Sprouting and regeneration after pyramidotomy and blockade of the myelin-associated neurite growth inhibitors NI 35/250 in adult rats

After a selective unilateral lesion of the corticospinal tract (CST) at the level of the brainstem (pyramidotomy) and neutralization of the myelin associated neurite growth inhibitors NI-35/250 with the monoclonal antibody (mAb) IN-1, we had previously observed a strong behavioural recovery in parallel with an enhanced structural plasticity of the lesioned as well as the unlesioned CST. The present study focuses on the regenerative response of the cut CST axons at the lesion site in these adult rats. The results show a strong enhancement of regenerative sprouting of CST fibres by treatment with the mAb IN-1. Successful elongation of these sprouts through the pyramidal decussation and into the cervical spinal cord was also dependent on the presence of this antibody. In the spinal cord, regenerating fibres were rarely found in the position of the former CST; most of the fibres were distributed seemingly randomly over the entire lateral extent of the spinal cord.     

Regenerating corticospinal fibers in the Marmoset (Callitrix jacchus) after spinal cord lesion and treatment with the anti-Nogo-A antibody IN-1

Neutralizing the myelin-associated growth inhibitor Nogo-A in adult spinal cord-injured rats can promote regeneration of injured and compensatory sprouting of uninjured axons. Nogo-A is present in humans, making its neutralization a possible novel treatment option for paraplegic patients. In this study we examined the effects of an extensively used anti-Nogo-A antibody (mAb IN-1) on the regenerative capabilities of lesioned corticospinal tract (CST) axons in a primate, the Marmoset monkey. Unilateral thoracic lesions of the CST were performed in six adult Marmosets, followed by the application of mAb IN-1 into the cerebrospinal fluid circulation by a graft of hybridoma cells. A unilateral injection of biotin dextran amine into the motor cortex was performed to analyse sprouting and regeneration of the lesioned axons. In the control antibody-treated animal CST fibers stopped rostral to the lesion site and often showed retraction bulbs. In contrast, in four out of five mAb IN-1-treated animals fine labeled neurites had grown into, through and around the lesion site. Thus, this study provides first anatomical evidence that in primates, the neutralization of the myelin-associated inhibitor Nogo-A results in increased regenerative sprouting and growth of lesioned spinal cord axons.     

Reorganization of descending motor tracts in the rat spinal cord

Following lesion of the central nervous system (CNS), reinnervation of denervated areas may occur via two distinct processes: regeneration of the lesioned fibres or/and sprouting from adjacent intact fibres into the deafferented zone. Both regeneration and axonal sprouting are very limited in the fully mature CNS of higher vertebrates, but can be enhanced by neutralizing the neurite outgrowth inhibitory protein Nogo-A. This study takes advantage of the distinct spinal projection pattern of two descending tracts, the corticospinal tract (CST) and the rubrospinal tract (RST), to investigate if re-innervation of denervated targets can occur by sprouting of anatomically separate, undamaged tracts in the adult rat spinal cord. The CST was transected bilaterally at its entry into the pyramidal decussation. Anatomical studies of the RST in IN-1 antibody-treated rats showed a reorganization of the RST projection pattern after neutralization of the myelin associated neurite growth inhibitor Nogo-A. The terminal arborizations of the rubrospinal fibres, which are normally restricted to the intermediate layers of the spinal cord, invaded the ventral horn but not the dorsal horn of the cervical spinal cord. Moreover, new close appositions were observed, in the ventral horn, onto motoneurons normally receiving CST projections. Red nucleus microstimulation experiments confirmed the reorganization of the RST system. These observations indicate that mature descending motor tracts are capable of significant intraspinal reorganization following lesion and suggests the expression of cues guiding and/or stabilizing newly formed sprouts in the adult, denervated spinal cord.     

Regeneration and Sprouting of Chronically Injured Corticospinal Tract Fibers in Adult Rats Promoted by NT-3 and the mAb IN-1, Which Neutralizes Myelin-Associated Neurite Growth Inhibitors

Myelin-associated inhibitors of neurite growth play an important role in the regenerative failure after injury in the adult mammalian CNS. The application of the mAb IN-1, which efficiently neutralizes the NI-250/35 inhibitory proteins, alone or in combination with neurotrophin-3 (NT-3), has been shown to promote axonal regeneration when applied in acute injury models. To test whether IN-1 application can induce axonal growth also in a chronic injury model, we treated rats with IN-1 and NT-3 starting 2 or 8 weeks after injury. Rats underwent bilateral dorsal hemisection of the spinal cord at the age of 5–6 weeks. Regeneration of corticospinal (CST) fibers into the caudal spinal cord was observed in three of eight of those animals with a 2-week delay between lesion and treatment. CST fibers regenerated for 2–11.4 mm. In the control group sprouting occurred rostral to the lesion but no long-distance regeneration occurred. In animals where treatment started at 8 weeks after injury the longest fibers observed grew up to 2 mm into the caudal spinal cord. The results show that transected corticospinal axons retain the ability to regenerate at least for a few weeks after injury. Functional analysis of these animals showed a slight improvement of functional recovery.     

Improving axonal growth and functional recovery after experimental spinal cord injury by neutralizing myelin associated inhibitors

Injuries of the spinal cord often result in an irretrievable loss of motor and sensory functions of all body parts situated below the lesion site. Functional recovery is restricted mainly due to the limited regeneration and plasticity of injured axons in the adult central nervous system. Over the last few years different experimental approaches have led to axonal growth and functional benefits in animal models. This review focuses on the effects of the neutralization of myelin-associated neurite growth inhibitors, in particular Nogo-A, using the monoclonal antibody IN-1. Acute mAb IN-1 treatment of adult CNS lesioned rats results in extensive plastic changes of neuronal connections and regenerative fiber growth. In two different lesion paradigms (i.e. pyramidal tract lesion and incomplete spinal cord lesion in adult rats), the mAb IN-1-treated animals always showed a higher degree of recovery in various behavioral tests. These observations, together with electrophysiological results, suggest that neuronal CNS circuits of mAb IN-1-treated animals can be rearranged, and that sprouting and regenerating axons form functionally meaningful connections.     

Immune activation is required for NT-3-induced axonal plasticity in chronic spinal cord injury

After an unilateral lesion of the corticospinal tract (CST) at the level of the medulla over-expression of Neurotrophin-3 (NT-3) in lumbar spinal cord motoneurons induced axonal sprouting of the intact CST in the acutely injured but not uninjured or chronically injured spinal cord in rats. This suggested that processes associated with immune-mediated wound healing may act with NT-3 to induce neuroplasticity. To test whether immune processes were involved we measured NT-3-induced axonal sprouting in immunosuppressed compared to immunocompetent rats. Rats were immunosuppressed with anti-leukocyte antibodies 1 day before receiving a CST lesion and then 2 weeks later NT-3 was over-expressed in the lumbar spinal motoneurons with an adenoviral vector carrying the NT-3 gene targeted to the motoneurons by retrograde transport. At 35 days post-lesion no axonal sprouting was measured in immunosuppressed rats whereas axonal sprouting was measured in the immunocompetent rats. We then tested whether re-evoking an immune response in chronically lesioned rats would induce neuroplasticity. Rats received CST lesions and then 4 months later were treated with systemic injections of lipopolysaccharide (LPS) 7 days before NT-3 was over-expressed in the lumbar spinal motoneurons. Axonal sprouting was observed in the LPS treated rats but not in control animals that were not treated with LPS. Further studies showed that lesioning the CST activated and LPS reactivated microglia and CD4(+) T-cells in the acutely lesioned and chronically lesioned rats, respectively. However, immunosuppression only decreased the number of activated CD4(+) T-cells suggesting they were responsible for the support of axonal growth. These observations demonstrate that processes associated with immune-mediated wound healing play a role in NT-3-induced neuroplasticity after injury.     

Myelin-associated inhibitors of neurite growth and regeneration in the CNS.

Axons often respond to lesions by spontaneous sprouting which, in the PNS, can be followed by elongation over long distances. In contrast, in the CNS, regenerative axon growth in most fibre systems subsides after 0.5-1.0 mm. The observation that an identical situation can be found in tissue culture in the presence of trophic factors argued for the existence of inhibitory mechanisms within the CNS tissue. Detailed cell biological and biochemical studies have provided evidence for two membrane proteins localized selectively in oligodendrocytes and CNS myelin and which exert a powerful inhibitory effect on neurite growth. Antibodies raised against these neurite growth inhibitors (NI-35 and NI-250) and applied to rats with complete transections of the corticospinal tract (CST) resulted in CST axon regeneration over five to ten mm from the lesion site within two to three weeks. Analogous results were obtained in rats lacking myelin and oligodendrocytes in the spinal cord. During development, the 'fuzzy' appearance of the CST grown in the absence of oligodendrocytes or in the presence of anti-inhibitor antibodies indicates a boundary and guidance function of these inhibitors for late growing CNS tracts.     

Increased lesion‐induced sprouting of corticospinal fibres in the myelin‐free rat spinal cord

Myelin contains potent inhibitors of neurite growth which have been implicated in the failure of long-distance regeneration of nerve fibres within the CNS. These myelin-associated neurite growth inhibitors may also be involved in the stabilization of neural connections by suppressing sprouting and fibre growth. After lesions of the CNS in neonatal animals, extensive rearrangements of the remaining fibre systems have been observed. In the rat, this plasticity of neuronal connections is severely restricted following the first few weeks of postnatal life, coincident with the progression of myelination of the nervous system. A well-studied example of postnatal plasticity is the sprouting of one corticospinal tract (CST) into the denervated half of the spinal cord after unilateral motor cortex or pyramidal lesions. In the hamster and rat, significant CST sprouting is restricted to the first 10 postnatal days. Here we show that very extensive sprouting of corticospinal fibres occurs after deafferentations as late as P21 if myelination is prevented by neonatal X-irradiation in the rat lumbar spinal cord. Sprouted fibres from the intact CST cross the midline and develop large terminal arbors in the denervated spinal cord, suggesting the establishment of synaptic connections. Our results suggest that myelin and its associated neurite growth inhibitors play an important role in the termination of neurite growth permissive periods during postnatal CNS development. Corticospinal sprouting subsequent to lesions early in life, i.e. in the absence of myelin-associated neurite growth inhibitors may explain the frequent occurrence of mirror movements in patients with hemiplegic cerebral palsy.     

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

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

NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord

The regeneration capacity of spinal cord axons is severely limited. Recently, much attention has focused on promoting regeneration of descending spinal cord pathways, but little is known about the regenerative capacity of ascending axons. Here we have assessed the ability of neurotrophic factors to promote regeneration of sensory neurons whose central axons ascend in the dorsal columns. The dorsal columns of adult rats were crushed and either brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) or a vehicle solution was delivered continuously to the lesion site for 4 weeks. Transganglionic labelling with cholera toxin beta subunit (CTB) was used to selectively label large myelinated Abeta fibres. In lesioned rats treated with vehicle, CTB-labelled fibres were observed ascending in the gracile fasciculus, but these stopped abruptly at the lesion site, with no evidence of sprouting or growth into lesioned tissue. No CTB-labelled terminals were observed in the gracile nucleus, indicating that the lesion successfully severed all ascending dorsal column axons. Treatment with BDNF did not promote axonal regeneration. In GDNF-treated rats fibres grew around cavities in caudal degenerated tissue but did not approach the lesion epicentre. NT-3, in contrast, had a striking effect on promoting growth of lesioned dorsal column axons with an abundance of fibre sprouting apparent at the lesion site, and many fibres extending into and beyond the lesion epicentre. Quantification of fibre growth confirmed that only in NT-3-treated rats did fibres grow into the crush site and beyond. No evidence of terminal staining in the gracile nucleus was apparent following any treatment. Thus, although NT-3 promotes extensive growth of lesioned axons, other factors may be required for complete regeneration of these long ascending projections back to the dorsal column nuclei. The intrathecal delivery of NT-3 or other neurotrophic molecules has obvious advantages in clinical applications, as we show for the first time that dorsal column axonal regeneration can be achieved without the use of graft implantation or nerve lesions.     

Corticostriatal plasticity is restricted by myelin-associated neurite growth inhibitors in the adult rat.

After unilateral cortical lesions in neonatal rats, the spared unablated hemisphere is known to demonstrate remarkable neuroanatomical plasticity in corticofugal connectivity. This same type of structural plasticity is not seen after similar lesions in adult rats. One possibility for the lack of such a plastic response in the adult central nervous system may be the presence of myelin-associated neurite growth inhibitory proteins NI-35/NI-250. These proteins have previously been found to play a crucial role in preventing axotomized fibers from regenerating after adult rat spinal cord lesions. The aim of this study was to determine if blocking these inhibitory proteins by the application of the specific monoclonal antibody IN-1 would enhance corticostriatal plasticity from the spared hemisphere after unilateral cortical lesions in adult rats. Six- to 8-week-old Lewis rats underwent unilateral aspiration lesion of the sensorimotor cortex. Animals were immediately treated with either monoclonal antibody IN-1 or a control antibody released from hybridoma cells in Millipore filter capsules. After a survival period of 12 weeks, the opposite sensorimotor cortex was stereotaxically injected with the anterograde tracer biotinylated dextran amine, and biotinylated dextran amine-positive corticostriatal fibers were analyzed. The monoclonal antibody IN-1-treated animals showed an increase in corticostriatal fibers in the dorsolateral striatum contralateral to the injection site compared with control antibody-treated animals or normal controls, indicating a specific sprouting response in the deafferented zone. These results support the idea that through blockade of myelin-associated neurite inhibitory proteins, lesion-induced corticofugal plasticity is possible even in the adult central nervous system.     

Increased corticofugal plasticity after unilateral cortical lesions combined with neutralization of the IN-1 antigen in adult rats.

If damage to the central nervous system (CNS) occurs early in life, extensive rearrangements of the remaining fiber systems as well as regeneration of lesioned fibers take place. In the rat or hamster, newly grown projections have been described only if the lesion occurred within the first two weeks postnatally. This decreasing growth ability correlates with CNS maturation and the progression of myelination. Myelin contains the potent neurite growth inhibitors NI-35/250 that are crucially involved in the failure of long-distance regeneration and the lack of compensatory structural plasticity after adult CNS lesions. In this study, we show that extensive remodeling occurs well after the termination of the growth permissive period in the adult rat if we neutralize the inhibitory properties of myelin with the monoclonal antibody IN-1. After ablation of one motor cortex and treatment with the antibody IN-1, we observed that the remaining corticospinal tract (CST) from the spared hemisphere sprouted into the denervated, contralateral red nucleus and pons. In the pons, these fibers terminated in a typical somatotopic pattern. For comparison with neonatal plasticity, we performed the same lesion in two-day-old rats (no antibody). This lesion led as well to sprouting of the remaining CST into denervated brainstem nuclei, resulting in a bilateral corticofugal projection. Our results show that neutralization of myelin-associated neurite-growth inhibitors after CNS lesions leads to a structural remodeling of the spared corticofugal fibers in adult rats, a process normally restricted to a short postnatal period.     

Neuroimmune processes associated with Wallerian degeneration support neurotrophin‐3‐induced axonal sprouting in the injured spinal cord

Lesions of the spinal cord cause two distinctive types of neuroimmune responses, a response at the lesion site that leads to additional tissue destruction and a more subtle response, termed Wallerian degeneration (WD), that occurs distal to the lesion site. We have evidence that the neuroimmune response associated with WD may support tissue repair. Previously, we found that overexpression of neurotrophin‐3 (NT‐3) induced axonal growth in the spinal cord after a unilateral corticospinal tract (CST) lesion, but only if the immune system was intact and activated. We reasoned that a neuroimmune response associated with WD was involved in this neuroplasticity. To test this, we compared NT‐3‐induced axonal sprouting in athymic nude rats that lack functional T cells with rats with functional T cells and in nude rats grafted with CD4⁺ T cells or CD8⁺ T cells. There was no sprouting in nude rats and in nude rats grafted with CD8⁺ T cells. However, nude rats grafted with CD4⁺ T cells mounted a sprouting response. To determine which CD4⁺ subtype, type 1 T helper (Th1) or type 2 T helper (Th2) cells, was responsible, we grafted Th1 and Th2 cells into nude rats and tested whether they would support sprouting. Axonal sprouting was greater in rats grafted with Th2 cells, demonstrating that the Th2 subtype was responsible for supporting axonal sprouting. These data suggest that WD activates Th2 cells that, along with the direct effects of NT‐3 on CST axons, act to support axonal sprouting in the lesioned spinal cord. © 2013 Wiley Periodicals, Inc.     

Channeling of developing rat corticospinal tract axons by myelin-associated neurite growth inhibitors.

CNS myelin contains 2 membrane proteins that are potent inhibitors of neurite growth (NI-35 and NI-250). Because myelin formation starts at different times in different regions and tracts of the CNS, this inhibitory property of myelin could serve boundary and guidance functions for late-growing fiber tracts. In the rat, the corticospinal tract (CST) grows into and down the spinal cord during the first 10 postnatal days, in close proximity to the sensory tracts fasciculus cuneatus and gracilis. Immunofluorescence for myelin constituents showed that, in the rostral half of the spinal cord, the myelinating tissue of these ascending tracts surrounds the growing, myelin-free CST in a channellike fashion. Elimination of oligodendrocytes by x-irradiation of the newborn rats, or application of antibody IN-1, which neutralizes the inhibitory substrate property of CNS myelin, resulted in significant anatomical aberration of CST fibers. In particular, the tract was larger in cross-section, and aberrant CST fibers and fascicles intermixed with the neighboring sensory ascending tracts. These results assign an important channeling and "guard-rail" function to the oligodendrocyte-associated neurite growth inhibitors for the developing CST in the rat spinal cord.     

Structural plasticity of the adult CNS. Negative control by neurite growth inhibitory signals

The neuronal network of the adult central nervous system (CNS) retains a limited capacity for growth and structural change. This structural plasticity has been best studied in the context of lesion-induced growth and repair. More recently, structural changes underlying functional plasticity occurring under specific physiological conditions have also been documented, in particular in the cortex and the hippocampus. Areas known for their adult plastic potential retain high levels of the growth associated protein GAP-43, suggesting a persistence of important components of the intracellular growth machinery throughout life. Interestingly, a pronounced negative correlation exists between the levels of GAP-43 and myelination in the adult CNS. Because CNS myelin contains potent neurite growth inhibitory membrane proteins, neurite growth, sprouting and plasticity were investigated in the spinal cord and brain in areas where oligodendrocyte development and myelin formation was experimentally prevented, or in the presence of an inhibitor neutralizing antibody (mAB-IN-1). In all areas, lesion-induced or spontaneous sprouting was enhanced, in parallel with persistent high levels of GAP-43. Thus, spontaneous sprouting of side branches occurred from retinal axons in the optic nerve in the absence of myelin, and target-deprived retinal axons showed increased sprouting and innervation of the contralateral optic tectum in the presence of mAB IN-1. In experimentally myelin-free spinal cords collaterals from intact dorsal roots grew over long distances to innervate deafferented target regions following the section of three dorsal roots. Similarly, the corticospinal tract sprouted across the the midline and re-established a dense plexus of fibres on the contralateral side of the spinal cord following section of one corticospinal tract in juvenile rats. Following bilateral dorsal hemisection of the spinal cord including both corticospinal tracts in young and adult rats, long distance regeneration of corticospinal fibres leading to significant functional improvements of locomotion and certain reflexes was induced by the neurite growth inhibitor neutralizing antibody IN-1.     

Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats

Nogo-66 receptor (NgR), a common receptor for the three known myelin-associated inhibitors, i.e., Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), plays a key role in the failure of axonal regeneration in the adult mammalian central nervous system (CNS). Here we report a novel vaccine approach that stimulates the production of anti-NgR antibody to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). We showed that adult rats immunized with recombinant NgR produced high titers of the anti-NgR antibody and that antisera obtained from the immunized rats promoted neurite outgrowth of rat cerebellar neurons on the inhibitory MAG substrate in vitro. In a spinal cord dorsal hemisection model, NgR immunization promoted regeneration of lesioned corticospinal tract (CST) axons, anterogradely labeled with biotin dextran amine (BDA), beyond the lesion site. In a contusive SCI model, NgR immunization markedly reduced the total lesion volume and improved Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking performance. Thus, the NgR vaccine approach may represent a promising repair strategy to promote structural and functional recovery following SCI.     

Myelin-associated inhibitors of neurite growth

CNS myelin and oligondendrocyte membranes contain two minor proteins with strong inhibitory effects on growing neurites (neurite growth inhibitors NI-35 and NI-250). Monoclonal antibodies (IN-1, IN-2) were obtained that neutralize this activity in a variety of culture assays including adult rat optic nerve explants, which are invaded by growing neurites under the influence of these antibodies. In vivo, corticospinal tract lesions in young rats are known to be followed by abortive sprouting, not exceeding 1 mm of elongation. In contrast, the presence of antibody IN-1 led to regrowth of corticospinal axons over more than 5 mm within 2-3 weeks. In development, a negative guidance or channeling function may be associated with these inhibitors for late growing CNS tracts. In fact, application of antibodies or absence of oligodendrocytes during the first postnatal week led to severe anatomical disturbance of the developing rat corticospinal tract. Additional, e.g., stabilizing functions for these inhibitors in the adult CNS remain to be investigated.     

Regeneration of Lesioned Septohippocampal Acetylcholinesterase-positive Axons is Improved by Antibodies Against the Myelin-associated Neurite Growth Inhibitors NI-35/250

Two oligodendrocyte membrane proteins, NI-35 and NI-250, have been shown to be highly inhibitory for neurite growth. Upon neutralization of these components with the specific monoclonal antibody IN-1, lesioned corticospinal tract fibres were able to regenerate over long distances. In the present study, we have investigated the behaviour of regenerating cholinergic septohippocampal tract fibres. Large fimbria/fornix aspiration lesions were bridged by human amnion extracellular matrix material containing nerve growth factor, and the inhibitor-neutralizing antibody IN-1 or a control antibody were applied. After 3 - 5 weeks survival time, acetylcholinesterase (AchE)-positive fibres had crossed the bridge and, upon entering the hippocampus, had developed a profuse fibre plexus. In the controls (antibody against peroxidase) the fibre growth within the hippocampal tissue remained limited to maximally 1 mm in the caudal and lateral directions. In the presence of the antibody IN-1, however, AchE-positive fibres were seen to grow for 2 - 4 mm both in the caudal and lateral directions. Interestingly, the regenerated fibres preferably grew to their original terminal areas in the infra- and suprapyramidal layers of the hippocampus proper and the hilus, and in the supragranular layer of the dentate gyrus. These data show that the neurite growth inhibitors severely impede regenerative axon growth also for the cholinergic fibres in the hippocampus, and that their neutralization increases axon growth and leads to partial reconstitution of the original anatomical fibre distribution.     

Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice

Chondroitin sulphate proteoglycans (CSPGs) are extracellular matrix molecules whose inhibitory activity is attenuated by the enzyme chondroitinase ABC (ChABC). Here we assess whether CSPG degradation can promote compensatory sprouting of the intact corticospinal tract (CST) following unilateral injury and restore function to the denervated forelimb. Adult C57BL/6 mice underwent unilateral pyramidotomy and treatment with either ChABC or a vehicle control. Significant impairments in forepaw symmetry were observed following pyramidotomy, with injured mice preferentially using their intact paw during spontaneous vertical exploration of a cylinder. No recovery on this task was observed in vehicle‐treated mice. However, ChABC‐treated mice showed a marked recovery of function, with forelimb symmetry fully restored by 5 weeks post‐injury. Functional recovery was associated with robust sprouting of the uninjured CST, with numerous axons observed crossing the midline in the brainstem and spinal cord and terminating in denervated grey matter. CST fibres in the denervated side of the spinal cord following ChABC treatment were closely associated with the synaptic marker vGlut1. Immunohistochemical assessment of chondroitin‐4‐sulphate revealed that CSPGs were heavily digested around lamina X, alongside midline crossing axons and in grey matter regions where sprouting axons and reduced peri‐neuronal net staining was observed. Thus, we demonstrate that CSPG degradation promotes midline crossing and reinnervation of denervated target regions by intact CST axons and leads to restored function in the denervated forepaw. Enhancing compensatory sprouting using ChABC provides a route to restore function that could be applied to disorders such as spinal cord injury and stroke.     

Expression of myelin proteins in the opossum optic nerve: Late appearance of inhibitors implicates an earlier non-myelin factor in preventing ganglion cell regeneration

The pattern of appearance of myelin-associated proteins in the visual system of the Brazilian opossum Monodelphis domestica is described. Whole mounts of optic nerve, chiasm, and optic tract were sectioned horizontally and incubated with antibodies to myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein, (MAG), “Rip,” and the neurite inhibitory protein (IN-1), followed by visualization with diaminobenzidine and a peroxidase-conjugated secondary antibody. PLP is first detectable 24 days after birth (P24) at the centre of the optic chiasm. MBP, MAG, Rip, and IN-1 appear first in the same area at P26. By P28 the distribution of all proteins is similar, occupying the entire chiasm, optic tracts, and prechiasmatic portion of the optic nerves. Protein expression progresses along the optic nerve to reach the lamina cribrosa by P34, coincident with the time of eye opening. A critical period in which the retinofugal pathway has a regenerative capacity has recently been observed in Monodelphis. This period ends at P12, 2 weeks before the appearance of the myelin-associated inhibitory proteins MAG and IN-1. These results therefore suggest that regeneration in the developing retinofugal projection of the opossum is restricted by an earlier non-myelin factor, which is in contrast to current literature on the spinal cord. © 1996 Wiley-Liss, Inc.