Robert A. Rush

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.

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.

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.

Distribution of trkB tyrosine kinase immunoreactivity in the rat central nervous system

Recent evidence suggests that trkB tyrosine kinase is a high affinity receptor for brain-derived neurotrophic factor (BDNF). BDNF can act as a survival factor for several neuronal subgroups and its mRNA is distributed widely throughout the central nervous system. However, the functional targets of BDNF are poorly defined. We have used immunochemical and immunohistochemical techniques to determine the regional distribution and cellular localization of trkB tyrosine kinase-like immunoreactivity. The staining pattern indicates that the trkB-like antigen is widely distributed and present within both glia and neurons. Astrocytes were the most intensively labelled but many neuronal populations were also stained. In some regions including brain stem, spinal cord, hippocampus and diagonal band of Broca, neurons were stained at varying intensities. In other areas such as the cortex of the forebrain and amygdaloid nucleus, the stain was intense but diffuse, preventing positive identification of the cell types involved. Immunoblot results indicated two separate protein bands in all brain and spinal cord regions examined, of molecular weights 145 and 85 kDa, respectively. These findings aid the definition of neuronal and glial subpopulations of the central nervous system that may utilize BDNF.

Elevated Nerve Growth Factor Levels in the Synovial Fluid of Patients with Inflammatory Joint Disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean ± SEM NGF concentration in normal synovial fluids was 95 ± 33.2 pg/ml (range 39.1–143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 ± 123.8 pg/ml (range 152–1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 ± 90 pg/ml; range 89–1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.

Localization of neurotrophin-3-like immunoreactivity in peripheral tissues of the rat

Neurotrophin-3 (NT-3) mRNA is widely distributed in both the peripheral and central nervous systems but neither the distribution of the native factor nor its physiological function is known. In the present study we produced and characterized an antibody to a synthetic peptide and showed that it specifically recognised endogenous rat and recombinant human NT-3 (rNT-3), but not mouse nerve growth factor and recombinant brain derived-neurotrophic factor. NT-3-like immunoreactivity (NT-3-ir) was detected within the distal tubular cells of the kidney, the zona glomerulosa and reticularis of the adrenal cortex, ganglion cells of the adrenal medulla, red pulp of the spleen, epithelial cells of the intestinal villi and parenchymal cells of the liver. Within peripheral ganglia, NT-3-ir was observed in a subpopulation of large sensory neurons of dorsal root, trigeminal and cochleovestibular ganglia but not in principle neurons of the sympathetic ganglia. These results provide the first evidence for the localization of NT-3-ir and indicate its presence in various peripheral organs and large sensory neurons. We conclude that NT-3 may function outside the nervous system in addition to a neurotrophic role within large sensory neurons.

Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos

Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75NTR-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75NTR-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.