Jin-Hua Zhong

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.

Injured primary sensory neurons switch phenotype for brain-derived neurotrophic factor in the rat

Peripheral nerve injury results in plastic changes in the dorsal root ganglia and spinal cord, and is often complicated with neuropathic pain. The mechanisms underlying these changes are not known. We have now investigated the expression of brain-derived neurotrophic factor in the dorsal root ganglia with histochemical and biochemical methods following sciatic nerve lesion in the rat. The percentage of neurons immunoreactive for brain-derived neurotrophic factor in the ipsilateral dorsal root ganglia was significantly increased as early as 24 h after the nerve lesion and the increase lasted for at least two weeks. The level of brain-derived neurotrophic factor messenger RNA was also significantly increased in the ipsibut not contralateral dorsal root ganglia. Both neurons and satellite cells in the lesioned dorsal root ganglia synthesized brain-derived neurotrophic factor messenger RNA after the nerve lesion. There was a dramatic shift in size distribution of positive neurons towards large sizes seven days after sciatic nerve lesion. Morphometric analysis and retrograde tracing studies showed that no injured neurons smaller than 600 microm2 were immunoreactive for brain-derived neurotrophic factor, whereas the majority of large injured neurons were immunoreactive in the ipsilateral dorsal root ganglia seven days postlesion. The brain-derived neurotrophic factor-immunoreactive nerve terminals in the ipsilateral spinal cord were reduced in the central region of lamina II, but increased in more medial regions or deeper into laminae III/IV. These studies indicate that sciatic nerve injury results in a differential regulation of brain-derived neurotrophic factor in different subpopulations of sensory neurons in the dorsal root ganglia. Small neurons switched off their normal synthesis of brain-derived neurotrophic factor, whereas larger ones switched to a brain-derived neurotrophic factor phenotype. The phenotypic switch may have functional implications in neuronal plasticity and generation of neuropathic pain after nerve injury.

Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats

Injury to peripheral nerves often results in chronic pain which is difficult to relieve. The mechanism underlying the pain syndrome remains largely unknown. In previous studies we showed that neurotrophins are up‐regulated in satellite cells around sensory neurons following sciatic nerve lesion. In the present study, we have examined whether the neurotrophins in the dorsal root ganglia play any role in allodynia after nerve injury. Antibodies to different neurotrophins, directly delivered to injured dorsal root ganglia, significantly reduced (with different time sequences) the percentage of foot withdrawal responses evoked by von Frey hairs. The antibodies to nerve growth factor acted during the early phase but antibodies to neurotrophin‐3 and brain‐derived neurotrophic factor were effective during the later phase. Exogenous nerve growth factor or brain‐derived neurotrophic factor, but not neurotrophin‐3, directly delivered to intact dorsal root ganglia, trigger a persistent mechanical allodynia. Our results showed that neurotrophins within the dorsal root ganglia after peripheral nerve lesion are involved in the generation of allodynia at different stages. These studies provide the first evidence that ganglia‐derived neurotrophins are a source of nociceptive stimuli for neuropathic pain after peripheral nerve injury.

Suppression of p75NTR Does Not Promote Regeneration of Injured Spinal Cord in Mice

The neurotrophin receptor p75NTR is the coreceptor for Nogo receptor, mediating growth cone collapse in vitro by MAG, myelin oligodendrocyte glycoprotein (Omgp), and Nogo. Whether p75NTR plays any role in the failure of nerve regeneration in vivo is not known. Immunohistochemical data showed that p75NTR was expressed in only a very small subset of ascending sensory axons but not in any corticospinal axons in the dorsal column of either normal or injured spinal cord. Using p75NTR-deficient mice, we showed that the depletion of the functional p75NTR did not promote the regeneration of the descending corticospinal tract and ascending sensory neurons in the spinal cord 2 weeks after spinal cord injury. Local administration of p75NTR-Fc fusion molecule, the dominant-negative receptor to block the function of neurite outgrowth inhibitors, did not improve regeneration of ascending sensory neurons in the injured spinal cord. Our results suggest that p75NTR may not be a critical molecule mediating the function of myelin-associated inhibitory factors in vivo.

Effect of lumbar 5 ventral root transection on pain behaviors: A novel rat model for neuropathic pain without axotomy of primary sensory neurons

A peripheral nerve injury often causes neuropathic pain but the underlying mechanisms remain obscure. Several established animal models of peripheral neuropathic pain have greatly advanced our understanding of the diverse mechanisms of neuropathic pain. A common feature of these models is primary sensory neuron injury and the commingle of intact axons with degenerating axons in the sciatic nerve. Here we investigated whether neuropathic pain could be induced without sensory neuron injury following exposure of their peripheral axons to the milieu of Wallerian degeneration. We developed a unilateral lumbar 5 ventral root transection (L5 VRT) model in adult rats, in which L5 ventral root fibers entering the sciatic nerve were sectioned in the spinal canal. This model differs from previous ones in that DRG neurons and their afferents are kept uninjured and intact afferents expose to products of degenerating efferent ventral root fibers in the sciatic nerve and the denervated muscles. We found that the L5 VRT produced rapid (24 h after transection), robust and prolonged (56 days) bilateral mechanical allodynia, to a similar extent to that in rats with L5 spinal nerve transection (L5 SNT), cold allodynia and short-term thermal hyperalgesia (14 days). Furthermore, L5 VRT led to significant inflammation as demonstrated by infiltration of ED-1-positive monocytes/macrophages in the DRG, sciatic nerve and muscle fibers. These findings demonstrated that L5 VRT produced behavioral signs of neuropathic pain with high mechanical sensitivity and thermal responsiveness, and suggested that neuropathic pain can be induced without damage to sensory neurons. We propose that neuropathic pain in this model may be mediated by primed intact sensory neurons, which run through the milieu of Wallerian degeneration and inflammation after nerve injury. The L5 VRT model manifests the complex regional pain syndrome in some human patients, and it may provide an additional dimension to dissect out the mechanisms underlying neuropathic pain.

ProBDNF Collapses Neurite Outgrowth of Primary Neurons by Activating RhoA

Background Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized. Methodology/Principal Findings Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action. Conclusions proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.

Effects of endogenous neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury

Peripheral nerve injury is often complicated by a chronic pain syndrome that is difficult to treat. In animal models of peripheral nerve injury, sympathetic nerve terminals in the dorsal root ganglia (DRG) sprout to form baskets around large diameter neurons, an anatomical change that has been implicated in the induction of neuropathic pain. In the present study, we have investigated whether neurotrophins derived from peripheral sources play any roles in sympathetic sprouting and neuropathic pain in a rat model of peripheral nerve injury. After transection of the left lumbar (L) 5 spinal nerve, antisera specific to neurotrophins were injected intraperitoneally twice a week for 2 weeks. The foot withdrawal response to von Frey hairs was examined on days 1, 3, 7, 10, and 14 postlesion. After completion of behavioral tests, sympathetic sprouting in DRG was examined by tyrosine hydroxylase (TH) immunohistochemistry. The number of TH-immunoreactive (ir) fibers and baskets around large neurons within the lesioned DRG was dramatically increased in the rats treated with control normal sheep serum. Antisera specific to nerve growth factor (NGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) significantly reduced the sympathetic sprouting and the formation of baskets. L5 spinal nerve lesion induced a significant increase in foot withdrawal responses to von Frey hair stimuli, which was attenuated by treatment of antisera to neurotrophins with a different time sequential. The effect of BDNF antiserum occurred earlier and lasted longer than those of NGF and NT3 antisera. These results implicate that peripherally derived neurotrophins are involved in the induction of sympathetic sprouting and neuropathic pain following peripheral nerve injury.

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.

Distribution and localization of pro-brain-derived neurotrophic factor-like immunoreactivity in the peripheral and central nervous system of the adult rat.

The precursors for neurotrophins are proteolytically cleaved to form biologically active mature molecules which activate their receptors p75NTR and trks. A recent study showed that the precursor for nerve growth factor (NGF) can bind to p75NTR with a high affinity and induces apoptosis of neurons in vitro. Mutation in Val66Met of brain‐derived neurotrophic factor (BDNF) results in reduction in hippocampal function in learning and in the dysfunction of intracellular BDNF sorting and secretion. To examine the functions of pro‐neurotrophins in vivo, it is essential to know where they are expressed in the nervous system. In the present study, we have raised and characterized rabbit polyclonal antibodies against a peptide coding for the precursor region of the BDNF gene. The antibody specifically recognizes the precursor for BDNF by western blot. With the affinity purified precursor antibody, we have mapped the distribution and localization of the precursor for BDNF. The results showed that, like mature BDNF, pro‐BDNF is localized to nerve terminals in the superficial layers of dorsal horn, trigeminal nuclei, nuclei tractus solitarius, amygdaloid complex, hippocampus, hypothalamus and some peripheral tissues. These results suggest that pro‐BDNF, like mature BDNF, is anterogradely transported to nerve terminals and may have important functions in synaptic transmission in the spinal cord and brain.

Knockout of p75NTR impairs re‐myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75NTR, in activated Schwann cells in the Wallerian degenerated nerve is up‐regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75NTR in remyelination of the sciatic nerve was investigated in p75NTR mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild‐type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild‐type mice. Real‐time RT–PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75NTR mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75NTR is important for the myelinogenesis during the regeneration of peripheral nerves after injury.