Neonatal Nerve Research Articles

Observations at the CNS–PNS Border of Ventral Roots Connected to a Neuroma

Previous studies have shown that numerous sprouts originating from a neuroma, after nerve injury in neonatal animals, can invade spinal nerve roots. However, no study with a focus on how such sprouts behave when they reach the border between the central and peripheral nervous system (CNS–PNS border) has been published. In this study we have in detail examined the CNS–PNS border of ventral roots in kittens with light and electron microscopy after early postnatal sciatic nerve resection. A transient ingrowth of substance P positive axons was observed into the CNS, but no spouts remained 6 weeks after the injury. Using serial sections and electron microscopy it was possible to identify small bundles of unmyelinated axons that penetrated from the root fascicles for a short distance into the CNS. These axons ended blindly, sometimes with a growth cone-like terminal swelling filled with vesicles. The axon bundles were accompanied by p75 positive cells in both the root fascicles and the pia mater, but not in the CNS. It may thus be suggested that neurotrophin presenting p75 positive cells could facilitate axonal growth into the pia mater and that the lack of such cells in the CNS compartment might contribute to the failure of growth into the CNS. A maldevelopment of myelin sheaths at the CNS–PNS border of motor axons was observed and it seems possible that this could have consequences for the propagation of action potential across this region after neonatal nerve injury. Thus, in this first detailed study on the behavior of recurrent sprouts at the CNS–PNS border.

Development of a scaffoldless three-dimensional engineered nerve using a nerve-fibroblast co-culture

Nerve grafts are often required to replace tissue damaged by disease, surgery, or extensive trauma. Limitations such as graft availability, donor site morbidity, and immune rejection have led investigators to develop strategies to engineer nerve tissue. The goal of this study was to fabricate a scaffoldless three-dimensional (3D) nerve construct using a co-culture of fetal nerve cells with a fibroblast monolayer and allow the co-culture to remodel into a 3D construct with an external fibroblast layer and an internal core of interconnected neuronal cells. Primary fibroblasts were seeded on laminin-coated plates and allowed to form a confluent monolayer. Neural cells isolated from E-15 spinal cords were seeded on top of the fibroblast monolayer and allowed to form a networked monolayer across the monolayer of fibroblasts. Media shifts initiated contraction of the fibroblast monolayer and a remodeling of the co-culture into a 3D construct held statically in place by the two constraint pins. Immunohistochemistry using S100 (Schwann cell), beta3-tubulin, DAPI, and collagen I indicated an inner core of nerve cells surrounded by an external layer of fibroblasts. Conduction velocities of the 3D nerve and control (fibroblast-only) constructs were measured in vitro and compared to in vivo measures of neonatal sciatic nerve. The conduction velocities of the nerve constructs were comparable to 24-d-old neonatal nerve. The presence of Schwann cells and the ability to conduct neuronal signals in vitro suggest the scaffoldless 3D nerve constructs will be a viable option for nerve repair.

MHC class I upregulation is not sufficient to rescue neonatal alpha motoneurons after peripheral axotomy

Associated with neuronal death, profound synaptic changes occur in the spinal cord during the apoptotic process triggered after axotomy in neonatal rats. With respect to this, the major histocompatibility complex of class I (MHC class I) has recently emerged as a new mechanism related to synaptic stripping and plasticity. The present study investigated the impact of upregulating MHC class I expression by treatment with beta interferon (beta INF) on motoneuron survival, synaptic plasticity and astrogliosis after neonatal sciatic nerve injury. P2 rats were subjected to unilateral axotomy followed by three days of beta INF treatment. The results were analyzed by counting Nissl stained motoneurons, immunohistochemistry (anti-synaptophysin, MHC class I, GFAP and Iba-1) and transmission electron microscopy. INF treatment induced an increased expression of MHC class I, which resulted in a stronger synaptic elimination process in the spinal cord, as seen by the synaptophysin labeling. GFAP and Iba-1 upregulation were not significantly altered by the INF treatment, displaying the same degree of enhanced reactivity as compared to the placebo group. The ultrastructural analysis showed that, apart from the overall reduction of inputs in the neuropil, no statistical differences were present when comparing the INF and placebo treated animals. Also, neuronal survival was not altered by cytokine administration. The present results provide evidence that MHC class I upregulation after neonatal injury does not change the fate of lesioned motoneurons. In this way, the lack of neurotrophic support may cause broader synaptic loss, which superposes the more subtle effects of the upregulation of MHC class I.

New 3D hyaluronan-based scaffold for in vitro reconstruction of the rat sciatic nerve

Objective: For peripheral nerve regeneration, three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance. The purpose of this study was to test in vitro a novel hyaluronic acid-based tubular conduit (HYAFF-11 biomaterials: 1 × 10 mm) as a nerve guide.Methods: Human fibroblasts, RN22 Schwann cell lines, human umbilical vein endothelial cells and primary nerve cells, obtained from neonatal rat sciatic nerve, were harvested and seeded on HYAFF-11 devices. Histologic (hematoxylin–eosin), immunohistochemical (antibodies to S100, CD31 and Von Willebrand factor) and PCR analyses were performed after 7 and 14 days from cell seeding onto biomaterials. MTT-based (thiazolyl blue) and DELFIA cell proliferation kit tests were performed to observe the biocompatibility of the cells cultured within the biomaterial devices.Results: We concluded that the conduits were not cytotoxic and demonstrated that cultured RN22 Schwann cells and rat Schwann cells grow in vitro on new artificial nerve conduits. We thus inferred that the HYAFF-11 conduit was a suitable biomaterial able to support nerve cell growth in vitro and after 14 days of cultivation, remained circular with a round lumen, maintaining the size and shape of its original architecture. Finally, attachment and proliferation of endothelial cells attested to the feasibility of developing a coculture system to promote in vivo integration of a microvascularized nerve substitute.Discussion: HYAFF-11 pre-seeded with Schwann and endothelial cells has the potential to be an alternative to autografting for the repair of long peripheral nerve defects.

Hsp27 overexpression in the R6/2 mouse model of Huntington's disease: chronic neurodegeneration does not induce Hsp27 activation

Huntington's disease (HD) is caused by an expanded polyglutamine tract in the huntingtin protein. Mitochondrial dysfunction and free radical damage occur in both R6/2 mice and HD patient brains and might play a role in disease pathogenesis. In cell culture systems, heat-shock protein 27 (Hsp27), a small molecular chaperone, suppresses mutant huntingtin-induced reactive oxygen species formation and cell death. To investigate this in vivo, we conducted an extensive phenotypic characterization of mice arising from a cross between R6/2 mice and Hsp27 transgenic mice but did not observe an improvement of the R6/2 phenotype. Hsp27 overexpression had no effect in reducing oxidative stress in the R6/2 brain, assessed by measuring striatal aconitase activity and protein carbonylation levels. Native protein gel analysis revealed that transgenic Hsp27 forms active, large oligomeric species in heat-shocked brain lysates, demonstrating that it is efficiently activated upon stress. In contrast, Hsp27 in double transgenic brains exists predominantly as a low molecular weight, inactive species. This suggests that Hsp27, which is otherwise activatable upon heat shock, remains inactive in the R6/2 model of chronic neurodegeneration. Hsp27 transgenics had been previously shown to be protected from acute stresses such as kainate administration, ischemia/reperfusion heart injury and neonatal nerve injury. Our study is the first to suggest a differential modulation of Hsp27 activation in vivo and, importantly, it illustrates the diverse effect of Hsp27 on acute versus chronic models of disease.

Optic nerve transection affects development and use-dependent plasticity in neocortex of the rat: Quantitative acetylcholinesterase imaging

We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.

The slow Wallerian degeneration gene in vivo protects motor axons but not their cell bodies after avulsion and neonatal axotomy

The slow Wallerian degeneration gene (Wld(S)) delays Wallerian degeneration and axon pathology for several weeks in mice and rats. Interestingly, neuronal cell death is also delayed in some in vivo models, most strikingly in the progressive motoneuronopathy mouse. Here, we tested the hypothesis that Wld(S) has a direct protective effect on motoneurone cell bodies in vivo. Cell death was induced in rat L4 motoneurones by intravertebral avulsion of the corresponding ventral roots. This simultaneously removed most of the motor axon, minimizing the possibility that the protective effect toward axons could rescue cell bodies secondarily. There was no significant difference between the survival of motoneurones in control and Wld(S) rats, suggesting that the Wld(S) gene has no direct protective effect on cell bodies. We also tested for any delay in apoptotic motoneurone death following neonatal nerve injury in Wld(S) rats and found that, unlike Wld(S) mice, Wld(S) rats show no delay in cell death. However, the corresponding distal axons were preserved, confirming that motoneurone cell bodies and motor axons die by different mechanisms. Thus, Wld(S) does not directly prevent death of motoneurone cell bodies. It follows that the protection of neuronal cell bodies observed in several disease and injury models where axons or significant axonal stumps remain is most probably secondary to axonal protection.

Reelin is transiently expressed in the peripheral nerve during development and is upregulated following nerve crush

Reelin is an extracellular matrix protein which is critical for the positioning of migrating post-mitotic neurons and the laminar organization of several brain structures during development. We investigated the expression and localization of Reelin in the rodent peripheral nerve during postnatal development and following crush injury in the adult stage. As shown with Western blotting, immunocytochemistry and RT-PCR, Schwann cells in the developing peripheral nerve and in primary cultures from neonatal nerves produce and secrete Reelin. While Reelin levels are downregulated in adult stages, they are again induced following sciatic nerve injury. A morphometric analysis of sciatic nerve sections of reeler mice suggests that Reelin is not essential for axonal ensheathment by Schwann cells, however, it influences the caliber of myelinated axons and the absolute number of fibers per unit area. This indicates that Reelin may play a role in peripheral nervous system development and repair by regulating Schwann cell–axon interactions.

Heat shock protein 27 rescues motor neurons following nerve injury and preserves muscle function

Heat shock proteins (HSPs) are a family of ubiquitously expressed proteins that are up-regulated in response to a range of stresses and play an important role in cellular defence mechanisms. In previous studies, we demonstrated that overexpression of heat shock protein 27 (HSP27) in transgenic mice has significant cytoprotective properties in vivo, reducing caspase-3-mediated cell death in the hippocampus associated with limbic seizures and reducing infarct size in cardiac ischaemia. In motor neurons, HSP27 is also implicated as a survival promoting factor; however, it remains to be established whether HSP27 is able to exert long-term neuroprotective effects following neonatal nerve injury. We now show that, following neonatal nerve crush, HSP27 overexpression in vivo provides a substantial rescue of motor neurons 5–6 months following nerve injury. Furthermore, in vivo isometric tension recordings demonstrate that surviving motor neurons were able to regenerate, resulting in a 90% improvement ( P < 0.0005) in motor unit number in HSP27 mice. Moreover, this increase in motor unit number was associated with improved muscle weight, muscle force, contractile speeds, and histochemical markers of muscle activity. These properties of HSP27 therefore have considerable potential for improving long-term muscle function in motor neuron disorders.

TNFα mediates Schwann cell death by upregulating p75NTR expression without sustained activation of NFκB

Administration of tumour necrosis factor alpha (TNFalpha) to axotomised mouse neonatal sciatic nerves increased Schwann cell apoptosis in the distal nerve segments, 5-fold greater than axotomy alone. TNFalpha upregulated the low affinity neurotrophin receptor, p75NTR, indicative of phenotype reversion in Schwann cells. Furthermore, re-expression of p75NTR and downregulation of the pro-myelinating transcription factor, Oct 6, in Schwann cells occurred by treatment with TNFalpha, even after the maturation of these cells with brain derived neurotrophic factor (BDNF). TNFalpha treatment of Schwann cells produced only a transient activation of NFkappaB. More importantly, in NFkappaB (p65) mutant mice, axotomy increased Schwann cell apoptosis further than that seen in mice expressing NFkappaB (p65), implicating a survival role for NFkappaB. Collectively, these data suggest that TNFalpha can potentiate Schwann cell death through the modulation of their phenotype. Immature Schwann cells express a high level of p75NTR and as a consequence are susceptible to extracellular death stimuli because of the lack of sustained NFkappaB translocation.

The influence of GDNF on the timecourse and extent of motoneuron loss in the cervical spinal cord after brachial plexus injury in the neonate

Injuries of the peripheral nerve in the early post-natal period are known to cause massive loss in the motoneuron pools of the spinal cord. However, the exact time frame and extent of motoneuron death in the cervical spinal cord after a brachial plexus lesion and the altered course after neuroprotection with different trophic factors is not known. In the present study, the time course of induced motoneuron death after a neonatal peripheral nerve injury and the effect of GDNF was investigated over a 4 week time period to determine the window of opportunity for possible therapeutic interventions in obstetrical plexus palsy.The brachial plexus of a total of 70 animals was explored within 12 hours after birth and divided at trunc level. The plexus was then labeled with a fluorescent tracer to identify the corresponding motoneuron pool. Two groups were prepared: Group I remained untreated to assess the natural course of induced neuronal death. Group II received GDNF immediately after the lesion. Post-operatively the animals were evaluated sequentially over 29 days. Surviving motoneurons were evaluated quantitatively counting the nucleoli. The entire brachial plexus of the rat is supplied by a total of about 4000 motoneurons. After injury the number of motoneurons steadily diminished within the first 10 days to reach a plateau of about 20% of the original number.At this time the GDNF treated group still had 85% (3330± 247) of motoneurons viable. This further decreased so that at the termination of the experiment at day 29 there were still 2527± 285 motoneurons alive. This study clearly shows that pathology after a brachial plexus injury in the newborn is not restricted to the peripheral nerve alone. In this model 64% of motoneurons underwent apoptosis within the first week after injury, reaching a plateau after 10 days at 20%. GDNF successfully rescued motoneurons so that after 4 weeks still 65% were present. We conclude that GDNF leads to enhanced motoneuron survival so that exogenous trophic support of motoneurons might havea role in the treatment of all types of severe neonatal plexopathies, maintaining the viability of motoneurons until reconstructive surgery provides them with a pathway for regeneration and endogenous trophic support.

BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury.

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system.

KCNQ2 is a nodal K+ channel.

Mutations in the gene encoding the K+ channel KCNQ2 cause neonatal epilepsy and myokymia, indicating that KCNQ2 regulates the excitability of CNS neurons and motor axons, respectively. We show here that KCNQ2 channels are functional components of axon initial segments and nodes of Ranvier, colocalizing with ankyrin-G and voltage-dependent Na+ channels throughout the CNS and PNS. Retigabine, which opens KCNQ channels, diminishes axonal excitability. Linopirdine, which blocks KCNQ channels, prolongs the repolarization of the action potential in neonatal nerves. The clustering of KCNQ2 at nodes and initial segments lags that of ankyrin-G during development, and both ankyrin-G and KCNQ2 can be coimmunoprecipitated in the brain. KCNQ3 is also a component of some initial segments and nodes in the brain. The diminished activity of mutant KCNQ2 channels accounts for neonatal epilepsy and myokymia; the cellular locus of these effects may be axonal initial segments and nodes.

Evidence that perinatal and adult NG2-glia are not conventional oligodendrocyte progenitors and do not depend on axons for their survival

In the adult CNS, cells that express the NG2 chondroitin sulphate proteoglycan are considered oligodendrocyte progenitor cells (OPCs). However, adult NG2-glia are highly complex cells, suggesting they may be a mature glial cell type distinct from conventional OPCs. To test this possibility, we have determined the response of NG2-glia to axon loss in the neonatal rat optic nerve, which has been shown previously to result in an almost complete loss of OPCs. We show here that NG2-glia are not lost following enucleation of the neonatal or juvenile optic nerve and that at all ages NG2-glia become “reactive” in response to axon degeneration, in direct contrast to previous studies on OPCs. We provide evidence that NG2-glia with an adult morphological phenotype develop postnatally, at the same time as oligodendrocytes. In the neonatal nerve, we show that the loss of axons blocks the development of oligodendrocytes, although reactive NG2-glia are abundant and are able to generate oligodendrocytes when placed in culture. Conversely, axon transection did not result in the loss of mature oligodendrocytes, indicating that the dependence of oligodendrocytes on axon-derived survival factors decreases with differentiation. This study shows that NG2-glia are a highly reactive cell type and do not generate oligodendrocytes in vivo in the absence of appropriate axon-derived signals. This may underlie in part the failure of remyelination in chronic multiple sclerosis, despite the abundance of NG2+ expressing glial cells within demyelinated lesions. It is clear either that OPCs do not depend on axons for their survival, in total contradiction to previous studies, or that in vivo a population of NG2 expressing glia are not perinatal or adult OPCs in the accepted sense. We suggest that a population of NG2-glia diverge postnatally into a novel glial type that is functionally and behaviourally distinct from conventional OPCs.

Changes in the Expression of Parvalbumin Immunoreactivity in the Lumbar Spinal Cord of the Rat following Neonatal Nerve Injury

Nerve injury in newborn animals results in the loss of motoneurons and dorsal root ganglion neurons and long-term changes in reflex activation of surviving motoneurons. Parvalbumin has been previously shown to be found in large-diameter primary afferent axons and interneurons in the spinal cord, and was used here to study the changes in parvalbumin-immunoreactive appositions onto identified tibialis anterior/extensor digitorum longus (TA/EDL) motoneurons, during both normal development and following neonatal nerve injury in the rat spinal cord. During normal development, there was a decrease in the number of parvalbumin-immunoreactive appositions onto TA/EDL motoneurons. Thus, at postnatal day 7 (P7), there were 72.8 ± 17.5 (mean ± SD) appositions per motoneuron and by P14, it had decreased to 38.8 ± 13.2 (mean ± SD; p > 0.05). Following neonatal nerve injury at P2, there were fewer parvalbumin-positive afferent appositions close to the TA/EDL motoneurons than normal, so that at P7, there were 53.5 ± 17.1 (mean ± SD), and at P14, it further decreased to 25.8 ± 8.6 (mean ± SD; p > 0.05). This injury-induced reduction in the number of parvalbumin-immunoreactive boutons apposing TA/EDL motoneurons may result, at least in part, from the death of dorsal root ganglion cells with the consequent loss of their central projections. The alterations in the number of parvalbumin-positive appositions close to motoneurons observed in this study may contribute to the changes in the pattern of reflex activity observed in the developing spinal cord both during normal development and following neonatal injury.

Neurotrophic factors enhance the survival of muscle fibers in EDL, but not SOL, after neonatal nerve injury.

Neonatal sciatic nerve crush results in a sustained reduction of the mass of both extensor digitorum longus (EDL) and soleus (SOL) muscles in the rat. Type IIB fibers are selectively lost from EDL. We have investigated the effects of ciliary neurotrophic factor (CNTF) combined with neurotrophin (NT)-3 or NT-4 on muscle mass, as well as the number, cross-sectional area, and distribution of muscle fiber types and the number of motor neurons innervating EDL and SOL 3 mo after transient axotomy 5 days after birth. Both NT treatments prevented the axotomy-induced loss of muscle mass in both EDL and SOL and of total number of muscle fibers in EDL but not in SOL. Although IIB fiber loss was not prevented, both NT treatments resulted in altered fiber type distribution. Both NT combinations also reduced the loss of EDL motor neurons. These data suggest that a differential distribution of NT receptors on either motor neurons or muscle fibers may lead to different levels of susceptibility to neonatal axotomy.

The Effect of Neonatal Nerve Injury on the Expression of Heat Shock Proteins in Developing Rat Motoneurones

The expression of the heat shock proteins hsp27 and hsp70 was examined in the spinal cord and sciatic nerves of developing rats. Using immunohistochemistry, we found that hsp27 is present in many motoneurones at birth. With development, the intensity of staining increases, reaching adult levels by 21 days, when all sciatic motoneurones express hsp27. In the sciatic nerve, hsp27 is strongly expressed throughout postnatal development. In contrast, hsp70 immunoreactivity in motoneurones and the sciatic nerve is weak at birth and does not change with development. The expression of heat shock proteins has been shown to increase in cells under conditions of stress, where they have beneficial effects on cell survival. The effect of neonatal nerve injury on hsp27 and hsp70 expression was also examined in this study. Four days after injury, staining for hsp27 increases in motoneurones, whereas hsp70 does not change. However, there is a significant increase in hsp70 staining in glial cells surrounding the injured motor pool, predominantly in astrocytes. Since neonatal nerve injury induces apoptotic motoneurone death, we also studied the co-expression of hsp27 with markers of apoptosis. No hsp27-positive motoneurones were found to be apoptotic, as assessed by both TUNEL and caspase-3 immunoreactivity. Therefore, it is possible that the upregulation of hsp27 observed in injured motoneurones may play a role in protecting motoneurones from apoptotic cell death following nerve injury.

Treatment of the neuromuscular junction with 4-aminopyridine results in improved reinnervation following nerve injury in neonatal rats.

During early postnatal development, nerve injury results in the death of a large proportion of motoneurones and poor recovery of muscle function. Our previous results have shown that premature enhancement of transmitter release from nerve terminals prevents the death of motoneurones following neonatal nerve injury. Whether this increase in motoneurone survival is reflected in an improvement in the reinnervation of muscle was studied here. The muscles in one hindlimb of newborn rats were treated with 4-aminopyridine. Three days later, the sciatic nerve was crushed in the treated leg. When the animals were seven, 14 and 21days of age, the soleus and extensor digitorum longus muscles were removed and processed for GAP-43 (a 43-kDa growth-associated protein) and synaptophysin immunocytochemistry. Both GAP-43 and synaptophysin were expressed in normal soleus and extensor digitorum longus muscles at seven days. Synaptophysin was still expressed at 14 days, but GAP-43 expression had declined. Following nerve injury at three days of age, there was no GAP-43 or synaptophysin immunoreactivity in nerve terminals at seven days. By 21 days, there were 17.3+/-2.1 GAP-43-positive terminals per section in the soleus and 17.7+/-1.4 in the extensor digitorum longus, with mean terminal areas of 47.5+/-3.3 and 49.8+/-2.6 microm(2), respectively. In animals in which nerve crush was preceded by 4-aminopyridine treatment, at 21 days there were 32.9+/-2.6 GAP-43-immunoreactive terminals in the soleus and 44.9+/-2.3 in the extensor digitorum longus, with a mean area of 122.7+/-6.6 microm(2) in the soleus and 136.2+/-9.7 microm(2) in the extensor digitorum longus. These results indicate that in muscles pretreated with 4-aminopyridine, prior to nerve crush at three days, there are significantly more terminals, which occupy a larger area than in untreated muscles. Thus, increasing transmitter release prior to nerve injury significantly improved the ability of axons to reinnervate muscle.

Prevention of galanin upregulation following neonatal infraorbital nerve transection or attenuation of axoplasmic transport does not rescue central vibrissae-related patterns in the rat

Neonatal transection of, or blockade of axoplasmic transport in, the infraorbital nerve [ION, the trigeminal (V) branch that supplies the mystacial vibrissae follicles] results in a loss of all central patterns corresponding to the vibrissae follicles in the brainstem, thalamus and cortex except for those of the central terminal arbors of ION primary afferents that survive this lesion. Both of these manipulations also result in a rapid and dramatic upregulation of at least two peptides, galanin and neuropeptide Y, in surviving vibrissae-related primary afferents. Galanin is of particular interest, because this peptide has effects on neuronal activity and growth, both factors which may be involved in the disappearance of central vibrissae-related patterns in rats that have sustained neonatal ION transection or axoplasmic transport blockade. The present study used antisense technology to determine whether the upregulation of galanin in the central terminals of ION primary afferents is necessary for the loss of central vibrissae-related patterns in rats. Newborn rats had their left ION transected or axoplasmic transport in this nerve blocked by application of a vinblastine-impregnated implant, and at the same time received an injection of commercially synthesized phosphorothioate oligodeoxynucleotide sequences (15-20 bases) directly into the V ganglion in order to block galanin upregulation. These injections effectively prevented the upregulation of this peptide which is normally associated with ION transection or axoplasmic transport blockade. Preventing galanin upregulation, however, did not prevent or attenuate the loss of central vibrissae-related patterns in the brainstem or cortex normally observed following ION transection or axoplasmic transport blockade in this nerve. These results are thus consistent with the conclusion that the upregulation of galanin in the central terminals of V primary afferents, observed after damage to or attenuation of axoplasmic transport in the ION, is not necessary for the reorganization that results in a disappearance of central vibrissae-related patterns in the V neuraxis.

Prevention of galanin upregulation following neonatal infraorbital nerve transection or attenuation of axoplasmic transport does not rescue central vibrissae-related patterns in the rat

Prevention of galanin upregulation following neonatal infraorbital nerve transection or attenuation of axoplasmic transport does not rescue central vibrissae-related patterns in the rat