Requirement of myeloid cells for axon regeneration.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TK(mt-30) mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TK(mt-30) mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TK(mt-30) mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.

Anti-Nogo-A antibody treatment enhances sprouting of corticospinal axons rostral to a unilateral cervical spinal cord lesion in adult macaque monkey.

After injury, regrowth of axons in mammalian adult central nervous system is highly limited. However, in monkeys subjected to unilateral cervical lesion (C7-C8 level), neutralization of an important neurite outgrowth inhibitor, Nogo-A, stimulated axonal sprouting caudal to the lesion, accompanied by enhanced functional recovery of manual dexterity, compared with lesioned monkeys treated with a control antibody (Freund et al. [2006] Nat. Med. 12:790-792). The present study aimed at comparing the same two groups of monkeys for axonal sprouting rostral to the cervical lesion. The corticospinal tract was labeled by injecting the anterograde tracer biotinylated dextran amine into the contralesional motor cortex. The corticospinal axons were interrupted at the level of the lesion, accompanied by retrograde axonal degeneration (axon dieback), reflected by the presence of terminal retraction bulbs. The number of terminal retraction bulbs was lower in anti-Nogo-A antibody treated monkeys, and, when present, they were found closer to the lesion than in control-antibody treated monkeys. Compared with control antibody treated monkeys, the anti-Nogo-A antibody treated monkeys exhibited an increased cumulated axon arbor length and a higher number of axon arbors going in the medial direction from the white to the gray matter. Higher in the cervical cord (at C5 level), the anti-Nogo-A treatment enhanced the number of corticospinal fibers crossing the midline, suggesting axonal sprouting. Thus, the anti-Nogo-A antibody treatment enhanced axonal sprouting rostral to the cervical lesion; some of these fibers grew around the lesion and into the caudal spinal segments. These processes paralleled the observed improved functional recovery.

Neuron-binding human monoclonal antibodies support central nervous system neurite extension.

Two human IgMs (sHIgM12 and sHIgM42) were identified that supported in vitro central nervous system (CNS) neurite extension equal to the potent neurite stimulatory molecule laminin. Both IgMs bound to multiple cell types in unfixed CNS tissue and to the surface of neurons in culture. Both monoclonal antibodies (mAbs) overrode the inhibitory effect of CNS mouse myelin on granule cell neurite extension. Neither mAb bound to the surface of mature oligodendrocytes or strictly colocalized with myelin proteins. Sialidase treatment eliminated the neuronal surface binding of both mAbs, whereas blocking sphingolipid synthesis with Fumonisin B1 or removing GPI-linked proteins with PIPLC did not. When used as substrates for mixed neuron/glia aggregates, sHIgM12 and sHIgM42 supported robust neurite extension while astrocytes remained in the aggregates. In contrast, laminin supported astrocyte migration and spreading. Human mAbs that support neurite extension are novel factors that may be of use in encouraging axon repair following injury while minimizing glial cell infiltration. Both human mAbs were isolated from individuals with monoclonal gammopathy. Each individual has carried high mAb titers in circulation for years without detriment. sHIgM12 and sHIgM42 are therefore unlikely to be systemically pathogenic.

Nervous system injury: focus on the inflammatory cytokine 'granulocyte-macrophage colony stimulating factor'.

Any lesion in the nervous system, be it infectious, immunopathological, ischemic or traumatic, is followed by an inflammatory process that induces rapid activation of glial cells and additional recruitment of granulocytes, T-cells and monocytes/macrophages from the blood stream. Neuroinflammation is a double-sided sword. It can cause neuronal damage and participate in neuropathic pain, but it also has neuroprotective and neurotrophic effects at some stages. Cytokines are the main molecular actors of this 'network of inflammation'. Among them, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory hematopoietic cytokine widely used in haematological disorders to stimulate proliferation and differentiation of neutrophilic, eosinophilic and monocytic lineages. GM-CSF and its receptor are expressed in the brain and the cytokine can cross the blood-brain barrier. It is thus likely to affect various nervous system functions. This review will focus on the role of GM-CSF in nervous system disorders and their experimental models with particular emphasis on its possible beneficial effect on axonal regeneration after PNS and CNS injury.

Vaccination stimulates retinal ganglion cell regeneration in the adult optic nerve.

We examined whether vaccination of adult rats with spinal cord homogenate (SCH) can promote regeneration of retinal ganglion cells (RGCs) after microcrush lesion of the optic nerve. Injured animals vaccinated with SCH showed axon growth into the optic nerve and such regeneration was not observed in animals vaccinated with liver homogenate (LH). Regeneration was not a consequence of neuroprotection since our vaccine did not protect RGCs from axotomy-induced cell death. Sera of vaccinated animals were tested for antibodies against myelin-associated glycoprotein, NogoA, Nogo-66 receptor, or chondroitin sulphate proteoglycans (CSPG), but no significant levels were detected. Antibodies to myelin basic protein were present in the serum of some SCH-vaccinated animals. In culture, serum from SCH-vaccinated animals promoted RGC growth on myelin but not on CSPG. Our results show that the effect of the pro-regenerative vaccine is mediated by antibodies to SCH. However, we were not able to detect a significant immune reaction to growth inhibitory proteins, suggesting alternative mechanisms for the success of vaccination to promote regeneration.

Focal application of neutralizing antibodies to soluble neurotrophic factors reduces collateral axonal branching after peripheral nerve lesion.

A major reason for the insufficient recovery of function after motor nerve injury are the numerous axonal branches which often re-innervate muscles with completely different functions. We hypothesized that a neutralization of diffusable neurotrophic factors at the lesion site in rats could reduce the branching of transected axons. Following analysis of local protein expression by immunocytochemistry and by in situ hybridization, we transected the facial nerve trunk of adult rats and inserted both ends into a silicon tube containing (i) collagen gel with neutralizing concentrations of antibodies to NGF, BDNF, bFGF, IGF-I, CNTF and GDNF; (ii) five-fold higher concentrations of the antibodies and (iii) combination of antibodies. Two months later, retrograde labelling was used to estimate the portion of motoneurons the axons of which had branched and projected into three major branches of the facial trunk. After control entubulation in collagen gel containing non-immune mouse IgG 85% of all motoneurons projecting along the zygomatic branch sprouted and sent at least one twin axon to the buccal and/or marginal-mandibular branches of the facial nerve. Neutralizing concentrations of anti-NGF, anti-BDNF and anti-IGF-I significantly reduced sprouting. The most pronounced effect was achieved after application of anti-BDNF, which reduced the portion of branched neurons to 18%. All effects after a single application of antibodies were concentration-dependent and superior to those observed after combined treatment. This first report on improved quality of reinnervation by antibody-therapy implies that, in rats, the post-transectional collateral axonal branching can be reduced without obvious harmful effects on neuronal survival and axonal elongation.

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.

Interleukin-6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6-deficient mice.

Nerve injury triggers numerous changes in the injured neurons and surrounding non-neuronal cells. Of particular interest are molecular signals that play a role in the overall orchestration of this multifaceted cellular response. Here we investigated the function of interleukin-6 (IL6), a multifunctional neurotrophin and cytokine rapidly expressed in the injured nervous system, using the facial axotomy model in IL6-deficient mice and wild-type controls. Transgenic deletion of IL6 caused a massive decrease in the recruitment of CD3-positive T-lymphocytes and early microglial activation during the first 4 days after injury in the axotomized facial nucleus. This was accompanied by a more moderate reduction in peripheral regeneration at day 4, lymphocyte recruitment (day 14) and enhanced perikaryal sprouting (day 14). Motoneuron cell death, phagocytosis by microglial cells and recruitment of granulocytes and macrophages into injured peripheral nerve were not affected. In summary, IL6 lead to a variety of effects on the cellular response to neural trauma. However, the particularly strong actions on lymphocytes and microglia suggest that this cytokine plays a central role in the initiation of immune surveillance in the injured central nervous system.

Direct interactions between immunocytes and neurons after axotomy in Aplysia.

Axon growth during development and after injury has processes in common, but also differs in that regeneration requires the participation of cells of the immune system. To investigate how neuron-immunocyte interactions might influence regeneration, we developed an in vitro model whereby neurons and hemocytes from Aplysia californica were cocultured. The hemocytes, which behave like vertebrate macrophages, migrated randomly throughout the dish. When a neuron was encountered, some hemocytes exhibited an avoidance response, whereas others formed stable contacts. Hemocytes did not distinguish between neurons from different animals. Stable contacts occurred on neurites and growth cones, but not the cell soma, and were benign in that the hemocytes did not impede neurite growth. When hemocytes attached to the cell body, it presaged the destruction of the neuron. Destruction was a dynamic process that was initiated when groups of one to three hemocytes adhered to various regions of the cell soma. Each group was then joined by other hemocytes. They did not contact the neuron, but interconnected the initial groups, forming a network around the neuron. The network then contracted to dismember the cell. Once a neuron was destroyed, hemocytes removed the debris by phagocytosis. Both damaged neurons and those without apparent damage were targets for destruction. Severing neurites with a needle resulted in the destruction of only one of six cells. Our studies suggest that hemocytes, and by extrapolation, vertebrate macrophages, exhibit highly complex interactions with neurons that can exert a variety of influences on the course of nerve regeneration.

Monoclonal antibody 2G13, a new axonal growth cone marker.

Growth cones are specialized sensorimotor structures at the tips of neurites implicated in pathfinding decisions and axonal outgrowth during neuronal development. We generated a mouse monoclonal antibody (mAb 2G13) against chick tectum and found that the antibody exclusively labelled axonal growth cones, particularly their filopodia and lamellipodia, in developing rat CNS and in embryonic neurons in culture. The high fidelity of the staining of growth cones by mAb 2G13 means that the antibody will be a useful marker for identifying growth cones. In growth cones of cultured neurons, mAb 2G13 labelling is intracellular and mainly associated with the filamentous actin cytoskeleton. Experiments with cytochalasins, which depolymerise filamentous actin, showed that 2G13p (the protein recognised by mAb 2G13) is physically associated with filamentous actin in growth cones. These properties of 2G13p suggest a role in growth cone motility.