脑源性神经生长因子与脊髓全横断大鼠的神经可塑性

The present study employed a rat model of T10 spinal cord transection.Western blot analyses revealed increased brain-derived neurotrophic factor(BDNF) expression in spinal cord segments caudal to the transection site following injection of replication incompetent herpes simplex virus vector(HSV-BDNF) into the subarachnoid space.In addition,hindlimb locomotor functions were improved.In contrast,BDNF levels decreased following treatment with replication defective herpes simplex virus vector construct small interference BDNF(HSV-siBDNF).Moreover,hindlimb locomotor functions gradually worsened.Compared with the replication incompetent herpes simplex virus vector control group,extracellular signal regulated kinase1/2 expression increased in the HSV-BDNF group on days 14 and 28 after spinal cord transection,but expression was reduced in the HSV-siBDNF group.These results suggested that BDNF plays an important role in neural plasticity via extracellular signal regulated kinase1/2 signaling pathway in a rat model of adult spinal cord transection.     

Contrasting neuropathology and functional recovery after spinal cord injury in developing and adult rats

Conflicting findings exist regarding the link between functional recovery and the regrowth of spinal tracts across the lesion leading to the restoration of functional contacts. In the present study, we investigated whether functional locomotor recovery was attributable to anatomical regeneration at postnatal day 1 (PN1), PN7, PN14 and in adult rats two months after transection injury at the tenth thoracic segment of the spinal cord. The Basso, Beattie, and Bresnahan scores showed that transection led to a failure of hindlimb locomotor function in PN14 and adult rats. However, PN1 and PN7 rats showed a significant level of stepping function after complete spinal cord transection. Unexpectedly, unlike the transected PN14 and adult rats in which the spinal cord underwent limited secondary degeneration and showed a scar at the lesion site, the rats transected at PN1 and PN7 showed massive secondary degeneration both anterograde and retrograde, leaving a >5-mm gap between the two stumps. Furthermore, retrograde tracing with fluorogold (FG) also showed that FG did not cross the transection site in PN1 and PN7 rats as in PN14 and adult rats, and re-transection of the cord caused no apparent loss in locomotor performance in the rats transected at PN1. Thus, these three lines of evidence strongly indicated that the functional recovery after transection in neonatal rats is independent of regrowth of spinal tracts across the lesion site. Our results support the notion that the recovery of locomotor function in developing rats may be due to intrinsic adaptations in the spinal circuitry below the lesion that control hindlimb locomotor activity rather than the regrowth of spinal tracts across the lesion. The difference in secondary degeneration between neonatal and adult rats remains to be explored.     

Effects of exercise and fetal spinal cord implants on the H-reflex in chronically spinalized adult rats

This study investigated the modulation of hindlimb reflex excitability after transection of the spinal cord in adult rats. After transection, the H-reflex exhibited decreased depression at high stimulation frequencies compared to intact animals. Groups of animals which received a spinal cord transection followed by either an exercise regimen for the hindlimbs or a fetal spinal cord implant, showed high stimulation frequency depression similar to controls. This suggests that each of these palliative strategies helped to ‘normalize’ the excitability of specific spinal reflexes.     

Functional repair of transected spinal cord in embryonic chick

The purpose of this study was to determine the developmental stage of the chick embryo when descending spinal tracts lose the capacity for anatomical and functional repair after complete transection of the thoracic spinal cord. Previous studies have demonstrated that the first reticulospinal projections descend to the lumbar cord by embryonic day (E) 5. A comparison of the distribution and density of retrogradely labelled brainstem-spinal neurons in embryos versus hatchling chicks suggests that the descent of all brainstem-spinal projections is essentially complete to lumbar levels between E10 and El2. Transections and control sham operations were performed on different embryos from E3 through E14 of development. After a recovery period of 5-18 days, the extent of anatomical repair was assessed by injecting a small volume of a retrograde tract-tracing chemical into the upper lumbar spinal cord, caudal to the transection site. The brainstem nuclei were then examined for the number and distribution of retrogradely labelled brainstem-spinal neurons. In comparison to control animals, anatomical recovery appeared to be complete for embryos transected as late as E12, whereas thoracic cord transections conducted on E13-E14 resulted in reduced labelling of most brainstem-spinal nuclei. In addition, a number of E3-E6 transected embryos were allowed to hatch and with some assistance a few E7-E14 transected embryos also hatched. Functional recovery was assessed by behavioral observations and by focal electrical stimulation of brainstem locomotor regions (known to have direct projections to the lumbar spinal cord). Brainstem stimulation experiments were undertaken on transected and control embryos, either in ovo on E18-E20 or after hatching. Leg and wing muscle electromyographic recordings were used to monitor any brainstem evoked motor activity. Voluntary open-field locomotion (hatchling chicks) or brainstem evoked locomotion (embryonic or hatchling) in animals transected on or before E12 was indistinguishable from that observed in control (i.e. sham-operated or unoperated) chicks, indicating that complete functional recovery had occurred. In contrast, chicks transected on or after El3 showed reduced functional recovery. Since a previous study has shown that neurogenesis in chick brainstem-spinal neurons is complete prior to E5, the possible intrinsic neuronal mechanisms underlying the repair of descending supraspinal pathways are: (1) subsequent projections from later developing (undamaged) neurons, or (2) regrowth of previously axotomized projections (regeneration). For the E5-E12 chick embryos examined in this study, significant descending supraspinal fibers are present within the thoracic cord at the time of transection. Even if the transection is made at E12, when descending projections have completed their development to the lumbar cord, there is still a similar number and distribution of brainstem-spinal neurons labelled afterward (when compared to controls). This suggests that regeneration of previously axotomized projections may account for some of the observed anatomical and functional repair of brainstem-spinal pathways.     

Recovery of bimodal locomotion in the spinal-transected salamander, Pleurodeles waltlii

Electromyographic (EMG) analysis was used to provide an assessment of the recovery of locomotion in spinal-transected adult salamanders (Pleurodeles waltlii). EMG recordings were performed during swimming and overground stepping in the same animal before and at various times (up to 500 days) after a mid-trunk spinalization. Two-three weeks after spinalization, locomotor EMG activity was limited to the forelimbs and the body rostral to the transection. Thereafter, there was a return of the locomotor EMG activity at progressively more caudal levels below the transection. The animals reached stable locomotor patterns 3-4 months post-transection. Several locomotor parameters (cycle duration, burst duration, burst proportion, intersegmental phase lag, interlimb coupling) measured at various recovery times after spinalization were compared with those in intact animals. These comparisons revealed transient and long-term alterations in the locomotor parameters both above and below the transection site. These alterations were much more pronounced for swimming than for stepping and revealed differences in adaptive plasticity between the two locomotor networks. Recovered locomotor activity was immediately abolished by retransection at the site of the original spinalization, suggesting that the spinal cord caudal to the transection was reinnervated by descending brain and/or propriospinal axons, and that this regeneration contributed to the restoration of locomotor activity. Anatomical studies conducted in parallel further demonstrated that some of the regenerated axons came from glutamatergic and serotoninergic immunoreactive cells within the reticular formation.     

Motor recovery and anatomical evidence of axonal regrowth in spinal cord-repaired adult rats

Behavioral assessments of hindlimb motor recovery and anatomical assessments of extended axons of long spinal tracts were conducted in adult rats following complete spinal cord transection. Rats were randomly divided into 3 groups: 1) sham control group (laminectomy only; n = 12); 2) transection-only group, spinal cord transection at T8 (n = 20); and 3) experimental treatment group, spinal cord transection at T8, with peripheral nerve grafts (PNG) and application of acidic fibroblast growth factor (aFGF) (n = 14). The locomotor behavior and stepping of all rats were analyzed over a 6-month survival time using the Basso, Beattie, Bresnahan (BBB) open field locomotor test and the contact placing test. Immunohistochemistry for serotonin (5-HT), anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluoro-gold were used to evaluate the presence of axons below the damage site following treatment. When compared with the transection-only group, the nerve graft with the aFGF group showed 1) significant improvement in hindlimb locomotion and stepping, 2) the presence of 5-HT-labeled axons below the lesion site at lumbar cord level (these were interpreted as regenerated axons from the raphe nuclei), 3) the presence of anterograde BDA labeling of corticospinal tract axons at the graft site and below, and 4) fluoro-gold retrograde labeling of neuron populations in motor cortex and in red nucleus, reticulospinal nuclei, raphe nuclei, and vestibular nuclei. We conclude that peripheral nerve grafts and aFGF treatments facilitate the regrowth of the spinal axons and improve hindlimb function in a T-8 spinal cord-transected rat model.     

Serotonergic systems in the spinal cord of the amphibian urodele Pleurodeles waltl

The role of the monoamine serotonin (5-HT) in modulating the neural networks underlying axial locomotor movements was studied in an adult amphibian urodele, Pleurodeles waltl. 5-HT was applied to an in vitro brainstem-spinal cord preparation of P. waltl, which displayed fictive axial locomotor patterns following bath application of N-methyl-D-aspartate (5 microM) with D-serine (10 microM). Our results showed that 5-HT (1-25 microM) produces a reversible increase in the cycle duration and the duration of rhythmic bursting activity recorded extracellularly from ventral roots innervating the axial musculature. When applied alone, 5-HT does not trigger axial locomotor activity. The distribution pattern of 5-HT immunoreactive (5-HT-ir) cells along the spinal cord was investigated both in intact and in chronic spinal animals. The number of 5-HT-ir cell bodies is higher at brachial levels and decreases through crural levels. Sparse oval or fusiform 5-HT-ir somata are present within the gray matter, just ventrolateral to the central canal. Longitudinal fibers were detected throughout the entire white matter, except in the medial part of the dorsal funiculi. Two columns of intensely labeled and profusely branching thick and thin fibers associated with numerous varicosities run continuously along the ventrolateral surface of the spinal cord. Three weeks following full spinal cord transection at the level of the second spinal root, all longitudinal processes had disappeared, indicating their supraspinal origin, whereas the ventrolateral plexes remained, suggesting that they originated from intraspinal 5-HT-ir cell bodies. Our data showing that spinal 5-HT is organized according to a rostrocaudal gradient suggest that the 5-HT systems of P. waltl are not related to the presence of limb motor pools but more likely are related to axial central pattern generators (CPGs) networks down the length of the spinal cord. The possible involvement of these two sources (descending vs. intraspinal) of 5-HT innervation in the modulation of the axial CPGs is discussed.     

Different effects of spinalization and locomotor training of spinal animals on cholinergic innervation of the soleus and tibialis anterior motoneurons

Cholinergic input modulates excitability of motoneurons and plays an important role in the control of locomotion in both intact and spinalized animals. However, spinal cord transection in adult rats affects cholinergic innervation of only some hindlimb motoneurons, suggesting that specificity of this response is related to functional differences between motoneurons. Our aim was therefore to compare cholinergic input to motoneurons innervating the soleus (Sol) and tibialis anterior (TA) motoneurons following spinal cord transection at a low-thoracic level. The second aim was to investigate whether deficits in cholinergic input to these motoneurons could be modified by locomotor training. The Sol and TA motoneurons were identified by retrograde labelling with fluorescent dyes injected intramuscularly. Cholinergic terminals were detected using anti-vesicular acetylcholine transporter (VAChT) antibody. Overall innervation of motoneurons was evaluated with anti-synaptophysin antibody. After spinalization we found a decrease in the number of VAChT-positive boutons apposing perikarya of the Sol (to 49%) but not TA motoneurons. Locomotor training, resulting in moderate functional improvement, partly reduced the deficit in cholinergic innervation of Sol motoneurons by increasing the number of VAChT-positive boutons. However, the optical density of VAChT-positive boutons terminating on various motoneurons, which decreased after spinalization, continued to decrease despite the training, suggesting an impairment of acetylcholine availability in the terminals. Different effects of spinal cord transection on cholinergic innervation of motoneurons controlling the ankle extensor and flexor muscles point to different functional states of these muscles in paraplegia as a possible source of activity-dependent signaling regulating cholinergic input to the motoneurons.     

Regeneration of adult rat spinal cord is promoted by the soluble KDI domain of gamma1 laminin

Regeneration in the central nervous system (CNS) of adult mammals is hampered by formation of a glial scar and by proteins released from the myelin sheaths of injured neuronal pathways. Our recent data indicate that the KDI (Lys-Asp-Ile) domain of gamma1 laminin neutralizes both glial- and myelin-derived inhibitory signals and promotes survival and neurite outgrowth of cultured human spinal cord neurons. We show that after complete transection of the adult rat spinal cord, animals receiving onsite infusion of the KDI domain via osmotic mini-pumps recover and are able to sustain their body weights and walk with their hindlimbs. Animals treated with placebo suffer from irreversible hindlimb paralysis. Microscopic and molecular analyses of the spinal cords indicate that the KDI domain reduces tissue damage at the lesion site and enables neurite outgrowth through the injured area to effect functional recovery of the initially paralyzed animals. That the KDI domain enhances regeneration of acute spinal cord injuries in the adult rat suggests that it may be used to promote regeneration of spinal cord injuries in humans.     

Modulation of locomotion by a nerve graft across a spinal transection in rat

Adult rats received a complete mid-lower thoracic spinal cord transection and a peripheral nerve autograft was inserted across the transection site. Testing 3-4 months later showed that, after decerebration, stimulation of the mesencephalic locomotor region (MLR) induced forelimb but not hindlimb locomotion. However, in 5/7 animals, tail pinch interrupted MLR stimulation-induced forelimb stepping, while pinna pinch induced hindlimb muscle twitch. These effects were not present following complete section of the nerve graft or in 6 control animals which did not receive a graft. Exposure of the cut mid-portion of the grafts to DiI revealed the presence of labeled axons entering the spinal cord through both ends of the graft in those animals which showed the above effects. There was no transport in the 2 cases in which tail pinch interruption of MLR-induced stepping or pinna pinch-induced hindlimb muscle twitch did not occur. We conclude that non-specific information which can modulate locomotion may be flowing through the graft.     

Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats

We compared the effect of viral administration of brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT-3) on locomotor recovery in adult rats with complete thoracic (T10) spinal cord transection injuries, in order to determine the effect of chronic neurotrophin expression on spinal plasticity. At the time of injury, BDNF, NT-3 or green fluorescent protein (GFP) (control) was delivered to the lesion via adeno-associated virus (AAV) constructs. AAV-BDNF was significantly more effective than AAV-NT-3 in eliciting locomotion. In fact, AAV-BDNF-treated rats displayed plantar, weight-supported hindlimb stepping on a stationary platform, that is, without the assistance of a moving treadmill and without step training. Rats receiving AAV-NT-3 or AAV-GFP were incapable of hindlimb stepping during this task, despite provision of balance support. AAV-NT-3 treatment did promote the recovery of treadmill-assisted stepping, but this required continuous perineal stimulation. In addition, AAV-BDNF-treated rats were sensitized to noxious heat, whereas AAV-NT-3-treated and AAV-GFP-treated rats were not. Notably, AAV-BDNF-treated rats also developed hindlimb spasticity, detracting from its potential clinical applicability via the current viral delivery method. Intracellular recording from triceps surae motoneurons revealed that AAV-BDNF significantly reduced motoneuron rheobase, suggesting that AAV-BDNF promoted the recovery of over-ground stepping by enhancing neuronal excitability. Elevated nuclear c-Fos expression in interneurons located in the L2 intermediate zone after AAV-BDNF treatment indicated increased activation of interneurons in the vicinity of the locomotor central pattern generator. AAV-NT-3 treatment reduced motoneuron excitability, with little change in c-Fos expression. These results support the potential for BDNF delivery at the lesion site to reorganize locomotor circuits.     

Hindlimb loading determines stepping quantity and quality following spinal cord transection

We compared the bipedal hindlimb stepping ability of untrained and trained (step-trained 6 min/day) spinal rats (mid-thoracic spinal cord transection at post-natal day 5) at different levels of body weight support on a treadmill over a 40-day period, starting at 69 days of age. A robotic device provided precise levels of body weight support and recorded hindlimb movement. We assessed stepping ability using: (1) step quantity determined from the measured hindlimb movement, (2) ordinal scales of paw placement, weight-bearing, and limb flexion, and (3) the lowest level of body weight support at which stepping was maintained. Stepping quantity and quality depended strongly on the level of support provided. Stepping ability improved with time, but only at the higher levels of weight-bearing, and independently of training. Increasing limb loading by gradually decreasing body weight support altered the spatiotemporal properties of the steps, resulting in an increase in step length and stance duration and a decrease in swing and step cycle duration. The rats progressively improved their ability to support more load before collapsing from a maximum of about 42 g ( approximately 25% of body weight) at Day 1 to 73 g ( approximately 35% of body weight) at Day 40. We conclude that the level of hindlimb loading provided to a spinally transected rat strongly influences the quantity and quality of stepping. Furthermore, the relationship between stepping ability and loading conditions changes with time after spinal cord transection and is unaltered by small amounts of step training. Finally, load-bearing failure point can be a quantitative measure of locomotor recovery following spinal cord injury, especially for severely impaired animals that cannot step unassisted.     

Do propriospinal projections contribute to hindlimb recovery when all long tracts are cut in neonatal or weanling rats?

Lateral hemisection lesions separated by 1 to 3 spinal segments were made on opposite sides of the mithoracic spinal cord in 1-month-old (N = 15; weanling operates) and newborn albino rats (N = 16; neonatal operates). Hindlimb behavior was assessed between 1 and 6 months p.o. for both groups of operates using a protocol and rating system that have previously proved effective in differentiating behavioral recovery of the hindlimbs as a function of age of spinal transection. In addition, at the conclusion of behavioral testing, operates received spinal injections of [3H]proline and HRP caudal to the spinal lesions to determine if lesions were complete and if neurons within the region between the two lesions (interlesion zone) projected into the caudal spinal cord. In both groups of operates, neurons were retrogradely labeled within the interlesion zone bilaterally, primarily in laminae VII-VIII. When both lesions were complete lateral hemisections in weanling operates, little behavioral recovery was observed, similar to complete spinal cord transection (N = 3). However, much greater behavioral recovery was seen, including supporting reactions and locomotor responses, when one or both lesions spared axons along the ventrolateral rim of the white matter. Neurons were retrogradely labeled in the brain stem reticular formation (N = 12) in these cases. All lesions were complete lateral hemisections in neonatal operates but much greater behavioral recovery was seen than in weanling operates with the same lesions, including supporting, placing, and locomotor responses. In an additional group of eight neonatal operates, the spinal cord rostral to the spinal hemisections was transected at 1 month of age. Supportive, placing, and locomotor responses were seen immediately after recovery from anesthesia and responses returned to pretransection levels in six of eight operates over the 10-day survival period. Fink-Heimer impregnation showed that degeneration argyrophilia from the transection bilaterally filled the interlesion zone but little argyrophilia was seen caudal to this region. Our results indicate that an intact propriospinal circuit remains in both neonatal and weanling operates but does not appear to contribute to hindlimb response development or recovery. The greater behavioral recovery in neonatal operates appears due to intrinsic connections (doral root, interneuronal) continuing to be able to drive the spinal circuitry underlying the spared behaviors.     

Differences in the regenerative response of neuronal cell populations and indications for plasticity in intraspinal neurons after spinal cord transection in adult zebrafish

In zebrafish, the capacity to regenerate long axons varies among different populations of axotomized neurons after spinal cord transection. In specific brain nuclei, 84-92% of axotomized neurons upregulate expression of the growth-related genes GAP-43 and L1.1 and 32-51% of these neurons regrow their descending axons. In contrast, 16-31% of spinal neurons with axons ascending to the brainstem upregulate these genes and only 2-4% regrow their axons. Dorsal root ganglion (DRG) neurons were not observed to regrow their ascending axons or to increase expression of GAP-43 mRNA. Expression of L1.1 mRNA is high in unlesioned and axotomized DRG neurons. In the lesioned spinal cord, expression of growth-related molecules is increased in a substantial population of non-axotomized neurons, suggesting morphological plasticity in the spinal-intrinsic circuitry. We propose that locomotor recovery in spinal-transected adult zebrafish is influenced less by recovery of ascending pathways, but more by regrowth of descending tracts and rearrangement of intraspinal circuitry.     

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.     

From cortex to cord: motor circuit plasticity after spinal cord injury

Spinal cord injury is associated with chronic sensorimotor deficits due to the interruption of ascending and descending tracts between the brain and spinal cord. Functional recovery after anatomically complete spinal cord injury is limited due to the lack of long-distance axonal regeneration of severed fibers in the adult central nervous system. Most spinal cord injuries in humans, however, are anatomically incomplete.Although restorative treatment options for spinal cord injury remain currently limited, research from experimental models of spinal cord injury have revealed a tremendous capability for both spontaneous and treatment-induced plasticity of the corticospinal system that supports functional recovery. We review recent advances in the understanding of corticospinal circuit plasticity after spinal cord injury and concentrate mainly on the hindlimb motor cortex, its corticospinal projections, and the role of spinal mechanisms that support locomotor recovery. First, we discuss plasticity that occurs at the level of motor cortex and the reorganization of cortical movement representations. Next, we explore downstream plasticity in corticospinal projections. We then review the role of spinal mechanisms in locomotor recovery. We conclude with a perspective on harnessing neuroplasticity with therapeutic interventions to promote functional recovery.     

The use of behavioral methods to predict spinal cord plasticity

The mechanisms underlying recovery of function following damage to the CNS, although suspected, are virtually unknown. After damage to the adult cat spinal cord, recovery of motor behavior depends on which systems have been interrupted and which remain intact. For example, following hemisection, overground (voluntary) and reflex locomotion recover and, although a normal kinematic pattern recovers, accurate placement of the limb during locomotion does not return to normal levels. This recovery is associated with lowering of thresholds for postural reflexes suggesting that increased afferent input may compensate for diminished descending control. In contrast, after unilateral loss of afferent input by lumbosacral deafferentation, (L1-S2 dorsal roots cut) overground locomotion recovers but a permanently abnormal kinematic pattern is used; reflex locomotion (bipedal locomotion on a treadmill) does not recover at all in the deafferented hindlimb. The specificity of the recovery suggests that increased input from descending pathways, which is required for overground but not reflex locomotion may compensate for loss of afferent input. Anatomical sequellae of these two lesion types have been examined. Studies after hemisection support the notion of a permanently increased dorsal root input as mapped by monoclonal antibody 'rat 102'. This is associated with a transient increase in GAP-43 labeling in the dorsal horn. In contrast, after deafferentation an increase is found in the descending serotonergic input to the deafferented side. These observations suggest that recovery of specific locomotor behavior can be used to predict compensatory changes in spared pathways. For the study of the effects of transplants, we have used complete spinal transections in newborn kittens with transplantation of E26 cat spinal cord into the transection site. The normal kitten develops overground locomotion beginning the end of the first week postnatal but reflex locomotion is delayed until the end of the second week. After transection on the first day of life, with or without a transplant, reflex locomotion begins precociously. Overground locomotion fails to develop in transection-only animals but does develop in animals with transection and transplant. This locomotion although clearly abnormal, shows postnatal development in terms of weight support and lateral stability. Furthermore, there is some indication of coordination between fore and hind limbs. These observations suggest that the transplants permit the development of some descending control although the anatomical correlates of this sparing/recovery of function are uncertain; the transplant rescues neurons caudal to the transection and also permits regeneration of some descending pathways into the transplant and caudally into host spinal cord.     

Differential expression of class 3 and 4 semaphorins and netrin in the lamprey spinal cord during regeneration

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.     

Lipocalin 2 plays an immunomodulatory role and has detrimental effects after spinal cord injury

Lipocalin 2 (Lcn2) plays an important role in defense against bacterial infection by interfering with bacterial iron acquisition. Although Lcn2 is expressed in a number of aseptic inflammatory conditions, its role in these conditions remains unclear. We examined the expression and role of Lcn2 after spinal cord injury (SCI) in adult mice by using a contusion injury model. Lcn2 expression at the protein level is rapidly increased 12-fold at 1 d after SCI and decreases gradually thereafter, being three times as high as control levels at 21 d after injury. Lcn2 expression is strongly induced after contusion injury in astrocytes, neurons, and neutrophils. The Lcn2 receptor (Lcn2R), which has been shown to influence cell survival, is also expressed after SCI in the same cell types. Lcn2-deficient (Lcn2?/?) mice showed significantly better locomotor recovery after spinal cord contusion injury than wild-type (Lcn2?/?) mice. Histological assessments indicate improved neuronal and tissue survival and greater sparing of myelin in Lcn2?/? mice after contusion injury. Flow cytometry showed a decrease in neutrophil influx and a small increase in the monocyte population in Lcn2?/? injured spinal cords. This change was accompanied by a reduction in the expression of several pro-inflammatory chemokines and cytokines as well as inducible nitric oxide synthase early after SCI in Lcn2?/? mice compared with wild-type animals. Our results, therefore, suggest a role for Lcn2 in regulating inflammation in the injured spinal cord and that lack of Lcn2 reduces secondary damage and improves locomotor recovery after spinal cord contusion injury.     

Observations on the development of brainstem-spinal systems in the north american opossum

The North American opossum is born 12 to 13 days after conception and is available for 90 days or more in an external pouch where it can be observed and experimentally manipulated. It is of particular interest that the hindlimbs of the newborn opossum are very immature and remain immobile for a week or more after birth. Degeneration techniques reveal that immature brainstem axons are present within the marginal zone of the lumbosacral cord before hindlimb movements begin (our stage I) and material processed for formaldehyde induced fluorescence shows that some of them transport monoamines. Several lines of evidence suggest that part of the fluorescent axons arise within the nucleus locus coeruleus. At this early stage the electron microscope reveals that all brainstem-spinal axons are small (0.1-0.4 μm in diameter) and unmyelinated. By the time random hindlimb movements can be observed (stage II), brainstem axons, including those transporting monoamines, can be demonstrated to have grown into limited areas of the intermediate zone of the lumbosacral cord and to arise from most of the areas contributing to them in the adult animal (horseradish peroxidase technique). Such axons are still immature and it is not yet clear that they have formed synaptic terminals. Brainstem axons continue to grow into the intermediate zone of the lumbosacral cord for some time and come to occupy all of their adult territories before thoracic transection produces obvious change in hindlimb motility (beginning of stage III). It is still another 20 days or so before thoracic transection produces spinal shock comparable to that in the adult animal. The relatively mature use of the hindlimbs and the full expression of spinal shock correlate with changes in the technique and survival time needed to demonstrate degenerating brainstem axons in experimental material.