the uterine cervix at parturition activates neural circuits involving primary sensory nerves and supraspinally projecting neurons of the lumbosacral spinal cord, resulting in output of hypothalamic neurohormones. Dorsal root ganglia (DRG) and spinal neurons of these circuits are not well-characterized. The objectives of this study were to detail the activation of DRG and spinal neurons of the L6/S1 levels that are stimulated at late pregnancy, verify hypothalamic projections of activated spinal neurons, and determine whether activated neurons express estrogen receptor-alpha (ERalpha). Expression of phosphorylated cyclic-AMP response element-binding protein (PCREB) and Fos immunohistochemistry were used to "mark" activated DRG and spinal neurons, respectively. Retrograde tracing identified uterine-cervix-related and spinohypothalamic neurons. Baseline PCREB expression in the DRG increased during pregnancy and peaked during the last trimester. Some PCREB-expressing neurons contained retrograde tracer identifying them as cervix-related neurons. Fos-expressing neurons were few in spinal cords of nonpregnant and day 22 pregnant rats but were numerous in parturient animals. Some Fos-expressing neurons located in the dorsal half of the spinal cord contained retrograde tracer identifying them as spinohypothalamic neurons. Some DRG neurons expressing PCREB also expressed ERalpha, and some spinal neurons activated at parturition projected axons to the hypothalamus and expressed ERalpha. These results indicate that DRG and spinal cord neurons are activated at parturition; that those in the spinal cord are present in areas involved in autonomic and sensory processing; that some spinal neurons project axons to the hypothalamus, ostensibly part of a neuroendocrine reflex; and that sensory and spinal neurons can respond to estrogens. Moreover, some activated sensory neurons may be involved in the animal's perception of labor pain.     

Expression of glial cell line-derived neurotrophic factor family of growth factors in peripheral nerve injury in rats

The glial cell line-derived neurotrophic factor (GDNF) family of growth factors may be involved in the regenerative support of neurons in the peripheral nervous system. In order to study the role of these growth factors and their receptors following rat peripheral nerve injury we examined the changes in their mRNA levels in the spinal cord, the dorsal root ganglia and the peripheral nerve trunk. Following transaction of the sciatic nerve GDNF mRNA was up-regulated rapidly in the denervated nerve distal to the cut along with the mRNA for one of its receptors, GFRalpha-1. GFRalpha-1 mRNA was also increased in the DRG ipsilateral to the nerve injury suggesting that GDNF may be involved in the trophic support of DRG sensory neurons. In contrast there were no analogous changes in the mRNA levels of neurturin, persephin and artemin following injury.     

Galectin-1 is involved in the potentiation of neuropathic pain in the dorsal horn

Galectin-1 is one of the endogenous-galactoside-binding lectins, suggested to be involved in a variety of functions, such as neurite outgrowth, synaptic connectivity, cell proliferation and apoptosis. This protein is expressed in the dorsal root ganglion (DRG) and the spinal cord in the developing and adult rats, especially intensely in small DRG neurons. In the present study, we examined whether galectin-1 is colocalized with TrkA or c-Ret mRNA in small DRG neurons and the effect of axotomy on the expression of galectin-1 in the spinal cord. About 20% of the DRG neurons showed intense galectin-1-immunoreactivity (IR). Of the intensely galectin-1-IR DRG neurons, 93.9% displayed c-Ret mRNA positive signals. On the other hand, only 6.8% displayed TrkA mRNA positive signals. Galectin-1-IR was increased in the dorsal horn at 1 to 2 weeks after axotomy. Intrathecal administration of anti-recombinant human galectin-1 antibody (anti-rhGAL-1 Ab) partially but significantly attenuated the upregulation of substance P receptor (SPR) in the spinal dorsal horn and the mechanical hypersensitivity induced by the peripheral nerve injury. These data suggest that endogenous galectin-1 may potentiate neuropathic pain after the peripheral nerve injury at least partly by increasing SPR in the dorsal horn.     

The behavioral and neuroanatomical effects of IB4-saporin treatment in rat models of nociceptive and neuropathic pain

One distinguishing feature of primary afferent neurons is their ability to bind the lectin IB(4). Previous work suggested that neurons in the inner part of lamina II (IIi), onto which IB(4)-positive sensory neurons project, facilitate nociceptive transmission following tissue or nerve injury. Using an IB(4)-saporin conjugate (IB(4)-SAP), we examined the contribution of IB(4)-positive neurons to nociceptive processing in rats with and without nerve injury. Intrasciatic injection of IB(4)-SAP (5 mug/5 mul) significantly decreased IB(4)-labeling and immunoreactive P(2)X(3) in the spinal cord and delayed the behavioral and neuroanatomical consequences of L5 spinal nerve ligation (SNL) injury. In the absence of injury, thermal and mechanical nociceptive thresholds increased 2 weeks post-treatment only in IB(4)-SAP-treated, but not control (saline or saporin only), rats. Acute NGF-induced hyperalgesia was also attenuated following IB(4)-SAP treatment. In the SNL model, mechanical allodynia failed to develop 1 and 2 weeks post-injury, but was fully established by 4 weeks. Moreover, neuropeptide Y immunoreactivity (NPY-ir), which increases in the spinal cord after nerve injury, was unchanged in IB(4)-SAP-treated animals whereas immunoreactive PKCgamma decreased 2, but not 4, weeks post-injury. Quantitative RT-PCR revealed a reduction in P(2)X(3) mRNA in L4 DRG of IB(4)-SAP-treated animals, but no change in TrkA expression. Our results suggest that IB(4)-positive neurons in L4 are required for the full expression of NGF-induced hyperalgesia and participate in the behavioral and anatomical consequences that follow injury to the L5 spinal nerve.     

A novel group of nerve growth factor receptor-immunoreactive neurons in the ventral horn of the lumbar spinal cord.

During development and following axonal injury in adults, neurons in the anterior horn of the spinal cord express nerve growth factor receptor (NGF-R) messenger ribonucleic acid (mRNA) and protein. To examine whether unlesioned anterior horn neurons show signs of responsiveness to NGF in adult animals, spinal cords from control rats and monkeys, as well as animals that had received NGF intraventricularly, were processed for NGF-R immunocytochemistry using monoclonal and polyclonal antibodies against NGF-R. In all animals, neurons located in central/ventral sectors of lamina IX in lumbar segments of the spinal cord expressed NGF-R-like immunoreactivity; this population of nerve cells appeared to increase in size after treatment with NGF. Our findings suggest that, in adults, a subset of spinal motor neurons may respond to NGF.     

GADD45A protects against cell death in dorsal root ganglion neurons following peripheral nerve injury

A significant loss of neurons in the dorsal root ganglia (DRG) has been reported in animal models of peripheral nerve injury. Neonatal sensory neurons are more susceptible than adult neurons to axotomy- or nerve growth factor (NGF) withdrawal-induced cell death. To develop therapies for preventing irreversible sensory cell loss, it is essential to understand the molecular mechanisms responsible for DRG cell death and survival. Here we describe how the expression of the growth arrest- and DNA damage-inducible gene 45α (GADD45A) is correlated with neuronal survival after axotomy in vivo and after NGF withdrawal in vitro. GADD45A expression is low at birth and does not change significantly after spinal nerve ligation (SNL). In contrast, GADD45A is robustly up-regulated in the adult rat DRG 24 hr after SNL, and this up-regulation persists as long as the injured fibers are prevented from regenerating. In vitro delivery of GADD45A protects neonatal rat DRG neurons from NGF withdrawal-induced cytochrome c release and cell death. In addition, in vivo knockdown of GADD45A expression in adult injured DRG by small hairpin RNA increased cell death. Our results indicate that GADD45A protects neuronal cells from SNL-induced cell death.