Xin-Fu Zhou

Endogenous brain-derived neurotrophic factor is anterogradely transported in primary sensory neurons

Neurotrophins are a family of proteins which act as survival and differentiative factors in the developing and mature nervous system. Extensive evidence has been provided for their retrograde action following incorporation into nerve terminals and transport to the cell body. In contrast, we now demonstrate that one neurotrophin, brain-derived neurotrophic factor, is transported anterogradely via both peripheral and central processes of spinal sensory neurons. Using newly generated antisera, we have examined the distribution of brain-derived neurotrophic factor immunoreactivity and found it to be present within a subpopulation of sensory somata, primarily those with a small-to-medium diameter. The immunoreactivity was accumulated on both the distal and proximal sides of a ligature on the sciatic nerve. The accumulation on the distal side, but not on the proximal side, was substantially reduced by pretreatment with brain-derived neurotrophic factor antibodies in vivo. In contrast to the periphery, the immunoreactivity only accumulated on the proximal side of a lesion of the dorsal root. In the spinal cord, most nerve terminals immunoreactive for brain-derived neurotrophic factor were identified in lamina II. Lesion of the dorsal root led to a reduction of these nerve terminals. These studies indicate that the factor is transported not only retrogradely to, but also anterogradely from, the spinal ganglia to terminals in the periphery and spinal cord. The findings add a new dimension to the role of neuronal growth factors, since anterograde transport has not been observed previously for any endogenous survival factor.

Deprivation of endogenous brain-derived neurotrophic factor results in impairment of spatial learning and memory in adult rats.

Brain-derived neurotrophic factor (BDNF) is abundantly expressed in the hippocampus and cerebral cortex and is involved in synaptic plasticity and long-term potentiation (LTP). The present study was under taken to investigate whether endogenous BDNF was required for spatial learning and memory in a rat model. Antibodies to BDNF (anti-BDNF, n=7) or control immunoglobulin G (control, n=6) were delivered into the rat brain continuously for 7 days with an osmotic pump. The rats were then subjected to a battery of behavioral tests. The results show that the average escape latencies in the BDNF antibody treated group were dramatically longer than those of the control (F=13.3, p<0.001). The rats treated with control IgG swam for a significantly longer distance in the P quadrant (where the escape plane had been placed) compared with the other three quadrants (p<0.05). In contrast, anti-BDNF-treated rats swam an equivalent distance in all four quadrants. The average percentage of swimming distance in the P quadrant by anti-BDNF-treated rats was much less than that by control IgG treated rats (p<0.001). These results suggest that endogenous BDNF is required for spatial learning and memory in adult rats.

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain‐derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody‐treated mice was reduced by 83%. In the BDNF antibody‐treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody‐treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.

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.

Differential expression of the p75 nerve growth factor receptor in glia and neurons of the rat dorsal root ganglia after peripheral nerve transection.

Sympathetic nerve terminals on blood vessels within the dorsal root ganglia sprout after sciatic nerve lesions in the rat. The mechanism underlying this phenomenon is not clear, but might be predicted to involve nerve growth factor or its homologs because these factors are known to trigger collateral sprouting of undamaged sympathetic noradrenergic terminals. We have found that sciatic nerve lesions lead to a decreased expression of neuronal p75, the low-affinity receptor for the neurotrophins, but an increased expression of glial p75 in ipsilateral dorsal root ganglia. Intriguingly, the increased expression of p75 was found primarily in association with glia surrounding large-diameter neurons, which are those associated with the noradrenergic sprouts. A smaller but significant glial response was also found in contralateral ganglia. The glial response in ipsilateral ganglia could be mimicked by ventral, but not dorsal, root transection. The dorsal root lesion-induced glial responses in contralateral ganglia were greater than those induced by ventral root or sciatic nerve lesions. Combined lesions of dorsal root and either ventral root or sciatic nerve did not prevent the glial responses of ipsilateral ganglia, suggesting that a peripheral signal is involved. Colocalization studies indicate that tyrosine hydroxylase-immunoreactive nerve sprouts were associated with p75-immunoreactive glial cells. Thus, increased glial synthesis of p75 might provide an explanation for the abnormal growth of sympathetic fibers in dorsal root ganglia after 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.

Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation

Our recent study showed that peripheral inflammation induced an increased expression of brain-derived neurotrophic factor (BDNF) mRNA which was mediated by nerve growth factor (NGF) in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF immunoreactivity in the DRG and spinal cord following peripheral inflammation by means of immunohistochemistry. Significant increases in the percentage of BDNF-immunoreactive (IR) neuron profiles in the L5 DRG and marked elevation in the expression of BDNF-IR terminals in the spinal dorsal horn were observed following peripheral tissue inflammation produced by an intraplantar injection of Freunds adjuvant into the rat paws. These findings suggest that peripheral tissue inflammation induces an increased BDNF synthesis in the DRG and an elevated anterograde transport of BDNF to the spinal dorsal horn. The functional role of this increased BDNF was discussed briefly.

Isolation and Characterization of Neural Crest Progenitors from Adult Dorsal Root Ganglia

After peripheral nerve injury, the number of sensory neurons in the adult dorsal root ganglia (DRG) is initially reduced but recovers to a normal level several months later. The mechanisms underlying the neuronal recovery after injury are not clear. Here, we showed that in the DRG explant culture, a subpopulation of cells that emigrated out from adult rat DRG expressed nestin and p75 neurotrophin receptor and formed clusters and spheres. They differentiated into neurons, glia, and smooth muscle cells in the presence or absence of serum and formed secondary and tertiary neurospheres in cloning assays. Molecular expression analysis demonstrated the characteristics of neural crest progenitors and their potential for neuronal differentiation by expressing a set of well‐defined genes related to adult stem cells niches and neuronal fate decision. Under the influence of neurotrophic factors, some of these progenitors gave rise to neuropeptide‐expressing cells and protein zero‐expressing Schwann cells. In a 5‐bromo‐2′‐deoxyuridine chasing study, we showed that these progenitors likely originate from satellite glial cells. Our study suggests that a subpopulation of glia in adult DRG is likely to be progenitors for neurons and glia and may play a role in neurogenesis after nerve injury.

Localization of neurotrophin-3-like immunoreactivity in the rat central nervous system

Neurotropin-3 (NT3) is a nerve growth factor (NGF) homologue whose function is presently unknown. The factor promotes the survival of a subpopulation of sensory and sympathetic neurons in vitro. NT3 mRNA is widely distributed in both the peripheral and central nervous system but the distribution of NT3 has not yet been examined. In the present study we have determined the regional distribution and cellular localization of NT3-like immunoreactivity (-IR) in the central nervous system by immunohistochemistry. Both glia and neurons were stained. NT3-IR glia were distributed in corpus callosum, substantia nigra, fimbria of hippocampus, subependymal areas of the ventricles and cerebellum. In the forebrain, NT3-IR was detected in a number of neuronal cells, including pyramidal cells in the fifth layer of the cerebral cortices, subpopulations of neurons in the septal nuclei, diagonal bands of Broca, olfactory primary cortex, amygdala and islands of Calleja. In the hippocampus, pyramidal cells in the CA1, CA2 and lateral regions of CA3 and granular cells in dorsal dentate gyrus were labelled with different intensities. Neurons in the bed nuclei of the striatum terminalis, mesencephalic trigeminal nuclei and motoneurons in the brain stem and spinal cord were intensively labelled. A subpopulation of neurons in the reticular thalamic nuclei and midbrain were moderately labelled. Finally, in the cerebellum, NT3-IR was also found in Purkinje cells and neurons in the deep cerebellar nuclei. In some brain regions such as hippocampus, the distribution of NT3-IR correlates with that of mRNANT3 as described by others. In contrast in other regions such as spinal cord and brain stem, little correlation was found between protein and mRNA. The results suggest that some NT3 immunoreactive neurons in the central nervous system accumulate NT3 in accord with a neurotrophic role for their maintenance or survival, while others may synthesize and secrete the factor to provide support for innervating neurons.

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.