Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS.

The MAG-deficient mouse was used to test whether MAG acts as a significant inhibitor of axonal regeneration in the adult mammalian CNS, as suggested by cell culture experiments. Cell spreading, neurite elongation, or growth cone collapse of different cell types in vitro was not significantly different when myelin preparations or optic nerve cryosections from either MAG-deficient or wild-type mice were used as a substrate. More importantly, the extent of axonal regrowth in lesioned optic nerve and corticospinal tract in vivo was similarly poor in MAG-deficient and wild-type mice. However, axonal regrowth increased significantly and to a similar extent in both genotypes after application of the IN-1 antibody directed against the neurite growth inhibitors NI-35 and NI-250. These observations do not support the view that MAG is a significant inhibitor of axonal regeneration in the adult CNS.

Cytokine-induced acute inflammation in the brain and spinal cord.

Different compartments in the central nervous system mount distinct inflammatory responses. The meninges and choroid plexus respond to pro-inflammatory stimuli in a manner reminiscent of a peripheral inflammatory response, whereas the brain parenchyma is refractory. Trauma-induced lesions in brain and in spinal cord are associated with leukocyte infiltration, blood-brain barrier (BBB) breakdown, and secondary tissue destruction. Unexpectedly, these phenomena are generally more pronounced in the parenchyma of the spinal cord than in the parenchyma of the brain. To investigate whether these differences between brain and spinal cord can be attributed, at least in part, to differing sensitivities to proinflammatory cytokines, we stereotactically injected recombinant rat (rr) TNFalpha or rrIL-1beta into the striatum or the spinal cord of Wistar rats. In the brain, the injection of rrTNFalpha failed to evoke BBB breakdown or leukocyte recruitment, whereas in the spinal cord injection of TNFalpha resulted in marked BBB breakdown and leukocyte recruitment. Similarly, the injection of rrIL-1beta into the brain parenchyma failed to induce BBB breakdown and gave rise to only minimal neutrophil recruitment, whereas the injection of rrIL-1beta into the spinal cord induced significant BBB breakdown and recruitment of neutrophils and lymphocytes. Thus, using a minimally invasive injection technique, equivalent in both circumstances, we have shown that there are marked differences in the inflammatory response between the brain parenchyma and spinal cord parenchyma. This observation has important implications for the treatment of spinal cord injuries.

AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats.

Microglial cells are among the first and dominant cell types to respond to CNS injury. Following calcium influx, microglial activation leads to a variety of cellular responses, such as proliferation and release of cytotoxic and neurotrophic mediators. Allograft inflammatory factor-1, AIF-1 is a highly conserved EF-handed, putative calcium binding peptide, associated with microglia activation in the brain. Here, we have analyzed the expression of AIF-1 following spinal cord injury at the lesion site and at remote brain regions. Following spinal cord injury, AIF-1+ cells accumulated in parenchymal pan-necrotic areas and perivascular Virchow-Robin spaces. Subsequent to culmination at day 3--a situation characterized by infiltrating blood borne macrophages and microglia activation--AIF-1+ cell numbers decreased until day 7. In remote areas of Wallerian degeneration and delayed neuronal death, a more discrete and delayed activation pattern of AIF-1+ microglia/macrophages reaching maximum levels at day 14 was observed. There was a considerable match between AIF-1+ cells and PCNA (proliferating cell nuclear antigen) or Ki-67+ labeled cells. AIF-1 expression preceded the expression of ED1, thus indicating a pre-phagocytic role. It appears that AIF-1+ microglia/macrophages are among the earliest cells to respond to spinal cord injury. Our results suggest a role of AIF-1 in the initiation of the early microglial response leading to activation and proliferation essential for the acute response to CNS injury. AIF-1 might modulate microgliosis influencing the efficacy of tissue debris removal, myelin degradation, recruitment of oligodendrocytes and re-organisation of the CNS architecture.

Region-specific appearance of myelin constituents in the developing rat spinal cord.

The appearance of myelin-specific glycolipids and of myelin basic protein was studied with regard to the detailed anatomy of the rat cervical spinal cord. The expression of these constituents in particular fibre tracts and regions occurs at specific times of development between postnatal days 1 and 14. This mosaic-like appearance started in the ventral funiculus (day 1) followed by fasciculus cuneatus and ventro-lateral funiculus (day 2), and fasciculus gracilis and dorso-lateral funiculus (days 3 to 4). Cortico-spinal tract (day 11), Lissauer tract (day 14) and the commissures started to acquire myelin very late. In the grey matter, myelin constituents appeared around days 11 to 14 in a patchy pattern. These results support a concept of highly local interactions regulating oligodendrocyte differentiation. In addition, a general rostro-caudal gradient of myelin development exists in the spinal cord, which is independent of the ascending or descending nature of the fibre tracts. Appearance of myelin constitutents in the caudal spinal cord was not prevented by a neonatal transection at mid-thoracic levels.

Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors.

After lesions in the differentiated central nervous system (CNS) of higher vertebrates, interrupted fibre tracts do not regrow and elongate by more than an initial sprout of approximately 1 mm. Transplantations of pieces of peripheral nerves into various parts of the CNS demonstrate the widespread capability of CNS neurons to regenerate lesioned axons over long distances in a peripheral nerve environment. CNS white matter, cultured oligodendrocytes (the myelin-producing cells of the CNS), and CNS myelin itself, are strong inhibitors of neuron growth in culture, a property associated with defined myelin membrane proteins of relative molecular mass (Mr) 35,000 (NI-35) and 250,000 (NI-250). We have now intracerebrally applied the monoclonal antibody IN-1, which neutralizes the inhibitory effect of both these proteins, to young rats by implanting antibody-producing tumours. In 2-6-week-old rats we made complete transections of the cortico-spinal tract, a major fibre tract of the spinal cord, the axons of which originate in the motor and sensory neocortex. Previous studies have shown a complete absence of cortico-spinal tract regeneration after the first postnatal week in rats, and in adult hamsters and cats. In IN-1-treated rats, massive sprouting occurred at the lesion site, and fine axons and fascicles could be observed up to 7-11 mm caudal to the lesion within 2-3 weeks. In control rats, a similar sprouting reaction occurred, but the maximal distance of elongation rarely exceeded 1 mm. These results demonstrate the capacity for CNS axons to regenerate and elongate within differentiated CNS tissue after the neutralization of myelin-associated neurite growth inhibitors.

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.

The effect of providing ipecac to families seeking poison-related services.

Although home availability of ipecac is recommended for families with young children in case of unintentional toxic ingestion, fewer than half actually have it. We designed a study to evaluate the efficacy of providing ipecac to families requiring poison-related services. Families (n = 100) contacting the Children's Memorial Hospital (CMH) emergency department (ED)/poison center were enrolled. Baseline general poison knowledge and self-report of ipecac availability were obtained. Ipecac was discussed, and families were mailed general safety and poison information, the ED telephone number, and a coded package of ipecac, with instructions. Approximately three months later a follow-up call was made to determine change in knowledge, access to our ED (or any poison center) phone number, and availability of ipecac. Initially 71% had heard of ipecac, 51% knew what it did, and 47% said they had it. Ninety families were contacted in follow-up, 82 by phone and eight by mail. Eighty-three of 90 (92%) knew what ipecac did (vs 51/100 initially; P < 0.0001). Sixty-eight of 90 (76%) knew the ED or a poison control phone number (vs 39/100 initially; P < 0.0001). Seventy-seven of 82 (94%) reached by phone read the ipecac code number (vs 47/100 initial self-reports of possession; P < 0.0001). The data indicate that providing ipecac to poison service users increases availability in the home for at least three months. Poison service users may be particularly amenable to anticipatory guidance and interventions related to poisoning prevention and preparedness.

Sprouting and regeneration of lesioned corticospinal tract fibres in the adult rat spinal cord.

We have studied the effects of tissue transplants and antibodies (IN-1) against the myelin-associated neurite growth inhibitory proteins on sprouting and regeneration of the rat corticospinal tract (CST). Transplantation of embryonic spinal cord tissue into bilateral transection lesions of the lower thoracic spinal cord in young adult rats resulted in a marked increase of the sprouting of the lesioned CST. This sprouting effect was probably elicited by soluble factors released from the transplants, and was enhanced by the IN-1 antibodies. The retraction of lesioned CST fibres normally observed with prolonged survival times was also reduced by the presence of transplants. In spite of these growth-promoting effects of the transplants, the regenerative elongation of CST sprouts into the caudal spinal cord was dependent upon the neutralization of the myelin-associated inhibitory proteins. In the controls (no antibodies or control antibodies) only 27% of the animals showed elongation of CST fibres exceeding the sprouting distance of 0.7 mm. These fibres grew to a maximal length of 1.8 mm (mean +/- SEM, 1.2 +/- 0.1). In contrast, 60% of the IN-1-treated, transplant-containing rats showed elongations of > 0.7 mm, and these fibres grew up to 10.1 mm (4.6 +/- 0.5). Regenerating fibres crossed the lesion site through remaining tissue bridges. Neither embryonic spinal cord transplants nor a variety of implanted bridge materials could serve as a substrate for the regenerating CST axons.

Reactions of oligodendrocytes to spinal cord injury: cell survival and myelin repair.

The aim of this study was to elucidate whether oligodendrocytes die in fiber tracts that are spared by a spinal cord injury but are in close vicinity of inflammatory cells. Adult rat spinal cords were studied histologically 1 day to 2 weeks after a contusion lesion that left the ventral white matter largely intact. Massive oligodendrocyte death occurred in the lesion center, along with the death of neurons, microglia, and astrocytes. Oligodendrocytes, specifically positive for proteolipid protein (PLP) mRNA, were counted in the ventral white matter where axons at the rostral and caudal edges of the lesion were histologically intact. Although these regions contained many macrophages and neutrophils hypothesized to contribute to secondary tissue loss, there was no significant loss of oligodendrocytes. In the ventral funiculus, 3 and 6 mm rostral and caudal to the lesion, oligodendrocyte numbers were also unchanged, in spite of the presence of many activated microglial cells. From day 7 on, oligodendrocytes in close vicinity to the lesion increased their expression of PLP mRNA. We conclude that, at least within the first 2 weeks after a spinal cord contusion lesion, there is no major devastating influence of inflammatory cells or their mediators on oligodendrocytes. When death occurs, it may be due to mechanical trauma, ischemia, or excitotoxicity within the lesion or it may occur as a result of axonal degeneration.

Lymphocyte recruitment following spinal cord injury in mice is altered by prior viral exposure.

The inflammatory response induced by mechanical lesion of the spinal cord is known to include the recruitment of neutrophils and macrophages, while the involvement of lymphocytes has been largely ignored. We have studied the pattern of lymphocyte recruitment following partial transection of the mouse spinal cord. Using immunohistochemical techniques, all three types of lymphocytes (CD4-positive T-cells, CD8-positive T-cells and B-cells) were found in the vicinity of the lesion site within hours and persisted for up to 7 days. There was a predominance of B-lymphocytes during the first 3 days. A second, late phase of cell infiltration, dominated by CD8-positive T-lymphocytes, occurred in mice that had been raised in a conventional breeding unit and had acquired antibody titres to a common murine virus (mouse hepatitis virus). In contrast, mice kept in specific pathogen-free facilities did not show this late-phase response. These findings suggest a possible role for lymphocytes in secondary tissue loss, local demyelination, scar formation, cytokine-mediated inflammatory responses or trophic processes. They also provide evidence that a virus infection can significantly enhance the reaction of T-cells to a spinal cord lesion.

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.

Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion.

The number of neurotrophic factors found in the central nervous system is rapidly growing, but their functions in vivo are largely unknown. In the peripheral nervous system they promote the survival of developing and lesioned neurons and enhance nerve fibre growth and regeneration. Here we study the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the largest tract system leading from the brain to the spinal cord, the corticospinal tract (CST). The developing CST grows down the spinal cord during the first postnatal days and innervates its targets after a waiting period by collateral sprouting. We find that NT-3 injected locally specifically enhances this sprouting, whereas BDNF has no effect. In adult rats, injection of NT-3 (but not BDNF) into the lesioned spinal cord increases the regenerative sprouting of the transected CST. The distance of growth of the sprouts is very restricted, but application of an antibody that neutralizes myelin-associated neurite growth inhibitory proteins results in long-distance regeneration of CST fibres.

Cholinergic innervation of fetal neocortical transplants is increased after neutralization of myelin-associated neurite growth inhibitors.

Fetal neocortical transplants placed into adult neocortical sensorimotor aspiration lesions are known to receive afferent input from the adult host rat brain. As this input is less dense than normal, the present study was designed to investigate whether neutralization of myelin-associated neurite growth inhibitors NI-35/250 might promote host derived cholinergic innervation of fetal neocortical transplants. Adult rats received unilateral sensorimotor cortical aspiration lesions, and block grafts from embryonic day 14-15 neocortical tissue were placed immediately into the lesion cavities. Mouse hybridoma cells secreting either the monoclonal antibody IN-1, which blocks neurite growth inhibitors NI-35/250, or a control antibody or medium without cells were applied in millipore filter capsules directly over the fetal graft tissue. The brains were processed 12 weeks later for the visualization of acetylcholinesterase-positive, presumptive cholinergic fibers. We found an enhancement in the cholinergic innervation of fetal grafts in the recipients treated with the antibody IN-1 both in terms of fibers growing into the graft and of density within the center of the grafts. These results indicate that myelin-associated neurite growth inhibitors are involved in the development of host-transplant connectivity in the adult brain.

Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord.

Lesion-induced inflammatory responses in both brain and spinal cord have recently become a topic of active investigation. Using C57BL/6J mice, we compared the tissue reaction in these two central nervous system (CNS) compartments with mechanical lesions of similar size involving both grey and white matter. This evaluation included the quantitative assessment of neutrophils, lymphocytes and activated macrophages/microglia, as well as astrocyte activation, upregulation of vascular cell adhesion molecules (ICAM-1, VCAM-1, PECAM) and the extent of blood-brain barrier (BBB) breakdown. Time points analysed post-lesioning included 1, 2, 4 and 7 days (as well as 10 and 14 days for the BBB). We found clear evidence that the acute inflammatory response to traumatic injury is significantly greater in the spinal cord than in the cerebral cortex. The numbers of both neutrophils and macrophages recruited to the lesion site were significantly higher in the spinal cord than in the brain, and the recruitment of these cells into the surrounding parenchyma was also more widespread in the cord. The area of BBB breakdown was substantially larger in the spinal cord and vascular damage persisted for a longer period. In the brain, as in spinal cord, the area to which neutrophils were recruited correlated well with the area of BBB breakdown. It will be of interest to determine the extent to which the infiltration of inflammatory cells contributes, either directly or indirectly, to the vascular permeability and secondary tissue damage or, conversely, to local tissue repair in the brain and the spinal cord.

Influence of IN-1 antibody and acidic FGF-fibrin glue on the response of injured corticospinal tract axons to human Schwann cell grafts.

Two strategies have been shown by others to improve CST regeneration following thoracic spinal cord injury: 1) the administration of a monoclonal antibody, IN-1, raised against a myelin-associated, neurite growth inhibitory protein, and 2) the delivery of acidic fibroblast growth factor (aFGF) in fibrin glue in association with peripheral nerve grafts. Because autologous transplantation of human Schwann cells (SCs) is a potential strategy for CNS repair, we evaluated the ability of these two molecular agents to induce CST regeneration into human SC grafts placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. IN-1 or control (HRP) antibodies were delivered to the injury/graft region by encapsulated hybridoma cells ("IN-1 ravioli") or daily infusion of hybridoma culture supernatant; aFGF-fibrin glue was placed in the same region in other animals. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), was performed. Thirty-five days after grafting, the CST response was evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provided information about axonal die-back. In control animals (graft only), the CST did not enter the SC graft and underwent axonal die-back [SI = 1.4 +/- 0.1, MTD = 2.0 +/- 0.2, LS = 1.3 +/- 0.3, (n = 3)]. Results of IN-1 delivery from ravioli did not differ from controls, but injections of IN-1-containing supernatant resulted in a significant degree of sprouting but did not prevent axonal die-back [SI = 1.9 +/- 0.1, MTD = 1.5 +/- 0.2, LS = 1.1 +/- 0.1, (n = 7)] and traced fibers did not enter grafts. Acidic FGF dramatically reduced axonal die-back and caused sprouting [SI = 2.0 +/- 0.1 (n = 5), MTD = 0.5 +/- 0.04 (n = 6), LS = 0.4 +/- 0.1 (n = 6)]. Some traced fibers entered SC grafts and in 2/6 cases entered the distal interface. We conclude that 1) human SC grafts alone do not support the regeneration of injured CST fibers and do not prevent die-back, 2) grafts plus IN-1 antibody-containing supernatant support some sprouting but die-back continues, and 3) grafts plus aFGF-fibrin glue support regeneration of some fibers into the grafts and reduce die-back.

Local changes in vascular architecture following partial spinal cord lesion in the rat.

Lesions of the CNS induce a complex cascade of tissue reactions. The purpose of this study was to determine the response of the vasculature to partial spinal cord transection. Adult rat spinal cords were lesioned and then examined during acute, subacute, and chronic periods for the presence of endothelial cells and blood vessels at the lesion site. The association of endothelial cells and astrocytes was examined immunohistochemically (RECA-1 and glial fibrillary associated protein, respectively). During the first 48 h following an incision lesion of the dorsal spinal cord, the vasculature was significantly decreased, concurrently with the tissue loss due to primary and secondary degeneration. Subsequently, at 4 days postlesion, vasculature repair processes were evidenced by a significant increase in the number of vessels present at the lesion center. Blood vessels even formed in areas densely packed with macrophages and tissue debris. After 1 week, the number of blood vessels declined in the lesion center and at the place of the forming caverns. These results show significant initial attempts at repair of the vasculature which do not, however, lead to the restoration of a compact tissue and cannot prevent the subsequent formation of caverns.