Treatment of chronically injured spinal cord with neurotrophic factors stimulates betaII-tubulin and GAP-43 expression in rubrospinal tract neurons

Exogenous neurotrophic factors provided at a spinal cord injury site promote regeneration of chronically injured rubrospinal tract (RST) neurons into a peripheral nerve graft. The present study tested whether the response to neurotrophins is associated with changes in the expression of two regeneration-associated genes, betaII-tubulin and growth-associated protein (GAP)-43. Adult female rats were subjected to a right full hemisection lesion via aspiration of the C3 spinal cord. A second aspiration lesion was made 4 weeks later and gel foam saturated in brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), or phosphate-buffered saline (PBS) was applied to the lesion site for 60 min. Using in situ hybridization, RST neurons were examined for changes in mRNA levels of betaII-tubulin and GAP-43 at 1, 3, and 7 days after treatment. Based on analysis of gene expression in single cells, there was no effect of BDNF treatment on either betaII-tubulin or GAP-43 mRNA expression at any time point. betaII-Tubulin mRNA levels were enhanced significantly at 1 and 3 days in animals treated with GDNF relative to levels in animals treated with PBS. Treatment with GDNF did not affect GAP-43 mRNA levels at 1 and 3 days, but at 7 days there was a significant increase in mRNA expression. Interestingly, 7 days after GDNF treatment, the mean cell size of chronically injured RST neurons was increased significantly. Although GDNF and BDNF both promote axonal regeneration by chronically injured neurons, only GDNF treatment is associated with upregulation of betaII-tubulin or GAP-43 mRNA. It is not clear from the present study how exogenous BDNF stimulates regrowth of injured axons.     

Glial fibrillary acidic protein expression in the hamster red nucleus: effects of axotomy and testosterone treatment

Testosterone propionate (TP) administration coincident with facial nerve axotomy in the hamster attenuates glial fibrillary acidic protein (GFAP) expression in the facial nucleus that is normally increased following axotomy alone. This ability of TP to modulate astrocyte activity has been linked to the ability of steroid hormones to enhance the regenerative response of injured motor neurons. In an ongoing study designed to examine the potential influences of steroid hormones on centrally projecting motoneurons, the astrocyte reaction in the red nucleus was examined. In the present study, in situ hybridization was used to assess changes in GFAP mRNA in the hamster red nucleus following spinal cord injury (SCI) and TP treatment. Castrated male hamsters were subjected to right rubrospinal tract (RST) transection at spinal cord level T1, with half the animals implanted subcutaneously with Silastic capsules containing 100% crystalline TP and the remainder sham implanted. The uninjured red nucleus served as an internal control. Postoperative survival times were 1, 2, 7, and 14 days. Qualitative-quantitative analyses of emulsion autoradiograms were accomplished. Axotomy alone resulted in a significant but transient increase in GFAP mRNA levels at 2 days postoperative in the injured red nucleus compared with the contralateral uninjured red nucleus. However, in TP-treated animals, GFAP mRNA levels were no different than control levels at 2 dpo but were significantly increased at 7 dpo relative to contralateral control. Additionally, the increase in GFAP mRNA levels following TP treatment was significantly smaller than following axotomy alone. These data suggest that testosterone both delays and reduces the astrocytic reaction in the red nucleus following rubrospinal tract axotomy, and confirms a difference between peripheral and central glial responses to axotomy and steroid administration.     

Combination of olfactory ensheathing cells with local versus systemic cAMP treatment after a cervical rubrospinal tract injury

The failure of CNS axons to regenerate following traumatic injury is due in part to a growth‐inhibitory environment in CNS as well as a weak intrinsic neuronal growth response. Olfactory ensheathing cell (OECs) transplants have been reported to create a favorable environment promoting axonal regeneration, remyelination, and functional recovery after spinal cord injury. However, in our previous experiments, OEC transplants failed to promote regeneration of rubrospinal axons through and beyond the site of a dorsolateral funiculus crush in rats. Rubrospinal neurons undergo massive cell atrophy and limited expression of regeneration‐associated genes after axotomy. Using the same injury model, we tested the hypothesis that treatment of the red nucleus with cAMP, known to stimulate the intrinsic growth response in other neurons, will promote rubrospinal regeneration in combination with OEC transplants. In addition, we assessed a systemic increase of cAMP using the phosphodiesterase inhibitor rolipram. OECs prevented cavity formation, attenuated astrocytic hypertrophy and the retraction of the axotomized rubrospinal axons, and tended to reduce the overall lesion size. OEC transplantation lowered the thresholds for thermal sensitivity of both forepaws. None of our treatments, alone or in combination, promoted rubrospinal regeneration through the lesion site. However, the systemic elevation of cAMP with rolipram resulted in greater numbers of OECs and axonal density within the graft and improved motor performance in a cylinder test in conjunction with enhanced rubrospinal branching and attenuated astrocytic hypertrophy. © 2010 Wiley‐Liss, Inc.     

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.     

Gonadal steroid control of preprocholecystokinin mRNA expression in the limbic-hypothalamic circuit: Comparison of adult with neonatal steroid treatments

The neuropeptide cholecystokinin (CCK) is involved in the regulation of female, but not male, reproductive behavior. In both sexes, estrogen regulates the expression of CCK in adulthood within the bed nucleus of the stria terminalis and medial amygdaloid nucleus. These areas are parts of an interconnected limbic system-hypothalamic circuit, the development of which is influenced by estrogen during the early postnatal period. This is the same period during which central nervous system (CNS) expression of CCK is dramatically increased, suggesting that the male and female patterns of CCK expression may be the result of early postnatal exposure to estrogen. In the present experiment, the expression of preprocholecystokinin (pCCK) mRNA was determined by in situ hybridization with an isotopically labeled pCCK complementary RNA and emulsion autoradiography in animals whose neonatal and adult gonadal steroid levels had been manipulated. The number of pCCK-expressing cells in animals that were gonadectomized as adults was determined by neonatal estrogen, but stimulation with steroids in adulthood induced a similar number of pCCK-expressing cells in both sexes in the medial amygdala and bed nucleus of the stria terminalis. Neonatal treatment of females with estrogen or testosterone, followed by ovariectomy in adulthood, eliminated the sex difference in pCCK mRNA expression. Males treated neonatally with the aromatase inhibitor androstenedione (to block metabolism of testosterone to estrogen) and orchidectomized in adulthood had a level of pCCK mRNA expression that was similar to that of ovariectomized females. These data suggest that, during neonatal development, estrogen determines the constitutive expression of pCCK mRNA in the medial amygdala and bed nucleus of the stria terminalis, resulting in higher levels of pCCK mRNA expression in males than in females. However, exogenous gonadal steroids induce the same levels of pCCK mRNA expression in adult females, indicating that the levels of gonadal steroids and the patterns of their secretion are the predominant influences on the sexually dimorphic adult levels of pCCK mRNA expression. © 1994 Wiley-Liss, Inc.     

Grafts of BDNF-producing fibroblasts rescue axotomized rubrospinal neurons and prevent their atrophy

We have reported that intraspinal transplants of fibroblasts genetically modified to express brain-derived neurotrophic factor (BDNF) promote rubrospinal axon regeneration and functional recovery following subtotal cervical hemisection that completely ablated the rubrospinal tract. In the present study we examined whether these transplants could prevent cell loss and/or atrophy of axotomized Red nucleus neurons. Adult rats received a subtotal spinal cord cervical hemisection followed by a graft of unmodified fibroblasts or fibroblasts producing BDNF into the lesion cavity. One or 2 months later, fluorogold was injected several segments caudal to the lesion-transplant site to retrogradely label those Red nucleus neurons whose axons have regenerated. Unmodified fibroblasts failed to protect against either cell loss or atrophy. Neuron counts and soma-size measurements in Nissl-stained preparations showed a 45% loss of recognizable neurons and 40% atrophy of the surviving neurons in the injured Red nucleus. Grafts of BDNF-producing fibroblasts reduced neuron loss to less than 15% and surviving neurons showed only a 20% decrease in mean soma size. Soma size analysis of fluorogold-labeled Red nucleus neurons indicated that the Red nucleus neurons whose axons regenerated caudal to the graft did not atrophy. We conclude that fibroblasts engineered ex vivo to secrete BDNF and grafted into a partial cervical hemisection promote axon regeneration while reducing cell loss and atrophy of neurons in the Red nucleus. These results suggest that transplants of genetically engineered cells could be an important tool for delivery of therapeutic factors that contribute to the repair of spinal cord injury.     

Evidence for new growth and regeneration of cut axons in developmental plasticity of the rubrospinal tract in the North American opossum.

We have shown previously that rubral axons can grow around a lesion of their spinal pathway in the developing opossum and that a critical period exists for that plasticity (Martin and Xu, Dev Brain Res 39:303, 1988). Since most rubrospinal neurons degenerate after axotomy during the critical period, we have proposed that plasticity results primarily from growth of late arriving axons around the lesion rather than regeneration of cut axons (Xu and Martin, J Comp Neurol 279:368, 1989). In the present study, we used a double-labeling paradigm to test that hypothesis. Four groups of pouch young opossums received bilateral or unilateral injections of Fast Blue (FB) into the caudal thoracic or rostral lumbar cord (T12-L2) at different ages in order to label rubrospinal neurons. Three or 4 days later, the rubrospinal tract was transected unilaterally, four to five segments rostral to the injection(s). If the injection was unilateral, the lesion was made ipsilateral to it. The animals were maintained for about 1 month before a second marker, Diamidino Yellow (DY), was injected, usually bilaterally, between the FB injection(s) and the lesion. The animals were maintained for about 5 days before sacrifice and sections through the red nucleus and spinal cord were examined with a fluorescence microscope. During the critical period for plasticity, only a few rubral neurons contralateral to the lesion were labeled by FB alone, supporting our previous contention that most axotomized neurons degenerate. In contrast, many neurons were labeled by DY alone, indicating that their axons were not present in the caudal cord at the time of the FB injection and that they grew around the lesion during the 1 month survival to incorporate DY. A few double-labeled neurons were also found. One interpretation of such neurons is that they survived axotomy, as evidenced by the presence of FB, and supported axons which grew around the lesion to take up DY. Another interpretation is that they supported late growing axons which incorporated residual FB as well as DY. In order to choose between these alternatives, a similar double-labeling paradigm was carried out, but with removal of FB at the time of the lesion. Since a few neurons were still double labeled, we conclude that regeneration of cut axons also contributed to rubrospinal plasticity. Our results support our previous suggestion that developmental plasticity of the rubrospinal tract results primarily from growth of late arriving axons around the lesion, but they also suggest that regeneration of cut axons occurs.     

Expression of β-calcitonin gene-related peptide in axotomized rubrospinal neurons and the effect of brain derived neurotrophic factor

The mRNA levels for α- and β-calcitonin gene-related peptide (CGRP) in rat rubrospinal neurons were studied by in situ hybridization 3, 7, 14, 28 and 56 days following cervical spinal hemisection. CGRP-like immunoreactivity (LI) in the rubrospinal neurons and the rubrospinal tract in cervical spinal cords were examined using immunohistochemistry. There was almost no signal for α- and β-CGRP mRNAs and undetectable level of CGRP-LI in the rubrospinal neurons ipsilateral to cervical spinal hemisection (control side). Fourteen days after spinal hemisection, the rubrospinal neurons contralateral to cervical hemisection (axotomized side) showed CGRP-LI in their cell bodies, and CGRP containing fibers were observed in the lateral funiculi just proximal, but not distal, to the injury sites. In situ hybridization showed upregulation of β-CGRP mRNA in a subpopulation of the rubrospinal neurons on the axotomized side. The proportion of β-CGRP mRNA-expressing neurons reached its maximum (approximately 19%) 4 days following axotomy and slowly decreased to about 5% 56 days after axotomy. The percentage of α-CGRP mRNA-expressing neurons was much lower than that of β-CGRP mRNA (maximum about 2.6% 4 days after axotomy) and not significantly different from the control side throughout the time period studied. These data indicate that axotomy induces de novo synthesis of the CGRP β-subtype in rubrospinal neurons and that the β-CGRP is transported to the injury site through the rubrospinal tract. In addition, we studied the effect of the intracerebral injections of brain derived neurotrophic factor (BDNF). BDNF treatment fully reversed the severe cell atrophy that followed axotomy and increased the number of neurons labeled for β-CGRP mRNA, but did not increase the percentage of rubrospinal neurons expressing β-CGRP mRNA. Thus, topical application of BDNF does not have direct modulatory effect on CGRP induction in axotomized neurons in the red nucleus.     

Chronic peripheral nerve section results in a rearrangement of the central axonal arborizations of axotomized A beta primary afferent neurons in the rat spinal cord

In order to investigate the reorganization of the neuropil of the dorsal horn following peripheral nerve injury, the central terminal arborizations of 35 A beta primary afferent neurons, chronically injured by a cut and ligation of the sural nerve 6–12 weeks previously, were studied by the intra-axonal injection of horseradish peroxidase. Their morphology was compared to 13 intact sural nerve hair follicle afferents. Following axotomy, three kinds of morphological abnormalities were observed in the collateral arbors of the 26 afferents that were hair follicle-like. Atrophy with thin stem axons and reduced terminal branch patterns with few boutons was seen in 5 afferents. Sprouting of bouton-containing terminals into lamina I and IIo was found in 8 afferents. Finally, abnormal arborization patterns in the deeper laminae were observed in 29% of the collateral arbors. Changes included the loss in some arbors of a flame-shaped appearance, which is characteristic of hair follicle afferents, atypical branching patterns and ventrally directed axons producing wider and deeper arbors, compared to normal. Axotomy also caused a disruption of the normal somatotopic organizaiton of sural nerve A beta afferents. This disruption manifested as a variability in the normally mediolaterally restricted terminal sheet, with a consequent loss of the strict somatotopic register in the rostrocaudal direction. Damage to the peripheral axon of A beta primary afferents induces a structural reorganization of their central terminals in the dorsal horn of the spinal cord, which may modify sensory input to the central nervous system.     

Developmental and regional expression of choline acetyltransferase mRNA in the rat central nervous system

The developmental and regional expression of choline acetyltransferase (ChAT) mRNA was examined in the rat brain and spinal cord by northern blot analysis and in situ hybridization. ChAT mRNA expression in the brain showed a biphasic increase during development, with a first peak at two weeks postnatally, a marked decrease by the third week, and a second increase between the third and fifth week after birth, indicating that emergence of the cholinergic phenotype occurs at different times in different brain regions. In the spinal cord, ChAT mRNA was detected at similar levels from embryonic stage 13 (E13) until birth, increasing thereafter until adulthood. In the adult rat central nervous system, high levels of ChAT mRNA were detected in the spinal cord and brain stem structures. Lower levels were seen in midbrain, septum, striatum, thalamus, and olfactory bulb. ChAT mRNA containing cells were identified by in situ hybridization in the olfactory tubercule, piriform cortex, striatum, several basal forebrain nuclei, and spinal cord. A nearly two-fold increase in adult spinal cord ChAT mRNA levels were seen one week after a bilateral crush lesion of the sciatic nerve, indicating that ChAT mRNA expression is regulated during motoneuron regeneration.     

Close axonal injury of rubrospinal neurons induced transient perineuronal astrocytic and microglial reaction that coincided with their massive degeneration

To learn more about the pathophysiology of axonal injury and the significance of axon collaterals on the survival of axotomized cord-projection central neurons, we studied the survival rate, surrounding astrocytic and microglial reactions, and bouton coverage on rat rubrospinal cell bodies following their axonal lesion at the brain stem and upper cervical level. The brain stem lesion disconnected most rubrospinal neurons from all their targets, while the upper cervical lesion spared their supraspinal collaterals. Much higher cell loss accompanied by robust astrocytic and microglial reaction was found following brain stem than upper cervical lesion starting 4 days postaxotomy. The reaction of astrocytes had subsided while microglial reaction remained relatively robust by 10 weeks postaxotomy when the cell loss had slowed down. Ultrastructural observation revealed that reactive astrocytes covered 40%, an increase from the 20% of control, of brain stem-axotomized rubrospinal cell body surface at 4 days and 2 weeks and returned to normal levels by 10 weeks postlesion. An increase of apposition by axons and dendrites and a moderate decrease of round and flattened vesicle-containing bouton contacts at 4 days and 2 weeks and returning to normal levels at 10 weeks postaxotomy accompanied this. It appears that although axotomy induced robust astrocytic reaction around cord-projection central neurons, this, unlike their periphery-projection counterparts, failed to effectively strip their somatic synapses. In effect, this might in part determine neuronal fate following axonal injury.     

Orexin-A regulates growth hormone-releasing hormone mRNA content in a nucleus-specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus

The orexins or hypocretins are two neuropeptides involved in the regulation of diverse biological processes such as feeding, sleep and neuroendocrine function. Recent findings suggest a possible functional interaction between orexins, somatostatin and growth hormone-releasing hormone (GHRH) in the rat hypothalamus. In order to understand the possible functional linkage between orexins and these neuropeptides, we determined the effects of intracerebroventricular orexin-A administration on hypothalamic somatostatin and GHRH mRNA levels. Furthermore, we examined whether growth hormone (GH) mediates these interactions by using two animal models that showed GH deficiency: hypophysectomized rats and dwarf Lewis rats. Using in situ hybridization, our data showed that GHRH mRNA levels in the paraventricular nucleus of the hypothalamus are decreased after orexin-A treatment, without changes in the arcuate nucleus of the hypothalamus. On the other hand, orexin-A treatment induces a GH-dependent stimulatory effect on somatostatin mRNA content in the periventricular nucleus of the hypothalamus. Finally, we demonstrated, for the first time, that hypophysectomized rats and dwarf Lewis rats, two classical models of GH deficiency with alterations in sleep patterns, showed a marked reduction in the GHRH mRNA levels in the paraventricular nucleus of the hypothalamus. These data improve our understanding of the interactions among the different systems involved in the control and pathophysiology of food intake, sleep and GH secretion.     

Down-regulation of mRNAs for synaptic adhesion molecules neuroligin-2 and -3 and synCAM1 in spinal motoneurons after axotomy

After peripheral axotomy, synapses are eliminated from the somata of spinal motoneurons. Recent evidence indicates that synaptic adhesion molecules play a role in maintenance of synaptic contacts, but so far such molecules have not been investigated in the context of synapse elimination after injury. In vitro, the neuroligins (NLGs) and SynCAM1 drive formation of synapses, and RNAi of NLGs results in decreased synaptic input, indicating an important role for these molecules in synaptic biology. To address potential involvement of NLGs and SynCAMs in postinjury synapse elimination, we investigated the mRNA expression of NLG1, -2, and -3; SynCAM1 and -3; and PSD-95--an intracellular NLG-binding scaffolding protein--in rat spinal motoneurons in control animals and after sciatic nerve transection (SNT). mRNA signals for NLG2, NLG3, SynCAM1, and SynCAM3, but not NLG1, were seen in uninjured motoneurons. Immunoreactivity for SynCAM was seen in close relation to synaptophysin immunoreactivity on the surface of motoneurons and in close relation to neurofilament immunoreactivity in the sciatic nerve. After axotomy, the signals for NLG2, NLG3, and SynCAM1 mRNAs decreased, whereas the signal for NLG1 mRNA remained undetectable and that for SynCAM3 remained at control levels. The signal for PSD-95 mRNA decreased gradually and reached approximately 50% of control values 2 weeks after axotomy. Thus the retrograde response to axotomy of spinal motoneurons involves a rapid down-regulation of NLG2, NLG3, and SynCAM1 mRNAs and a gradual decrease in PSD-95 mRNA. This indicates that down-regulation of synaptic adhesion molecules plays a role in postinjury synapse elimination.     

Expression of heat shock genes (hsp70) in the rabbit spinal cord: localization of constitutive and hyperthermia-inducible mRNA species.

We have previously reported that hyperthermia induces the expression of a heat shock gene in the rabbit brain (Sprang and Brown, Mol Brain Res 3:89-93, 1987). Striking regional and cell type differences in the pattern of induction of the hsp70 mRNA were noted. Tissue injury also induces the rapid induction of hsp70 mRNA in the mammalian brain (Brown et al., Neuron 2:1559-1564, 1989). In the present study, in situ hybridization with 35S-labelled riboprobes specific for constitutive and inducible hsp70 mRNA species was employed to investigate the effect of fever-like temperatures on hsp70 gene expression in the rabbit spinal cord. Expression of constitutive hsp70 mRNA was detected in large motor neurons of both control and hyperthermic animals. Within 1 hr after hyperthermia, a massive induction of inducible hsp70 mRNA was noted in fibre tracts of the spinal cord, a pattern consistent with a strong glial response to heat shock. Induction was not observed in the large motor neurons.     

Electrical stimulation accelerates and increases expression of BDNF and trkB mRNA in regenerating rat femoral motoneurons

Electrical stimulation promotes the speed and accuracy of motor axonal regeneration. The positive effects of stimulation are mediated at the cell body. Here we characterize the effect of electrical stimulation on motoneuronal expression of BDNF and its receptor, trkB, two genes whose expression levels in motoneurons correlate with regeneration and are regulated by electrical activity in a variety of neurons. We used semiquantitative in situ hybridization to measure expression of mRNA encoding BDNF and the full-length trkB receptor at intervals of 8 h, 2 days and 7 days after unilateral femoral nerve cut, suture, and stimulation. Expression in regenerating motoneurons was compared to that of contralateral intact motoneurons. BDNF and trkB signals were not significantly upregulated 8 h and 2 days after femoral nerve suture and sham stimulation. By 7 days, there was a 2-fold increase in both BDNF and trkB mRNA expression. In contrast, stimulation of cut and repaired nerves for only 1 h led to rapid upregulation of BDNF and trkB mRNA by 3-fold and 2-fold, respectively, within the first 8 h. The stimulation effect peaked at 2 days with 6-fold and 4-fold increases in the signals, respectively. Thereafter, the levels of BDNF and trkB mRNA expression declined to equal the 2-fold increase seen at 7 days after nerve repair and sham-stimulation. We conclude that brief electrical stimulation stimulates BDNF and trkB expression in regenerating motoneurons. Because electrical stimulation is known to accelerate axonal regeneration, we suggest that changes in the expression of BDNF and trkB correlate with acceleration of axonal regeneration.     

Neuropeptide role of both peptide YY and neuropeptide Y in vertebrates suggested by abundant expression of their mRNAS in a cyclostome brain

The evolution of the neuropeptide Y (NPY) family of peptides has been unclear despite sequence information from many vertebrates. We describe here two NPY-related peptides deduced from cDNA clones of the river lamprey (Lampetra fluviatilis), a cyclostome providing one of the best models of a primitive vertebrate brain. One peptide corresponds to NPY as it has 83% identity to human NPY and its mRNA is expressed in the lateral brainstem, dorsal spinal cord and retina. The second lamprey peptide corresponds anatomically to peptide YY (PYY) as its mRNA is found in gut cells and in medial brainstem neurons. Its sequence is 60–70% identical to both PYY and NPY of mammals. These data suggest that the gene duplication leading to NPY and PYY had already occurred in the ancestral vertebrate 450 million years ago. The expression of the presumed PYY homolog in both gut and central nervous system indicates that PYY has served the dual role as a hormone and a neuropeptide from an early stage in vertebrate evolution. The similarities in the location of NPY- and PYY-expressing cells between lamprey and mammals suggest that the functions of these peptides may have been conserved. © 1994 Wiley-Liss, Inc.