David Tonge

Effects of extracellular matrix components on axonal outgrowth from peripheral nerves of adult animals in vitro

Relatively little is known of the growth requirements for regenerating axons of the peripheral nervous system of adult animals. In the present study, we show that extracellular matrix material secreted by the Engelbreth-Holm-Swarm tumor cell line (matrigel) supports axonal growth from explanted peripheral nerve-dorsal root ganglia (DRG) preparations of adult mice and amphibia in serum-free media, without addition of growth factors. Axonal growth in matrigel was much more profuse than that in the more commonly used gels of type 1 collagen and, after some days in culture, was accompanied by migration of Schwann cells along axons. The most abundant protein in matrigel is laminin, which has been shown in many studies to support axonal growth but, surprisingly, antisera to laminin did not inhibit axonal growth in matrigel. To determine the ability of the major components of matrigel, laminin, type IV collagen, and heparan sulfate proteoglycan (HSPG), to support axonal growth, these proteins were added to preparations of mouse peripheral nerve-DRGs in type I collagen gels. Regenerating axons were significantly longer in the presence of laminin and type IV collagen than in control cultures, while HSPG had a slight inhibitory effect. In this assay system, however, diluted matrigel solution was even more effective in stimulating axonal growth than laminin or type IV collagen, either alone or in combination. The results suggest that in addition to laminin and type IV collagen, other components within matrigel may contribute to its ability to support axonal growth.

Upregulation of bradykinin B2 receptor expression by neurotrophic factors and nerve injury in mouse sensory neurons.

Bradykinin B2 receptor mRNA was detected at low levels, both by RT-PCR and by in situ hybridization, in freshly isolated dorsal root ganglia (DRG) and in ganglia cultured in the absence of neurotrophic factors, but was strongly upregulated by culture in the presence of nerve growth factor (NGF). The effect of NGF is mediated via TrkA receptors. The related neurotrophins, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, were ineffective in upregulating B2 mRNA, but a small upregulation was seen with the unrelated neurotrophin glial cell line-derived neurotrophic factor (GDNF). Surface membrane B2 receptor expression, detected by immunofluorescence using a B2-specific antibody, was low in outgrowing axons cultured in the absence of neurotrophic factors, but was elevated by addition of NGF or GDNF. Conditioned media prepared by incubating injured nerve, skin, or muscle had a similar effect to NGF in upregulating B2 mRNA and protein expression, and the activity was largely removed by neutralization of NGF in the conditioned medium with an anti-NGF antibody. After nerve crush injury in vivo an enhancement in B2 mRNA expression was seen, peaking after 7 days and returning to precrush levels after 14 days. In all conditions tested, the proportion of neurons expressing B2 mRNA remained the same at around 23% of small neurons, suggesting that upregulation only occurs in the B2-positive neurons. These experiments show that NGF, and to a lesser extent GDNF, upregulates the expression of bradykinin B2 mRNA and B2 receptor protein in the surface membrane of DRG neurons and that NGF is an important factor responsible for upregulating bradykinin B2 receptor expression after nerve crush injury in vivo.

Effective gene delivery to adult neurons by a modified form of electroporation

Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%). Neuronal GFP expression was observed within 18 h and continued for up to 14 days. This compares with values up to 60% of RGCs expressing GFP following infection with an HSV-1 vector. Adult rat dorsal root ganglion (DRG) neurons were also successfully transfected. Thus, in summary, Nucleofection provides the possibility for a fast and efficient method for cDNA delivery and study of gene function in adult mammalian neurons.

Effect of Lens Lesion on Neurite Outgrowth of Retinal Ganglion Cells in Vitro

Recent studies have shown that lens lesion promotes axonal regeneration in the optic nerve of adult rats. In the present investigations, dissociated retinal ganglion cells (RGC) from intact postnatal (P) 9-11 rats showed spontaneous neurite outgrowth on laminin-1, in contrast to RGC from intact P14-adult rats. Neurite outgrowth from P9-14 RGC on laminin-1 was promoted by prior lens lesion and also during coculture with lesioned lenses. Neurite outgrowth from adult RGC following prior lens lesion, or in cocultures with lesioned lenses, required the presence of laminin-2. In media conditioned by lesioned lenses, the stimulatory effect on neurite outgrowth was still observed in the presence of K252a (trk receptor blocker) and mAb 228 (which blocks the effects of leukemia inhibitory factor and ciliary neurotrophic factor). Together, these results suggest the existence of a neuritogenic factor(s) associated with the lesioned lens that belongs to neither the neurotrophin nor the gp130 cytokine family.

Axonal outgrowth and neuronal apoptosis in cultured adult mouse dorsal root ganglion preparations: effects of neurotrophins, of inhibition of neurotrophin actions and of prior axotomy.

Dorsal root ganglia (L4 and L5) with attached spinal roots and nerve stumps were isolated from young adult mice and cultured in a layer of extracellular matrix material (matrigel). Within one day, a large number of axons grew out from the cut ends of the nerve and the dorsal root. The average outgrowth length was more than doubled by nerve growth factor, which also strongly increased the number of fibres, showing extensive branching. There was also a significant outgrowth stimulation by neurotrophin-3, but no observable effect by brain-derived neurotrophic factor. In preparations isolated and cultured six days after peripheral nerve transection in vivo, there was an increase in both the outgrowth length (about 1.5- to 2-fold) and in the number of axons. Stimulation of axonal outgrowth, which concerned outgrowth from both the peripheral nerve and the dorsal root, could be further enhanced by the addition of nerve growth factor to the culture. K-252a, a selective inhibitor of neurotrophin receptor-associated tyrosine kinase activity, did not affect either the normal outgrowth or the increased outgrowth in pre-axotomized preparations, at a concentration which abolished the stimulating effects by exogenous nerve growth factor and neurotrophin-3. Under the culturing conditions used, spontaneous apoptosis occurred, but none of the neurotrophins tested, nor K-252a, affected the number of apoptotic neuronal cells analysed by nick-labelling DNA breaks at the end of a 48-h culturing period. Altogether, the present data suggest that for most dorsal root ganglia neurons, signalling through the trk receptors does not influence the apoptosis in vitro and is not required for either the spontaneous axonal outgrowth in matrigel or the increased outgrowth which occurs after prior axotomy in vivo.

Impaired Axonal Regeneration by Isolectin B4-Binding Dorsal Root Ganglion Neurons In Vitro

The subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, α7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system. In cultures of dissociated DRG neurons of adult mice on laminin, robust spontaneous neurite outgrowth from IB4-negative neurons occurs and is strongly enhanced by previous axotomy. In contrast, IB4-labeled neurons show little neurite outgrowth and do not express GAP 43, even after axotomy or culture with GDNF. Moreover, growth of their axons through collagen gels is impaired compared with other DRG neurons. To determine whether the sparse neurite outgrowth of IB4-labeled neurons is attributable to lack of integrin expression, DRG cultures were infected with a herpes simplex 1 vector encoding α7-integrin, but its forced expression failed to promote neurite outgrowth in either IB4-labeled or other DRG neurons or in cultured adult retinal ganglion cells. Forced coexpression of both α7-integrin and GAP 43 also failed to promote neurite outgrowth in IB4-labeled neurons. In addition, cultured sciatic nerve segments were found to release much lower levels of GDNF, demonstrated by ELISA, than nerve growth factor. These findings together with their impaired intrinsic axonal regeneration capacity may contribute to the known vulnerability of the IB4-labeled population of DRG neurons to peripheral nerve injury.

Involvement of alpha 7 beta 1 integrin in the conditioning-lesion effect on sensory axon regeneration

Conditioning lesions of peripheral nerves improve axonal regeneration after injury and involve changes in expression of proteins required for axonal growth. Integrin alpha7beta1 expression in motor and sensory neurons increases following nerve lesions and motor axon regeneration is impaired in alpha7 integrin KO mice (J. Neurosci. 20, 1822-1830). To investigate the role of alpha7beta1 integrin in sensory axon regeneration, dorsal root ganglia of adult mice were cultured in gels of laminin-rich extracellular matrix (Matrigel) or collagen. Normal dorsal root ganglia in Matrigel or collagen supplemented with laminin showed spontaneous axonal outgrowth, which was greatly increased in conditioned preparations, but only in the presence of laminin. Conditioned dorsal root ganglia from normal mice cultured with a blocking antibody to beta1 integrin and from alpha7 integrin KO mice showed reduced axonal growth in both Matrigel- and laminin-supplemented collagen gels. Enhanced axonal regeneration after conditioning lesions therefore involves increased responsiveness to laminin and integrin alpha7beta1 expression.

Effects of glial cell line-derived neurotrophic factor on axonal growth and apoptosis in adult mammalian sensory neurons in vitro

The effects of glial cell line-derived neurotrophic factor on axonal outgrowth and apoptosis were studied in vitro using explanted dorsal root ganglia-peripheral nerve preparations of adult mice. In gels of matrigel or collagen type 1, glial cell line-derived neurotrophic factor increased both the numbers and lengths of axons growing out of explanted preparations, although less effectively than nerve growth factor. Stimulation of axonal outgrowth by glial cell line-derived neurotrophic factor was unaffected by K252a, a protein kinase inhibitor which blocks the effects of nerve growth factor and other neurotrophins acting through trk receptors. To determine the phenotype of the axons responding to glial cell line-derived neurotrophic factor, preparations were stained using antibodies to trkA, calcitonin gene-related peptide, 200,000 mol. wt phosphorylated neurofilaments (monoclonal antibody RT97) and the lectin Bandeiraea simplicifolia 1B4. RT97 recognizes large diameter neurons whilst 1B4 labels small diameter neurons which broadly do not express neurotrophin receptors. In preparations cultured with glial cell line-derived neurotrophic factor, significant increases in the numbers of outgrowing axons labelled with RT97 and 1B4 were observed but the numbers of calcitonin gene-related peptide-positive axons were not significantly increased and their staining intensity was generally faint. In separate preparations it was found that in the presence of glial cell line-derived neurotrophic factor, the majority of the 1B4 labelled axons were trkA negative, indicating that this factor can stimulate axonal growth in this population of neurons which do not respond to the neurotrophins. Spontaneous apoptosis in neurons and satellite cells occurs in explanted preparations of the type used in the present investigations, but in cryostat sections of preparations cultured in the presence of glial cell line-derived neurotrophic factor, the incidence of apoptosis was lower than in control preparations which had been cultured in the absence of this factor. This suggests that glial cell line-derived neurotrophic factor may promote survival of some adult sensory neurons in vitro.

Use of explant cultures of peripheral nerves of adult vertebrates to study axonal regeneration in vitro.

Explanted preparations of peripheral nerves with attached dorsal root ganglia of adult mammals and amphibia survive for several days in serum-free medium and can be used to study axonal regeneration in vitro. This review outlines the methods which we routinely use and how they may be applied to study different aspects of axonal regeneration. When the peripheral nerves are crushed in vitro, axons regenerate through the crush site into the distal stump within 1 day (mouse) or 3 days (frog). The outgrowth distance of the leading sensory axons can be determined with the use of a simple method based on axonal transport of labelled proteins. A compartmentalised system permits selective application of drugs and other agents to either ganglia or peripheral nerve containing the regenerating axons and has been used to study selected aspects of regeneration including influence of non-neuronal cells, retrograde signalling, axonal release of proteins during regeneration and the role of phospholipase A2 activity. Explanted preparations may also be cultured in a layer of extracellular matrix material (matrigel), in which spontaneous outgrowth of a large number of naked axons from the cut ends of nerves starts within 1 day and continues for several days. This provides an opportunity to study the direct effects of different agents on axonal elongation. Preparations cultured in collagen gels show sparse spontaneous axonal growth, but this can be increased by addition of certain growth factors. The phenotype of the regenerating axons can be studied using immunohistochemical methods.

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons of the sciatic nerve

Monoclonal antibodies SMI-31 and 150 recognize phosphorylation epitopes on microtubule-associated protein 1B that have been shown to be developmentally down-regulated in the nervous system. We have used these antibodies to establish changes in the pattern of expression of their epitopes on microtubule-associated protein 1B in regenerating axons of the sciatic nerves in the adult mouse and rat. Immunohistochemical studies showed that, in the sciatic nerve, regenerating axons in both adult mice and rats were labelled with monoclonal antibody 150 in a proximodistal gradient which was highest at the growth cone. This is the first report of expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons. Immunoblotting showed that the expression of the isoform recognized by monoclonal antibody 150 is present in normal adult mouse sciatic nerve and in regenerating axons following crush or cut lesions, but was not detectable in the normal or regenerating adult rat peripheral nervous system. Regenerating axons were also labelled by monoclonal antibody SMI-31, but the labelling, unlike antibody 150 labelling, was uniform along the entire length of the axon and immunoblotting showed that it was due to recognition of neurofilament protein. We conclude that the phosphorylated isoforms of microtubule-associated protein 1B recognized by monoclonal antibody 150 that are developmentally down-regulated in the adult rat central and peripheral nervous systems and adult mouse cerebellum are maintained in the normal peripheral nervous system of the adult mouse. When peripheral axons regenerate in the adult mouse, the regenerating axons also contain these isoforms. Adult rat regenerating axons are stained by antibody 150 only in tissue sections, not in immunoblots. The maintenance of immature isoforms of microtubule-associated protein 1B in mouse peripheral axons may relate to a continual capacity for growth and remodelling. The immunohistochemical localization of the antibody 150 epitope in growth cone-like structures and sprouts in injured nerves shows that phosphorylation of microtubule-associated protein 1B is likely to be an integral part of the regenerative response. These results also show that the phosphorylation epitopes on microtubule-associated protein 1B recognized by monoclonal antibodies 150 and SMI-31 are different and that only expression of the former correlates with axonal regeneration.