Konstantin Wewetzer

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.

Transplantation of olfactory mucosa minimizes axonal branching and promotes the recovery of vibrissae motor performance after facial nerve repair in rats.

The occurrence of abnormally associated movements is inevitable after facial nerve transection. The reason for this post-paralytic syndrome is poor guidance of regrowing axons, whereby a given muscle group is reinnervated by misrouted axonal branches. Olfactory ensheathing glia have been shown to reduce axonal sprouting and stimulate axonal regeneration after transplantation into the spinal cord. In the present study, we asked whether transplantation of olfactory mucosa (OM) would also reduce sprouting of a damaged peripheral pure motor nerve. The adult facial nerve was transected, and the effect of the OM placed at the lesion site was analyzed with regard to the accuracy of target reinnervation, axonal sprouting of motoneurons, and vibrissal motor performance. Accuracy of target reinnervation and axonal sprouting were studied using preoperative/postoperative labeling and triple retrograde labeling of facial motoneurons, respectively. The vibrissal motor performance was monitored using a video-based motion analysis. We show here that implantation of OM, compared with simple facial–facial anastomosis, (1) improved the protraction, amplitude, angular velocity, and acceleration of vibrissal movements up to 80% of the control values, (2) reduced the percentage of branching motoneurons from 76 to 39%, and (3) improved the accuracy of reinnervation from 22 to 49%. Moreover, we present evidence, that transplanted OM but not buccal mucous membrane induced a sustained upregulation of trophic factors at the lesion site. It is concluded that transplantation of OM to the transected facial nerve significantly improves nerve regeneration.

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.

Remyelination of the nonhuman primate spinal cord by transplantation of H-transferase transgenic adult pig olfactory ensheathing cells

Olfactory ensheathing cells (OECs) have been shown to mediate remyelination and to stimulate axonal regeneration in a number of in vivo rodent spinal cord studies. However, whether OECs display similar properties in the primate model has not been tested so far. In the present study, we thus transplanted highly‐purified OECs isolated from transgenic pigs expressing the α1,2 fucosyltransferase gene (H‐transferase or HT) gene into a demyelinated lesion of the African green monkey spinal cord. Four weeks posttransplantation, robust remyelination was found in 62.5% of the lesion sites, whereas there was virtually no remyelination in the nontransplanted controls. This together with the immunohistochemical demonstration of the grafted cells within the lesioned area confirmed that remyelination was indeed achieved by OECs. Additional in vitro assays demonstrated l) that the applied cell suspension consisted of >98% OECs, 2) that the majority of the cells expressed the transgene, and 3) that expression of the HT gene reduced complement activation more than twofold compared with the nontransgenic control. This is the first demonstration that xenotransplantation of characterized OECs into the primate spinal cord results in remyelination.

Expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 messenger RNAs in spinal ganglia and sciatic nerve: regulation after peripheral nerve lesion.

In order to determine functional roles of basic fibroblast growth factor (FGF-2) in the peripheral nervous system we have analysed the expression of FGF-2 and FGF receptor 1 (FGFR1) in spinal ganglia and the sciatic nerve under normal conditions and after nerve crush using RNAse protection assay and in situ hybridization. In intact spinal ganglia, both FGF-2 and FGFR1 messenger RNAs are expressed, albeit at different levels. In situ hybridization identifies satellite cells as the source of FGF-2 and sensory neurons as the source of FGFR1 suggesting a paracrine mode of action of FGF-2 on sensory neurons. One day after crush lesion FGF-2 is significantly up-regulated in sensory ganglia L4-L6. Highest levels are found at day 7; control levels are approached after 28 days. FGFR1 messenger RNA, which is strongly expressed in intact spinal ganglia, displays no significant change after lesion. In the intact sciatic nerve, FGFR1 messenger RNA is detected at higher levels than FGF-2 messenger RNA. After injury, both transcripts display a time-dependent up-regulation in both the proximal and distal nerve stump. Schwann cells, as a putative source of the sciatic nerve-derived FGF-2, express both FGF-2 and FGFR1 messenger RNAs in vitro. The FGFR1 transcript level is increased in the presence of forskolin. FGF-2 does not affect expression of FGFR1 messenger RNA but stimulates its own expression. These results show that during peripheral nerve regeneration FGF-2 is up-regulated in both the crushed nerve and the respective spinal ganglia suggesting a possible physiological function of FGF-2 during the regeneration process.

Phagocytosis of O4+ axonal fragments in vitro by p75− neonatal rat olfactory ensheathing cells

Olfactory ensheathing cells (OECs) have gained wide interest because of their unique regeneration‐promoting capacity. However, despite their frequent use in regeneration studies, the characterization of the cells has remained fragmentary. In the present study, we analyzed freshly dissociated neonatal rat OECs at the light and electron microscopic level and studied their fate in vitro using a novel two‐step labeling protocol based on antibody internalization. We report the identification and characterization of two distinct OEC populations in situ and in primary cell suspensions that differed in number, p75 NGF receptor expression, and O4 immunoreactivity. The major OEC population in primary cells suspensions did not express p75 but stained positive for the glycolipid O4 (p75−/O4+). During culturing, these cells upregulated p75 expression and lost O4 immunoreactivity. Conversely, the minor OEC population consisted of p75+/O4− OECs that maintained p75 expression in vitro. Interestingly, ultrastructural analysis revealed not only that O4 immunoreactivity of p75− OECs was, in fact, due to O4+ axonal fragments adhering to the cell surface but also that p75− OECs rapidly phagocytosed these fragments in vitro. Taken together, the identification of two distinct OEC populations in the neonatal olfactory bulb that converge into single p75+ phenotype in vitro is reported. The observation that upregulation of p75 receptor expression in vitro was only apparent in those OECs closely associated with O4+ axonal processes may suggest that axonal signalling in vivo negatively regulates p75 receptor expression. The strong phagocytic activity of OECs in vitro may reflect one important aspect of their physiological function.

In vitro expression and regulation of ciliary neurotrophic factor and its α receptor subunit in neonatal rat olfactory ensheathing cells

During development and in the adult, ciliary neurotrophic factor (CNTF) is expressed at high levels in the olfactory system. In the present study, we asked whether neonatal rat olfactory ensheathing cells (OECs) express CNTF- and CNTF receptoralpha (CNTFRalpha)-mRNA in vitro and studied the regulation of both transcripts in response to growth factor and forskolin (FSK) treatment. We show here that OECs in vitro express CNTF and CNTFRalpha-mRNA under control conditions. Administration of FSK increased the expression of CNTFRalpha while lowering the levels of CNTF. Contrary to fibroblast growth factor-2, CNTF did not stimulate the proliferation of OECs. The observation that OECs express both the ligand and part of its receptor complex may indicate that CNTF exerts paracrine and/or autocrine effects in vivo, which apparently do not include the regulation of cell division.

Species-specific control of cellular proliferation and the impact of large animal models for the use of olfactory ensheathing cells and Schwann cells in spinal cord repair

Autologous transplantation of olfactory ensheathing cells (OECs) and Schwann cells (SCs) is considered a promising option to promote axonal regrowth and remyelination after spinal cord injury in humans. However, if the experimental data from the rodent model can be directly extrapolated to humans, as widely believed, remains to be established. While limitations of the rodent system have recently been discussed with regard to the distinct organization of the motor systems, the question whether OECs and SCs may display species-specific properties has not been fully addressed. Prompted by recent studies on canine and porcine glia, we performed a detailed analysis of the in vitro and in vivo properties of OECs and SCs and show that rodent but not human, monkey, porcine, and canine glia require mitogens for in vitro expansion, display a complex response to elevated intracellular cAMP, and undergo spontaneous immortalization upon prolonged mitogen stimulation. These data indicate fundamental inter-species differences of the control of cellular proliferation. Whether OECs and SCs from large animals and humans share growth-promoting in vivo properties with their rodent counterpart is not yet clear. Autologous implantation studies in humans did not reveal adverse effects of cell transplantation so far. However, in vivo studies of large animal or human glia and rodent recipients mainly focused on the remyelinating potential of the transplanted cells. Thus, further experimental in vivo studies in large animals are essential to fully define the axonal growth-promoting potential of OECs and SCs. Based on the homology of the in vitro growth control between porcine, canine and human glia, it is concluded that these species may serve as valuable translational models for scaling up human procedures. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Expression of interleukin-6 and its receptor in the sciatic nerve and cultured Schwann cells: relation to 18-kD fibroblast growth factor-2

Expression of interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2) in Schwann cells is modulated by external stimuli. To study possible interactions of both factors we have analyzed mutual effects of exogenous IL-6 and FGF-2 on the expression of each other and the corresponding receptor (R) molecules IL-6R and FGFR1 after peripheral nerve lesion in vivo and in vitro using cultured Schwann cells. Using rat Schwann cells we found that IL-6 did not exert any effects on the expression of FGF-2 and FGF receptor type 1 (R1) whereas exogenously applied 18-kD FGF-2 strongly increased the expression of the mRNAs of IL-6 and its receptor. In addition, immortalized Schwann cells over-expressing the 18-kD FGF-2 isoform showed elevated levels of IL-6 and IL-6R whereas immortalized Schwann cells over-expressing the high-molecular-weight isoforms (21 kD and 23 kD) displayed unaltered IL-6 and IL-6R expression levels. According to in situ hybridization studies of intact and crushed sciatic nerves in vivo, Schwann cells seems to be the main source of IL-6 and IL-6R. Following sciatic nerve crush, the FGF-2 and the IL-6 system are upregulated after the first hours. Furthermore, we showed that the early increase of the FGF-2 protein is mainly confined to the 18-kD isoform. These results are consistent with the idea of a functional coupling of FGF-2 and the IL-6 system in the early reaction of Schwann cells to nerve injury.

Prominent microglial activation in the early proinflammatory immune response in naturally occurring canine spinal cord injury.

Better understanding of the pathogenesis of spinal cord injury (SCI) is needed for the development of new therapeutic strategies. Spinal cord injury has been investigated in various rodent models, but extrapolation to humans requires the use of a large animal model that more closely mimics human SCI. Dogs frequently develop spontaneous SCI with features that bear a striking resemblance to the human counterpart. We investigated the temporal course of the immuneresponse during naturally occurring canine SCI and in organotypic canine spinal cord slice cultures that are devoid of peripheral immune cells. By immunohistochemistry, the inflammatory response in subacute canine SCI was largely restricted to resident immune cells as demonstrated by activation of major histocompatibility complex class II-expressing microglia/macrophages. By quantitative polymerase chain reaction, there was parallel upregulation of proinflammatorycytokine gene expression (i.e. of interleukin 6 [IL-6] and IL-8 witha trend toward upregulation of tumor necrosis factor) in acute canine SCI. Expression of neuroprotective cytokines (e.g. IL-10) remained unchanged, and transforming growth factor &bgr; upregulationwas delayed. In organotypic spinal cord slices, there was similar activation of major histocompatibility complex class II-positive microglia and prolonged upregulation of inflammatory cytokines, indicating that resident rather than infiltrating cells play major roles in the postinjury immune response. Thus, canine SCI represents a bridge between rodent models and human SCI that may be relevant for clinical and preclinical treatment studies.