Wolfram F. Neiss

Therapeutic vaccine for acute and chronic motor neuron diseases: Implications for amyotrophic lateral sclerosis

Therapeutic vaccination with Copaxone (glatiramer acetate, Cop-1) protects motor neurons against acute and chronic degenerative conditions. In acute degeneration after facial nerve axotomy, the number of surviving motor neurons was almost two times higher in Cop-1-vaccinated mice than in nonvaccinated mice, or in mice injected with PBS emulsified in complete Freunds adjuvant (P < 0.05). In mice that express the mutant human gene Cu/Zn superoxide dismutase G93A (SOD1), and therefore simulate the chronic human motor neuron disease amyotrophic lateral sclerosis, Cop-1 vaccination prolonged life span compared to untreated matched controls, from 211 ± 7 days (n = 15) to 263 ± 8 days (n = 14; P < 0.0001). Our studies show that vaccination significantly improved motor activity. In line with the experimentally based concept of protective autoimmunity, these findings suggest that Cop-1 vaccination boosts the local immune response needed to combat destructive self-compounds associated with motor neuron death. Its differential action in CNS autoimmune diseases and neurodegenerative disorders, depending on the regimen used, allows its use as a therapy for either condition. Daily administration of Cop-1 is an approved treatment for multiple sclerosis. The protocol for non-autoimmune neurodegenerative diseases such as amyotrophic lateral sclerosis, remains to be established by future studies.

Factors limiting motor recovery after facial nerve transection in the rat: combined structural and functional analyses

It is believed that a major reason for the poor functional recovery after peripheral nerve lesion is collateral branching and regrowth of axons to incorrect muscles. Using a facial nerve injury protocol in rats, we previously identified a novel and clinically feasible approach to combat axonal misguidance – the application of neutralizing antibodies against neurotrophic factors to the injured nerve. Here, we investigated whether reduced collateral branching at the lesion site leads to better functional recovery. Treatment of rats with antibodies against nerve growth factor, brain‐derived neurotrophic factor, fibroblast growth factor, insulin‐like neurotrophic factor I, ciliary neurotrophic factor or glial cell line‐derived neurotrophic factor increased the precision of reinnervation, as evaluated by multiple retrograde labelling of motoneurons, more than two‐fold as compared with control animals. However, biometric analysis of vibrissae movements did not show positive effects on functional recovery, suggesting that polyneuronal reinnervation – rather than collateral branching – may be the critical limiting factor. In support of this hypothesis, we found that motor end‐plates with morphological signs of multiple innervation were much more frequent in reinnervated muscles of rats that did not recover after injury (51% of all end‐plates) than in animals with good functional performance (10%). Because polyneuronal innervation of muscle fibres is activity‐dependent and can be manipulated, the present findings raise hopes that clinically feasible and effective therapies could be soon designed and tested.

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.

Superior muscle reinnervation after autologous nerve graft or poly-L-lactide-?-caprolactone (PLC) tube implantation in comparison to silicone tube repair

Recovery after peripheral nerve injury depends not only on the amount of reinnervation, but also on its accuracy. The rat sciatic nerve was subjected to an 8 mm long gap lesion repaired either by autograft (AG, n = 6) or tubulization with impermeable silicone tube (SIL, n = 6) or permeable tube of poly‐L‐lactide‐ϵ‐caprolactone (PLC, n = 8). Recordings of the compound muscle action potential (CMAP) from gastrocnemius (mGC), tibialis anterior (mTA) and plantar (mPL) muscles were performed 90 days after injury to assess the amount of muscle reinnervation. The CMAP amplitude achieved in mGC, mTA and mPL was similar in after nerve autograft (39%, 42%, 22% of control values) and PLC tube implantation (37%, 36%, 24%) but lower with SIL tube (29%, 30%, 14%). The nerve fascicles projecting into each of these muscles were then transected and retrograde tracers (Fluoro Gold, Fast Blue, DiI) were applied to quantify the percentage of motoneurons with single or multiple branches to different targets. The total number of labeled motoneurons for the three muscles did not differ in autografted rats (1186 ± 56; mean ± SEM) with respect to controls (1238 ± 82), but was reduced with PLC tube (802 ± 101) and SIL tube (935 ± 213). The percentage of neurons with multiple projections was lower after autograft and PLC tube (6%) than with SIL tube (10%). Considering the higher CMAP amplitude and lower number of neurons with multiple projections, PLC nerve conduits seem superior to SIL tubes and a suitable alternative to autografts for the repair of long gaps. J. Neurosci. Res. 63:214–223, 2001.

Transplantation of Olfactory Ensheathing Cells Stimulates the Collateral Sprouting from Axotomized Adult Rat Facial Motoneurons

Axon regrowth after CNS and PNS injury is only the first step toward complete functional recovery which depends largely on the specificity of the newly formed nerve-target projections. Since most of the studies involving the application of glial cells to the lesioned nervous system have focused primarily on the extent of neurite outgrowth, little is known regarding their effects on the accompanying processes of axonal sprouting and pathfinding. In this study, we analyzed the effects of transplanted olfactory ensheathing cells (OECs) on axonal sprouting of adult facial neurons by using triple fluorescent retrograde tracing and biometrical analysis of whisking behavior. We found that 2 months after facial nerve axotomy and immediate implantation of OECs in between both nerve stumps fixed in a silicon tube, the total number of labeled neurons was increased by about 100%, compared to animals with simple facial nerve suture or entubulation in an empty conduit. This change in the number of axon sprouts was not random. The highest increase in axon number was observed in the marginal mandibular branch, whereas no changes were detected in the zygomatic branch. This increased sprouting did not improve the whisking behavior as measured by biometric video analysis. Our results demonstrate that OECs are potent inducers of axonal sprouting in vivo. Hence OEC-filled nerve conduits may be a powerful tool to enforce regeneration of a peripheral nerve under adverse conditions, e.g., after long delay between injury and surgical repair. In mixed nerves, increased axonal sprouting will improve specificity since inappropriate nerve-target connections are pruned off during preferential motor innervation. In pure motor nerves, however, OEC-mediated axonal sprouting may result in polyneuronal innveration of target muscles.

Peripheral and spinal motor reorganization after nerve injury and repair.

Functional recovery after peripheral nerve injury depends on the amount as well as on the accuracy of reinnervation by regenerative axons. In this study, the rat sciatic nerve was subjected to crush injury or complete transection repaired by either (1) straight nerve suture, (2) crossed nerve suture of tibial and peroneal fascicles, or (3) silicone tubulization leaving a gap of 4 mm. The compound muscle action potentials (CMAP) of gastrocnemius, tibialis anterior and plantar muscles were recorded 90 days post operation to assess functional reinnervation and Fast Blue, Fluoro Gold and DiI were applied to the nerve branches projecting into these muscles to quantify morphological reinnervation. The CMAP amplitude achieved in gastrocnemius, tibialis anterior and plantar muscles was higher after nerve crush (86%, 82%, 65% of control) than after any surgical nerve repair (straight suture: 49%, 53%, 32%; crossed suture: 56%, 50%, 31%; silicone tube: 42%, 44%, 25%). The total number of labeled motoneurons, however, did not significantly differ between groups (control: 1238 +/- 82, crush: 1048 +/- 49, straight suture: 1175 +/- 106, crossed suture: 1085 +/- 84, silicone tube: 1250 +/- 182). The volume occupied by labeled motoneurons within the spinal cord was larger after surgical nerve repair than in crush or normal control animals, and fewer neurons showed abnormal multiple projections after crush (2.5%) or straight suture (2.2%) than following crossed suture (5%) or silicone tube (6%). In conclusion, nerve repair with a silicone tube leaving a short gap does not increase accuracy of reinnervation.

The hypoglossal-facial anastomosis as model of neuronal plasticity in the rat

Hypoglossal-facial cross anastomosis (HFA) causes regeneration with change of function, as the axotomized hypoglossal motoneurons sprout into the facial plexus and reinnervate the mimic musculature. Following HFA, hypoglossal-hypoglossal single anastomosis (HHA) and resection of 8-10 mm peripheral hypoglossal nerve in 190 female adult Wistar rats, we compared the axon reactions in the hypoglossal nucleus during 1) regeneration with change of function, 2) regeneration with restoration of original function and 3) degeneration of the nucleus. Following postoperative survival times of 1-16 weeks we estimated the volume of the hypoglossal nucleus and counted the number of hypoglossal neurons with the physical disector on both sides of the brainstem. Additional sections of the same animals were reacted with anti-synaptophysin, anti-GFAP and the isolectin Griffonia simplicifolia I-B4 (GSA I-B4) as cytochemical markers for presynaptic boutons, activated astroglia and microglia. After HHA and HFA all hypoglossal neurons survive and the volume of the hypoglossal nucleus remains constant. Resection of the hypoglossal nerve leads to the loss of one third of the hypoglossal neurons and of one third of the volume of the hypoglossal nucleus within 16 weeks post operation. Hypoglossal-facial anastomosis and hypoglossal-hypoglossal anastomosis differ in postoperative swelling of the hypoglossal nucleus, microglia and astroglia activation and the duration of synaptic stripping. All differences are limited to the acute growth phase during regeneration. It is concluded that hypoglossal-facial anastomosis provides more stimulation and facilitates faster recovery of the hypoglossal nucleus than does hypoglossal-hypoglossal anastomosis.

An example of neural plasticity evoked by putative behavioral demand and early use of vibrissal hairs after facial nerve transection.

Abnormally associated movements inevitably occur after surgical repair of the facial nerve. The reason for this postparalytic syndrome is poor navigation of regrowing axons. Despite the valuable functional advantage provided by the easily detected movement of vibrissae in rats, the major investigative tools for establishing the degree of misdirected reinnervation are still electrophysiologic recordings and retrograde tracing. In the present study we complemented data from pre- and postoperative retrograde labeling (FluoroGold, Fast Blue, DiI) of facial motoneurons with an evaluation of whisker movements. Using a video-based motion analysis system, we compared the recovery of vibrissae motor performance in visually normal and blind rats of the Sprague-Dawley strain. The analysis of whisker movement after facial nerve surgery revealed a striking discrepancy between morphologic and functional estimates. Whereas retrograde labeling displayed poor accuracy of target reinnervation and supernumerary axonal branching in both groups, the video-based motion analysis showed a perfect recovery of vibrissae movements in the blind rats. Attributing the complete recovery of whisker movement in the blind rats to an extraordinary plasticity of the facial motoneurons induced by putative behavioral demand and forced overuse, we conclude that the video-based analysis of whisker movement is a valuable tool for studying the progress in functional recovery.

DNA‐fragmentation and expression of apoptosis‐related proteins in experimentally denervated and reinnervated rat facial muscle

Muscle fibres may undergo apoptotic cell death in several neuromuscular disorders such as denervated muscle fibres in spinal muscular atrophies. We investigated DNA‐fragmentation (in situ by the TUNEL‐method) and expression of apoptosis‐associated proteins in experimentally denervated and reinnervated rat facial muscle up to 24 weeks after surgery to evaluate the rate and time lapse of apoptotic muscle fibre loss. While denervated muscle displayed constantly high rates of DNA‐fragmentation, denervated and immediately reinnervated muscle showed a distinct decrease of primarily elevated DNA‐cleavage, finally resembling rates of normal controls. Denervated muscle fibres revealed strong immunoreactivity of the anti‐apoptotic proteins bcl‐2 and bcl‐xL, and the pro‐apoptotic factor bax. In reinnervated muscle fibres, only bcl‐2 was constantly up‐regulated while bcl‐xL and bax diminished after the 7th week. The present findings indicate that denervation may prompt muscle fibres to activate an intrinsic ‘suicide’ programme to undergo apoptosis. High levels of bcl‐2 after denervation may sustain cell survival until reinnervation, e.g. after accidental nerve damage or in neurodegenerative disorders. Furthermore, increasing levels of bcl‐2 are able to neutralize high apoptosis‐promoting bax levels. Interventions modifying DNA‐fragmentation and the expression of apoptosis‐related proteins may lead to new therapeutic concepts in denervating disorders of muscle in the absence of other primary therapies.

Galectin‐3 is upregulated in microglial cells in response to ischemic brain lesions, but not to facial nerve axotomy

We have recently demonstrated that the β‐galactoside‐specific lectin galectin‐3 is expressed by microglial cells in vitro, but not by normal resting microglia in vivo. In the present study, we have analyzed the expression of galectin‐3 by microglia under traumatic conditions in vivo using two experimental rat models which substantially differ in the severity of lesion related to a breakdown of the blood‐brain barrier (BBB) and the occurrence of inflammatory processes. These two features are absent after peripheral nerve lesion and present after cerebral ischemia. Here we show that, following facial nerve axotomy under conditions allowing (nerve anastomosis) or not subsequent regeneration (nerve resection), galectin‐3 is not expressed by microglia in the corresponding facial nucleus 1–112 days after lesion. Galectin‐3 is also absent in microglia at sites of a defective BBB in the normal brain, such as the circumventricular organs. Following experimental ischemia (i.e., permanent occlusion of the middle cerebral artery), in contrast, galectin‐3 becomes strongly expressed by activated microglia as early as 48 hours after trauma, as determined by immunohistochemistry and Western blot analysis. Our findings suggest that the expression of galectin‐3 by microglia in vivo correlates with the state of microglial activation. J. Neurosci. Res. 61:430–435, 2000.