Protection of ischemic brain cells is dependent on astrocyte-derived growth factors and their receptors

An in vitro ischemia model (oxygen, glucose, and serum deprivation) is used to investigate the possible cellular and molecular mechanisms responsible for cerebral ischemia. We have previously demonstrated that supernatants derived from ischemic microglia can protect ischemic brain cells by releasing GDNF and TGF-beta1. In the present study, we investigate whether products of ischemic astrocytes can also protect ischemic microglia, astrocytes, and neurons in a similar manner. Supernatants from ischemic astrocytes were collected after various periods of ischemia and incubated with microglia, astrocytes, or neurons individually, under in vitro ischemic conditions. The components responsible for the protective effects of astrocyte-derived supernatants were then identified by Western blot, ELISA, trypan blue dye exclusion, and immunoblocking assays. Results showed that under conditions of in vitro ischemia the number of surviving microglia, astrocytes, and neurons was significantly increased by the incorporation of the astrocyte-derived supernatants. Astrocyte supernatant-mediated protection of ischemic microglia was dependent on TGF-beta1 and NT-3, ischemic astrocytes were protected by GDNF, and ischemic neurons were protected by NT-3. In addition, protein expression of TGF-beta1 and NT-3 receptors in microglia, GDNF receptors in astrocytes, and NT-3 receptors in neurons was increased by in vitro ischemia. These results suggest that astrocyte-derived protection of ischemic brain cells is dependent not only on factors released from the ischemic astrocytes, but also on the type of receptor present on the responding cells. Therapeutic potential of TGF-beta1, GDNF, and NT-3 in the control of cerebral ischemia is further suggested.     

Neurotrophic factors expressed in both cortex and spinal cord induce axonal plasticity after spinal cord injury

We reported recently that overexpression of neurotrophin-3 (NT-3) by motoneurons in the spinal cord of rats will induce sprouting of corticospinal tract (CST) axons (Zhou et al. [2003] J. Neurosci. 23:1424-1431). We now report that overexpression of brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF) in the rat sensorimotor cortex near the CST neuronal cell bodies together with overexpression of NT-3 in the lumbar spinal cord significantly increases axonal sprouting compared to that induced by NT-3 alone. Two weeks after unilaterally lesioning the CST at the level of the pyramids, we injected rats with saline or adenoviral vectors (Adv) carrying genes coding for BDNF (Adv.BDNF), GDNF (Adv.GDNF) or enhanced green fluorescent protein (Adv.EGFP) at six sites in the sensorimotor cortex, while delivering Adv.NT3 to motoneurons in each of these four groups on the lesioned side of the spinal cord by retrograde transport from the sciatic nerve. Four days later, biotinylated dextran amine (BDA) was injected into the sensorimotor cortex on the unlesioned side to mark CST axons in the spinal cord. Morphometric analysis of axonal sprouting 3 weeks after BDA injection showed that the number of CST axons crossing the midline in rats treated with Adv.BDNF or Adv.GDNF were 46% and 52% greater, respectively, than in rats treated with Adv.EGFP or PBS (P < 0.05). These data demonstrate that sustained local expression of neurotrophic factors in the sensorimotor cortex and spinal cord will promote increased axonal sprouting after spinal cord injury, providing a basis for continued development of neurotrophic factor therapy for central nervous system damage.     

Characterization of spinal HSP72 induction and development of ischemic tolerance after spinal ischemia in rats

Induction of heat shock protein (HSP72) has been implicated in the development of ischemic tolerance in several tissue organs including brain and spinal cord. In the present study, using an aortic balloon occlusion model in rats, we characterized the effect of transient noninjurious (3 or 6 min) or injurious intervals (10 min) of spinal ischemia followed by 4-72 h of reflow on spinal expression of HSP72 and GFAP protein. In a separate group of animals, the effect of ischemic preconditioning (3 or 6 min) on the recovery of function after injurious interval of spinal ischemia (10 min) was studied. After 3 min of ischemia, there was a modest increase in HSP72 protein immunoreactivity in the dorsal horn neurons at 12 h after reperfusion. After 6 min of ischemia, a more robust and wide spread HSP72 protein expression in both dorsal and ventral horn neurons was detected. The peak of the expression was seen at 24 h after ischemia. At the same time point, a significant increase in spinal tissue GFAP expression was measured with Western blots and corresponded morphologically with the presence of activated astrocytes in spinal segments that had been treated similarly. After 10 min of ischemia and 24 h of reflow, a significant increase in spinal neuronal HSP72 expression in perinecrotic regions was seen. Behaviorally, 3 min preconditioning ischemia led to the development of a biphasic ischemic tolerance (the first at 30 min and the second at 24 h after preconditioning) and was expressed as a significantly better recovery of motor function after exposure to a second 10-min interval of spinal ischemia. After 6 min ischemic preconditioning, a more robust ischemic tolerance at 24 h after preconditioning then seen after 3-min preconditioning was detected. These data indicate that 3 min of spinal ischemia represents a threshold for spinal neuronal HSP72 induction, however, a longer sublethal interval (6 min) of preconditioning ischemia is required for a potent neuronal HSP72 induction. More robust neurological protection, seen after 6 min of preconditioning ischemia, also indicates that HSP72 expression in spinal interneurons seen at 24 h after preconditioning may represent an important variable in modulating ischemic tolerance observed during this time frame.     

Differences in neurotrophic factor gene expression profiles between neonate and adult rat spinal cord after injury

The capacity of the central nervous system for axonal growth decreases as the age of the animal at the time of injury increases. Changes in the expression of neurotrophic factors within embryonic and early postnatal spinal cord suggest that a lack of trophic support contributes to this restrictive growth environment. We examined neurotrophic factor gene profiles by ribonuclease protection assay in normal neonate and normal adult spinal cord and in neonate and adult spinal cord after injury. Our results show that in the normal developing spinal cord between postnatal days 3 (P3) and P10, compared to the normal adult spinal cord, there are higher levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and glial-derived neurotrophic factor (GDNF) mRNA expression and a lower level of ciliary neurotrophic factor (CNTF) mRNA expression. Between P10 and P17, there is a significant decrease in the expression of NGF, BDNF, NT-3, and GDNF mRNA and a contrasting steady and significant increase in the level of CNTF mRNA expression. These findings show that there is a critical shift in neurotrophic factor expression in normal developing spinal cord between P10 and P17. In neonate spinal cord after injury, there is a significantly higher level of BDNF mRNA expression and a significantly lower level of CNTF mRNA expression compared to those observed in the adult spinal cord after injury. These findings suggest that high levels of BDNF mRNA expression and low levels of CNTF mRNA expression play important roles in axonal regrowth in early postnatal spinal cord after injury.     

Intrathecal gene delivery of glial cell line-derived neurotrophic factor ameliorated paraplegia in rats after spinal ischemia

Paraplegia is a catastrophic complication of thoracic aortic surgery. At present, there is no effective mean to prevent the ischemia-induced spinal cord trauma. Gene delivery of neurotrophic factors may hold promises for prevention of spinal injury. In the present study, we evaluated the effect of glial cell line-derived neurotrophic factor (GDNF) gene delivery on prevention of the pathological changes due to spinal ischemia. Recombinant adenovirus vectors encoding GDNF (Ad-GDNF) and green fluorescent protein (Ad-GFP) were used for gene transfer studies. Treatment with cobalt chloride induced dose-dependent bcl-2 and synaptophysin downregulation in spinal neuronal cells, which could be effectively reversed by GDNF gene transfer. Intrathecal injection of Ad-GDNF led to maximal GDNF expression in spinal cord within 2-7 days. Thus, after intrathecal administration of adenovirus vectors for 3 days, Sprague-Dawley rats received transient aortic occlusion to induce spinal ischemia and were monitored for behavior deficits. The Ad-GDNF-treated rats showed significantly lower paraplegia rate (40%) than that of Ad-GFP- or saline-treated groups (75-85%; P<0.01). In addition, the Ad-GDNF-treated rats exhibited significantly improved locomotor function comparing with rats of control groups (P<0.001). Histological analysis revealed that GDNF gene delivery profoundly attenuated the infiltration of leukocytes in spinal cord after ischemic insults. Furthermore, GDNF gene delivery prominently attenuated the ischemia-induced neuronal loss in dorsal horn lamina VI-VIII and reduction in synaptophysin expression in spinal cords. In conclusion, GDNF gene transfer confers protection to the neuronal cells and synapses networks, thereby alleviated the paraplegia due to spinal ischemia.     

Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.     

Neurotrophin-3 promotes cell death induced in cerebral ischemia, oxygen-glucose deprivation, and oxidative stress: possible involvement of oxygen free radicals

To explore the role of neurotrophin-3 (NT-3) during cerebral ischemia, NT-3-deficient brains were subjected to transient focal ischemia. Conditional mutant brains produced undetectable amounts of NT-3 mRNA, whereas the expression of the neurotrophin, BDNF, the NT-3 receptor, TrkC, and the nonselective, low-affinity neurotrophin receptor p75NTR, were comparable to wild-type. Baseline absolute blood flow, vascular and neuroanatomical features, as well as physiological measurements were also indistinguishable from wild-type. Interestingly, the absence of NT-3 led to a significantly decreased infarct volume 23 h after middle cerebral artery occlusion. Consistent with this, the addition of NT-3 to primary cortical cell cultures exacerbated neuronal death caused by oxygen-glucose deprivation. Coincubation with the oxygen free radical chelator, trolox, diminished potentiation of neuronal death. NT-3 also enhanced neuronal cell death and the production of reactive oxygen species caused by oxidative damage inducing agents. We conclude that endogenous NT-3 enhanced neuronal injury during acute stroke, possible by increasing oxygen-radical mediated cell death.     

Ischemia-related changes of glial-derived neurotrophic factor and phosphatidylinositol 3-kinase in the hippocampus: their possible correlation in astrocytes

In the present study, we observed the changes of endogenous expression of glial-cell-line-derived neurotrophic factor (GDNF) and phosphatidylinositol 3-kinase (PI-3 kinase) in the gerbil hippocampus after transient forebrain ischemia and investigated the correlation between GDNF and PI-3 kinase in the ischemic hippocampus. In the sham-operated group, GDNF and PI-3 kinase immunoreactivity was not found in any cells in the hippocampal CA1 region. GDNF, not PI-3 kinase, immunoreactivity was expressed in non-pyramidal cells in the CA1 region at 6 h after ischemic insult. At 12-24 h after ischemia, GDNF and PI-3 kinase immunoreactivity in the CA1 region was similar to that of the sham-operated group. From 2 days after ischemic insult, GDNF- and PI-3-kinase-immunoreactive astrocytes were detected in the CA1 region, and GDNF and PI-3 kinase immunoreactivity in astrocytes was highest in the CA1 region 4 days after ischemic insult. Moreover, at this time point, GDNF and PI-3 kinase were co-localized in some astrocytes. Western blotting showed that ischemia-related changes of GDNF and PI-3 kinase protein levels were similar to the immunohistochemical changes after ischemia. These results suggest that GDNF and PI-3 kinase may be related to delayed neuronal death and that GDNF and PI-3 kinase may be involved in activation of astrocytes.     

Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems

Viral gene delivery for spinal cord injury (SCI) is a promising approach for enhancing axonal regeneration and neuroprotection. An understanding of spatio-temporal transgene expression in the spinal cord is essential for future studies of SCI therapies. Commonly, intracellular marker proteins (e.g., EGFP) were used as indicators of transgene levels after viral delivery, which may not accurately reflect levels of secreted transgene. This study examined transgene expression using ELISA after viral delivery of D15A, a neurotrophin with BDNF and NT-3 activities, at 1, 2, and 4weeks after in vivo and ex vivo delivery using lentiviral, adenoviral, and retroviral vectors. Further, the inflammatory responses and viral infection patterns after in vivo delivery were examined. Lentiviral vectors had the most stable pattern of gene expression, with D15A levels of 536 +/- 38 and 363 +/- 47 pg/mg protein seen at 4 weeks after the in vivo and ex vivo delivery, respectively. Our results show that protein levels downregulate disproportionately to levels of EGFP after adenoviral vectors both in vivo and ex vivo. D15A dropped from initial levels of 422 +/- 87 to 153 +/- 18 pg/mg protein at 4 weeks after in vivo administration. Similarly, ex vivo retrovirus-mediated transgene expression exhibited rapid downregulation by 2 weeks post-grafting. Compared to adenoviral infection, macrophage activation was attenuated after lentiviral infection. These results suggest that lentiviral vectors are most suitable in situations where stable long-term transgene expression is needed. Retroviral ex vivo delivery is optional when transient expression within targeted spinal tissue is desired, with adenoviral vectors in between.     

Long-term locomotor training up-regulates TrkB(FL) receptor-like proteins,brain-derived neurotrophic factor,and neurotrophin 4 with different topographies of expression in oligodendroglia and neurons in the spinal cord

Neurotrophins are potent regulators of neuronal survival, maintenance, and synaptic strength. In particular, brain-derived neurotrophic factor (BDNF), acting through full-length TrkB receptor (TrkB(FL)), is implicated in the stimulation of neurotransmission. Physical activity has been reported to increase BDNF expression in the brain and spinal cord. In this study we have evaluated the hypothesis that activation of a spinal neuronal network, due to exercise, affects the entire spinal neurotrophin system acting via TrkB receptors by modulation of BDNF, neurotrophin 4 (NT-4), and their TrkB receptor proteins. We investigated the effect of treadmill walking (4 weeks, 1 km daily) on distribution patterns and response intensity of these proteins in the lumbar spinal cord of adult rats. Training enhanced immunoreactivity (IR) of both neurotrophins. BDNF IR increased in cell processes of spinal gray matter, mainly in dendrites. NT-4 IR was augmented in the white matter fibers, which were, in part, of astrocytic identity. Training strongly increased both staining intensity and number of TrkB(FL)-like IR small cells of the spinal gray matter. The majority of these small cells were oligodendrocytes, representing both their precursor and their mature forms. In contrast, training did not exert an effect on expression of the truncated form of TrkB receptor in the spinal cord. These results show that both neuronal and nonneuronal cells may be actively recruited to BDNF/NT-4/TrkB(FL) neurotrophin signaling which can be up-regulated by training. Oligodendrocytes of the spinal gray matter were particularly responsive to exercise, pointing to their involvement in activity-driven cross talk between neurons and glia.     

Increased expression of neurotrophins and their receptors in the mechanically compressed spinal cord of the spinal hyperostotic mouse (twy/twy)

The purpose of the present study was to identify any compensatory changes at the site of chronic compression of the spinal cord and neighboring segments. For this purpose, serial immunohistochemical and immunoblot analyses were performed for the expression levels of endogenous brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and their receptors, trkB and trkC in 24 tip-toe walking Yoshimura mice (twy/twy) aged 12-24 weeks. The twy mouse exhibits spontaneous calcified deposits posteriorly at the C1-C2 level, compressing the spinal cord. Immunoreactivities for BDNF, NT-3, trkB and trkC were preferentially localized in the gray matter, particularly in the anterior horn cells. In 24-week-old twy mice with severe compression, expression levels of these neurotrophins at the site of maximal compression were significantly lower than at the less- or non-compressed sites. In contrast, the expression levels of BDNF, NT-3, trkB and trkC were significantly higher at the rostral and caudal sites immediately adjacent to the maximal compression site. No such changes were noted in 12-week-old twy mice or in control Institute of Cancer Research mice. Our results suggest that overexpression of BDNF, NT-3, trkB and trkC in motoneuron areas neighboring the site of mechanical compression may represent compensatory changes in response to the compromised neuronal function at the level of compression, and that these proteins possibly contribute to neuronal survival and plasticity.     

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.     

Differential mRNA expression of neurotrophic factors GDNF, BDNF, and NT-3 in experimental herpes simplex virus encephalitis

Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNA levels were studied in the course of murine herpes simplex virus encephalitis. Induction of GNDF and NT-3 (both P < 0.05) was found during acute encephalitis. Despite absence of clinical impairment, both neurotrophic factors were overexpressed 2 months (NT-3) and 6 months (GDNF) following infection (both P < 0.05). Neurotrophic factors play an important role in neuronal survival and recovery after acute injury to the central nervous system (CNS) and may represent an additional therapeutic target for treatment of viral encephalitis.     

Changes in truncated trkB and p75 receptor expression in the rat spinal cord following spinal cord hemisection and spinal cord hemisection plus neurotrophin treatment

Although numerous studies have examined the effects of neurotrophin treatment following spinal cord injury, few have examined the changes that occur in the neurotrophin receptors following either such damage or neurotrophin treatment. To determine what changes occur in neurotrophin receptor expression following spinal cord damage, adult rats received a midthoracic spinal cord hemisection alone or in combination with intrathecal application of brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Using immunohistochemical and in situ hybridization techniques, p75, trkA, trkB, and trkC receptor expression was examined throughout the spinal cord. Results showed that trkA, full-length trkB, and trkC receptors were not present in the lesion site but had a normal expression pattern in uninjured parts of the spinal cord. In contrast, p75 receptor expression occurred on Schwann cells throughout the lesion site. BDNF and NT-3 (but not saline) applied to the lesion site increased this expression. In addition, the truncated trkB receptor was expressed in the border between the lesion and intact spinal cord. Truncated trkB receptor expression was also increased throughout the white matter ipsilateral to the lesion and BDNF (but not NT-3 or saline) prevented this increase. The study is the first to show changes in truncated trkB receptor expression that extend beyond the site of a spinal cord lesion and is one of the first to show that BDNF and NT-3 affect Schwann cells and/or p75 expression following spinal cord damage. These results indicate that changes in neurotrophin receptor expression following spinal cord injury could influence the availability of neurotrophins at the lesion site. In addition, neurotrophins may affect their own availability to damaged neurons by altering the expression of the p75 and truncated trkB receptor.     

Acute up-regulation of brain-derived neurotrophic factor expression resulting from experimentally induced injury in the rat spinal cord

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family of trophic factors, has multiple functions including a role in the promotion of neuronal survival and nerve fiber elongation in both the central and the peripheral nervous systems. We assessed the expression of endogenous BDNF following an experimentally induced compression injury to the spinal cord. Expression of BDNF mRNA was increased following the spinal cord injury; reaching maximum levels 24 h after the injury. Expression of BDNF mRNA returned to the levels observed in sham-operated control animals within 3 days of the injury. Using the in situ hybridization technique, we observed a wide distribution of BDNF expression among the different cell types in the spinal cord, including motor and sensory neurons, and in glia cells, including astrocytes. We also observed expression of BDNF in putative macrophages and/or microglia; however, this effect was not observed until day 7 following spinal cord injury. These results suggest that BDNF is synthesized in both neurons and astrocytes during the acute response to injury to the spinal cord, functioning in a mainly neuroprotective role. This is followed by a later phase of expression in which BDNF is produced by macrophages and/or microglia, apparently functioning in a restorative capacity.     

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat

After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.     

Survival effects of BDNF and NT-3 on axotomized rubrospinal neurons depend on the temporal pattern of neurotrophin administration

This study shows that both BDNF and NT-3 can prevent cell death in axotomized adult rat rubrospinal neurons (RSNs), but that the efficacy of neuroprotection depends on the temporal pattern of treatment. At 8 weeks after cervical spinal cord injury, 51% of the RSNs had died. Subarachnoidal BDNF infusion into the cisterna magna for 4 weeks resulted in neuronal hypertrophy and 71% survival. Continuous infusion for 8 weeks into the lumbar subarachnoidal space with either BDNF or NT-3 gave similar survival rates, while a combination of BDNF and NT-3 resulted in 96% survival, although the cells were atrophic. When administration of either BDNF or NT-3 was delayed and performed during postoperative weeks 5-8, the number of surviving neurons was increased compared to early treatment. Delayed treatment with a combination of BDNF and NT-3 resulted in complete survival and a reduction in neuronal atrophy. A decreased expression of TrkB receptors and microtubule-associated protein-2 in the RSNs after axotomy was counteracted by BDNF and NT-3. Microglial activity remained increased even when complete cell survival was achieved. Thus, the combination of neurotrophins as well as the temporal pattern of treatment need to be adequately defined to optimize survival of injured spinal tract neurons.     

Neurotrophin expression by spinal motoneurons in adult and developing rats

Expression of the neurotrophins NT-4, brain-derived neurotrophic factor (BDNF), and NT-3 in adult rat lumbosacral spinal cord motoneurons is reported. A sensitive in situ hybridization procedure demonstrates localization of the mRNA for each of these neurotrophins within spinal motoneurons of the adult and in early postnatal development. A majority of adult rat spinal cord lumbar motoneurons (approximately 63%) express NT-4 mRNA as assessed by counting motoneurons in the L4 and L5 segments of two adult rat spinal cords on adjacent cresyl violet-stained and in situ hybridization sections. Similarly, a majority of lumbar motoneurons (approximately 73%) express BDNF mRNA. Further analyses of adjacent lumbar spinal cord sections revealed that many, although not all motoneurons coexpress both NT-4 and BDNF mRNAs. At birth, the mRNA encoding NT-3 is expressed in motoneurons, but BDNF mRNA is not apparent until postnatal day 5 (P5) and NT-4 mRNA first appears at P9. The potential biological significance of neurotrophin mRNA expression in spinal motoneurons is supported by immunohistochemical localization of each neurotrophin protein in adult motoneurons. We discuss the potential role of spinal cord neurotrophins as autocrine or paracrine factors involved in modulating motoneuron synaptic function.     

Intrathecal injection of GDNF and BDNF induces immediate early gene expression in rat spinal dorsal horn

Glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are potent trophic factors for dorsal root ganglion cells. In addition, these factors are produced in subsets of dorsal root ganglion cells and transported anterogradely to their terminals in the superficial dorsal horn of the spinal cord, where they constitute the only source of GDNF and BDNF. We investigated the effect of 10 mug GDNF and BDNF injected by lumbar puncture on the expression of the immediate early gene (IEG) products c-Fos, c-Jun, and Krox-24 in the adult rat dorsal horn. In the dorsal horn of S1 spinal segments, GDNF and BDNF induced a strong increase in IEG expression, which was most pronounced in laminae I and II (2.9- to 4.5-fold). More distal from the injection site, in the dorsal horn of L1/L2 spinal segments, the increase in IEG expression was less pronounced, suggesting a concentration-dependent effect. In order to explain the effects of intrathecally injected GDNF, we investigated whether lumbo-sacral dorsal horn neurons expressed RET protein, the signal-transducing element of the receptor complex for GDNF. It was found that several of these neurons contained RET immunoreactivity and that some of the RET-labeled neurons had the appearance of nociceptive-specific cells, confirming their presumed role in pain transmission. Additionally, using double-labeling immunofluorescence combined with confocal microscopy, it was found that after intrathecal GDNF injection 35% of c-Fos-labeled cells were also labeled for RET. These results demonstrate that intrathecally administered GDNF and BDNF induce IEG expression in dorsal horn neurons in the adult rat, supposedly by way of their cognate receptors, which are present on these neurons. We further suggest that the endogenous release of GDNF and BDNF, triggered by nociceptive stimuli, is involved in the induction of changes in spinal nociceptive transmission as in various pain states.