Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway.

Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction between the root and spinal cord by reactive astrocytes. Do these cells stop axonal elongation by activating the physiological mechanisms that normally operate to stop axons during development, or do they physically obstruct the elongating axons? In order to distinguish these possibilities, the cytology of the axon tips of regenerating axons that were stopped by astrocytes was compared with the axon tips that were physically obstructed at a cul-de-sac produced by ligating a peripheral nerve. The terminals of the physically obstructed axon tips were distended with neurofilaments and other axonally transported structures that had accumulated when the axons stopped elongating. By contrast, neurofilaments did not accumulate in the tips of regenerating axons that were stopped by spinal cord astrocytes at the dorsal root transitional zone. These axo-glial terminals resembled the terminals that axons make on target neurons during normal development. On the basis of these observations, astrocytes appear to stop axons from regenerating in the mammalian spinal cord by activating the physiological stop pathway that is built into the axon and that normally operates when axons form stable terminals on target cells.

Diabetic neuropathies.

Diabetic neuropathy is a common complication of diabetes that may be associated both with considerable morbidity (painful polyneuropathy, neuropathic ulceration) and mortality (autonomic neuropathy). The epidemiology and natural history of diabetic neuropathy is clouded with uncertainty, largely caused by confusion in the definition and measurement of this disorder. We have reviewed various clinical manifestations associated with somatic and autonomic neuropathy, and we herein discuss current views related to the management of the various abnormalities. Although unproven, the best evidence suggests that near-normal control of blood glucose in the early years after diabetes onset may help delay the development of clinically significant nerve impairment. Intensive therapy to achieve normalization of blood glucose also may lead to reversibility of early diabetic neuropathy, but again, this is unproven. Our ability to manage successfully the many different manifestations of diabetic neuropathy depends ultimately on our success in uncovering the pathogenic processes underlying this disorder. The recent resurgence of interest in the vascular hypothesis, for example, has opened up new avenues of investigation for therapeutic intervention. Paralleling our increased understanding of the pathogenesis of diabetic neuropathy, refinements must be made in our ability to measure quantitatively the different types of defects that occur in this disorder. These tests must be validated and standardized to allow comparability between studies and more meaningful interpretation of study results.

Regeneration of lumbar dorsal root axons into the spinal cord of adult frogs (Rana pipiens), an HRP study.

Lumbar dorsal roots of adult frogs were crushed or cut and reanastomosed. Following survival times of up to 75 days, the regenerating dorsal roots were recut and anterogradely injury-filled with horseradish peroxidase. This revealed that in the adult frog, regenerating axons re-enter the spinal cord. Comparison of the distribution of these axons with that of normal dorsal root axons showed that there is a partial restoration of the segmental distribution in the gray matter. However, the long ascending sensory tract of the dorsal funiculus was not restored. The dorsal funiculus was markedly gliotic and had relatively few labelled, regenerated axons. The labelled axons that were seen in the dorsal funiculus either extended longitudinally for a distance just beneath the pia, apparently in association with the glia limitans, or traversed the region to enter the dorsal gray matter. Most of the large and small diameter axons that entered the gray matter did so by passing through the region of the dorsolateral fasciculus. Within the gray matter, small diameter, regenerated axons arborized in the region of the dorsal terminal field, a region that has been shown in the normal frog to receive cutaneous afferents only. Many large diameter axons, presumably muscle afferents, arborized in the ventral terminal field, a region shown in the normal frog to receive muscle afferents exclusively. However, many of these large diameter axons had arborizations that extended to both terminal fields, thus suggesting that some abberant connections are made during dorsal root regeneration in the adult frog.

Regeneration of motoneuron axons into the adult frog spinal cord after ventral-to-dorsal-root anastomosis.

Motoneuron axons routed into the adult frog spinal cord via a ventral-to-dorsal-root anastomosis regenerated into the white and the gray matters. The distribution, growth patterns, and arborizations of regenerated ventral root axons were compared to those of regenerated dorsal root axons within the same environment. Within the spinal white matter, regenerating ventral root axons behaved very similarly to regenerating dorsal root axons. Here, the regenerating ventral root axons grew longitudinally beneath the pia and radially toward the spinal gray matter, particularly within the dorsolateral fasciculus. The location of the regenerating axons and the patterns of their growth within the white matter suggest that glial endfeet and radial glial processes play a major role in the determination of these axonal growth patterns. When motor axons entered the gray matter, their arborizations were very similar to those of regenerated dorsal root axons, suggesting that these two very distinct populations of axons respond similarly to local cues within the spinal gray matter. One difference between the arborizations of these two populations of axons was the relative number of varicosities along axonal branches. Regenerated motoneuronal arborizations within the spinal gray matter had fewer en passant varicosities than regenerated dorsal root axonal arborizations. This difference may reflect the synaptogenetic response of the two types of axons to targets within the gray matter. The low number of en passant varicosities associated with the ventral root axonal aborizations suggests that these axons do not synapse with all available targets and that the rules governing synaptic specificity during development may apply during regeneration in the adult frog spinal cord.

Involvement of an axonal reflex in IgE-mediated inflammation in mouse skin.

A neurogenic component of IgE-mediated inflammation was demonstrated in mice by footpad denervation. Footpad swelling was reduced 26% following sciatic nerve transection, but unaffected by rhizotomy or spinal nerve transection. These data provide in vivo evidence that an axonal reflex is involved in IgE-mediated inflammation and completed distal to the cell bodies of the sensory neurons located in the lumbar spinal ganglia. Furthermore, depletion of neuropeptides with capsaicin also reduced IgE-mediated swelling by 26%, indicating that unmyelinated axons are involved in the neurogenic component of IgE-mediated inflammation.

The relationship of dorsal root afferents to motoneuron somata and dendrites in the adult bullfrog: a light and electron microscopic study using horseradish peroxidase.

The relationship of lumbar dorsal root afferents to lateral motor column motoneurons was studied using anterograde injury filling of dorsal roots and retrograde injury filling of ventral roots with horseradish peroxidase. At the light microscopic level, horseradish peroxidase labelled dorsal root axons were observed to separate into a medial division of large diameter axons which enter the dorsal funiculus and a lateral division of small diameter axons which form a compact bundle in the dorsolateral funiculus which may be homologous to the mammalian tract of Lissauer. Within the spinal gray, primary afferents terminate in two distinct regions. The more ventral of these terminal fields, which receives collaterals of primary afferent axons in the dorsal funiculus, overlaps the dendritic arborizations of the lateral motor column motoneurons. Some axons leave the ventral terminal field to enter the dorsal lateral motor column. Here they terminate on the primary dendrites and somata of lateral motor column motoneurons. At the electron microscopic level, labelled primary afferent terminals were seen to synapse upon lateral motor column motoneuron dendrites as well as upon the somata of dorsally positioned lateral motor column motoneurons. These terminals contain small spherical vesicles and occasional dense-cored vesicles. The synaptic specializations are characterized by a small amount of postsynaptic material. The lateral motor column may be divided into dorsal and ventral portions on the basis of the primary afferent distribution and this is in accord with functional, physiological and developmental data.

Neuronal nitric oxide synthase is induced in spinal neurons by traumatic injury.

Nitric oxide appears to mediate the immune functions of macrophages, the influence of endothelial cells on blood vessel relaxation, and also to serve as a neurotransmitter in the central and peripheral nervous system. Macrophage nitric oxide synthase is inducible with massive increases in new nitric oxide synthase protein synthesis following immune stimulation of macrophages. By contrast, endothelial nitric oxide synthase and neuronal nitric oxide synthase are thought to be constitutive with activation induced by calcium entry into cells in the absence of new protein synthesis. Developmental studies showing the transient expression of neuronal nitric oxide synthase in embryonic and early postnatal life in rodent spinal motoneurons and cerebral cortical plate neurons (Bredt and Snyder, unpublished observations) implies inducibility of neuronal nitric oxide synthase. Moreover, neuronal nitric oxide synthase expression is greatly enhanced in sensory ganglia following peripheral axotomy. Staining for NADPH diaphorase in spinal motoneurons is greatly increased following ventral root avulsion. In many parts of the Central Nervous System NADPH diaphorase staining reflects nitric oxide synthase. In the present study, we have combined in situ hybridization for neuronal nitric oxide synthase, immunohistochemical staining of neuronal nitric oxide synthase, and NADPH diaphorase staining to establish that neuronal nitric oxide synthase expression is markedly augmented in spinal motoneurons following avulsion. The generality of this effect is evident from augmented staining in nucleus dorsalis following spinal cord transection.

Proteolysis is a critical step in the physiological stop pathway: mechanisms involved in the blockade of axonal regeneration by mammalian astrocytes.

Regenerating axons of adult dorsal roots are stopped by reactive astrocytes at the PNS-CNS junction. While it has been suggested that the astrocytes might pose a physical barrier to axonal growth, based on ultrastructural comparisons of physically blocked and axo-glial endings, it was proposed that astrocytes in the root transitional zone block axonal growth by activating the physiological stop pathway within the growing axon tips. Part of the stop pathway involves the proteolytic breakdown and removal of neurofilaments as they enter the axon endings. Another component involves the establishment of anterograde-to-retrograde conversion for the removal of membranous elements from the axonal endings. Both of these components appear to be dependent upon the activation of proteases within the axon tips. Therefore, to further test our hypothesis we infused, by intrathecal catheterization, the region of the dorsal root transitional zone with the protease inhibitor leupeptin at a time when the majority of regenerating axons have terminated in the region. Ultrastructural analyses after leupeptin treatment revealed axo-glial endings distended by accumulations of neurofilaments and organelles, particularly tubulovesicular profiles. These observations further support the idea that astrocytes, like normal target cells, can activate the physiological stop pathway.

Radially oriented astrocytes in the normal adult rat spinal cord.

Glial cell organization in the adult rat spinal cord was studied using a modified Golgi technique and anti-GFAP immunofluorescence. Gray matter astrocytes appeared to be a homogeneous population, while in white matter, two morphologically distinct astrocyte subpopulations were seen. One astrocyte had the morphological characteristics of classically described fibrous astrocytes. However, the predominant astrocyte was a radially oriented cell which appeared to span the white matter from the pial surface to the gray-white interface.

Is CNS trauma a prerequisite for the elongation of CNS axons into denervated peripheral nerve?

The demonstration that some central nervous system (CNS) axons can regenerate when provided with a suitable environment raises the possibility of new treatments for CNS injury. However, at present the conditions for optimal regeneration are not well understood. For example, the methods used in previous studies have entailed CNS trauma as part of the research protocol (e.g. that resulting from the implantation of peripheral nerve grafts), and so the role of neuronal or axonal injury in the regrowth observed has been difficult to establish. To determine whether such injury is necessary for the central reinnervation of denervated peripheral nerve, the L5 dorsal root has been chronically denervated in rats by freeze-thawing its dorsal root ganglion (DRG), and the root has been left attached to either traumatized or non-traumatized spinal cord. The trauma induced was quite mild, and resulted from several vertical insertions of a fine needle. Two to 4 months later, retrogradely transported horseradish peroxidase (HRP) was used to label spinal neurons which sent axons into the denervated roots. HRP-labelled neurons were found in each of the spinal cords subjected to trauma, but no labelled neurons were observed in any of the non-traumatized cords. The number of HRP-labelled neurons in individual spinal cords was positively correlated with the degree of spinal cord trauma. We conclude first that the chronic and intimate presence of a denervated PNS tissue in continuity with the spinal cord is not, in itself, a sufficient stimulus to induce its reinnervation by CNS axons. Second, we conclude that under the conditions of this experiment CNS trauma is a prerequisite for the reinnervation of denervated peripheral nervous tissue by CNS axons.

Dorsal root afferents contact migrating motoneurons in the developing frog spinal cord.

Dorsal and ventral roots of Rana catesbeiana tadpole lumbar spinal cord were labeled with horseradish peroxidase during development of the hindlimb. Labeled motoneurons in the process of migrating were found in the region of the developing terminal arbors of dorsal root axons. Electron microscopy showed that some of these dorsal root terminals contacted the migrating motoneurons.

Axotomized frog sciatic nerve releases diffusible neurite-promoting factors.

Using the bullfrog (Rana catesbeiana) dorsal root ganglia (DRG) and its sciatic nerve (ScN) as a model system, we have previously described neuronal and non-neuronal molecular changes associated with the early regenerative response of DRG neurons to axotomy. Since diffusible molecular factors, released by axotomized ScN, might function to stimulate axon regrowth, we have assayed the ability of ScN-conditioned bath to promote in vitro neurite outgrowth from PC-12 cells. Diffusible ScN proteins were collected by incubating segments of normal or axotomized ScN in a small volume of RPMI media for 4 h (nerve bath). The nerve baths, supplemented with serum, were then added to PC-12 cell cultures to assay for the presence of neurite growth factors released by ScN. Results showed that nerve baths, collected from sham-operated or axotomized ScN, could not induce the differentiation of PC-12 into neurite-bearing cells. Therefore, in all subsequent neurite growth assay experiments, an exogenous source of nerve growth factor (NGF) (50 ng/ml) was added to the nerve baths or unconditioned media to generate and maintain PC-12 neuritic structure. We found that nerve baths, collected from previously axotomized (at least 3 days post-injury) nerve, contained diffusible factors which enhanced PC-12 neurite growth, relative to unconditioned media. No neurite growth factors were observed to be released by sham-operated ScN or 1-day post-axotomized ScN. Further experiments were conducted to identify the diffusible neurite growth factors released from axotomized ScN. We showed that the release (if any) of endogenous diffusible NGF or laminin from axotomized nerve could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed that the relative abundance of two diffusible proteins (M(r) approximately 35 and 70 kDa) in the nerve bath was directly correlated with the ability of the nerve bath to facilitate PC-12 neurite growth.

The development of the relationship between dorsal root afferents and motoneurons in the larval bullfrog spinal cord.

The relationship of dorsal root afferents to motoneuron somata and dendrites was studied by labelling dorsal and ventral roots of the tadpole lumbar enlargement with HRP at different stages of hindlimb development. Procedures were used which allowed for sequential light and electron microscopic analysis to determine whether close appositions between labelled elements represented synaptic contacts. Lateral motor column (LMC) motoneuron dendrites grow first into the lateral funiculus, and later begin arborizing within the spinal gray, concurrent with the arrival of developing dorsal root afferent fibers. Mature-appearing synaptic contacts between dorsal root afferents and motoneuron dendrites are established first on distal dendrites, and are observed on progressively more proximal dendrites as hindlimb development proceeds. Migrating motoneurons were also labelled in some animals. Distinct dorsal and ventral migratory pathways were noted; cells migrating dorsally were contacted by developing dorsal root afferents. Migrating motoneurons were associated with radially oriented processes, and were often closely apposed to other cells. The coincident development of dorsal root projections and the motoneuron dendrites which these fibers innervate in the adult, as well as the interaction between these two systems during cell migration, suggest that these two systems may be interdependent in establishing their normal relationship during development.

Peripheral nerve regeneration.

The success of peripheral nerve regeneration is dependent on the survival of axotomized neurons, the efficacy of axonal outgrowth from those neurons, and the specificity of reinnervation of peripheral targets by those neurons. Experimental evidence indicates that following peripheral injury, primary sensory (DRG) neurons and in some cases, motoneurons are lost. This cell death, which can involve one third or more of the axotomized neurons, suggests that some neurons in the adult are dependent on nerve or target-derived neurotrophic factors. One of these factors, NGF, when supplied to the cut proximal stump of the sciatic nerve, can save 100% of the DRG neurons that would normally succumb to axonal injury. But not all neurons are NGF-dependent, and other factors, including gonadal hormones, may be important to their survival following axotomy. Axonal elongation following peripheral nerve injury is dependent upon molecules in the extracellular matrix as well as secreted molecules from nonneuronal cells within the distal stump of the nerve. Extracellular matrix molecules such as laminin provide an adhesive substrate for axonal growth; but Schwann cells in the distal stump, which have been shown to synthesize increased amounts of NGF following peripheral nerve injury, appear to be essential for axonal elongation. Although neuronal survival and the efficacy of axonal elongation are important to peripheral nerve regeneration, the most important determinant of the success of peripheral nerve regeneration is the specificity of reinnervation. There remains some debate over whether regenerating axons are physically guided to the appropriate targets by mechanical guides in the form of basal laminar tubes, or whether they are lured by neurotropic factors derived from the distal nerve stump and targets. There is evidence that both factors are operative in the adult PNS. However, although recent data suggest that neurotropic factors within the adult nerve can influence the sorting of regenerating axons, clinical and experimental data indicate that physical constraints of nerve cytoarchitecture can override those tropic factors. Finally, although some degree of specificity of reinnervation of peripheral targets has been demonstrated, particularly for sensory receptors in skin and muscle, there are typically perturbations of sensation and movement due to axonal misrouting and aberrant reinnervation. Further laboratory research is needed to understand how neuron-target specificity is established during development of the PNS and to determine how the developmental mechanisms can be exploited to reestablish that specificity following peripheral nerve injury.

Neovascularization occurs in response to crush lesions of adult frog optic nerves.

The capacity of the adult frog optic nerve to regenerate following a crush lesion is well established and is in contrast to the lack of regeneration of mammalian optic nerves after similar lesions. One factor which may contribute to the enhanced regenerative capacity of amphibian optic nerves is the rapid removal of cellular debris from the nerve after injury. In this study the morphology of normal and crushed frog optic nerves has been compared. Although the intraorbital region of the normal adult frog optic nerve is avascular, new intraparenchymal blood vessels appear central to the crush site 24 h after the nerve lesion. The appearance of these blood vessels is coincident with the appearance of granulocytes and macrophages in the nerve. Successful regeneration of the adult frog optic nerve may depend on this neovascularization to facilitate the rapid removal of cellular debris and to supply regenerating axons with trophic substances.

Axo-glial interactions at the dorsal root transitional zone regulate neurofilament protein synthesis in axotomized sensory neurons.

After dorsal root crush, dramatic ultrastructural differences are observed between regenerated dorsal root axonal endings that are physically blocked at a ligation neuroma and those that are allowed to form axo-glial endings among the astrocytes at the dorsal root transitional zone (DRTZ). Physically blocked axonal endings swell immensely with membranous organelles and neurofilaments (NFs) while axo-glial endings do not, suggesting that DRTZ astrocytes stop axonal growth by activating a physiological stop pathway within those endings. Since protease-dependent NF degradation at axonal endings is a part of this pathway, this study addresses the question of whether NF subunit synthesis in the dorsal root ganglion (DRG) is regulated by the pathway. Lumbar dorsal roots were crushed and, at various postinjury times, the attached DRGs were removed and pulse-labeled in vitro with 35S-methionine for subsequent analysis of protein synthesis by electrophoresis and fluorography. Within 24 hr of axotomy, there was a down-regulation of the 68 kDa (NF-L) and 145 kDa (NF-M) NF subunits. At 14 d postcrush, a time when most of the regenerating axons have reached and been stopped by DRTZ astrocytes, NF protein synthesis returned to control levels. By contrast, when the axons were prevented from reaching the DRTZ by ligating or removing segments of the roots, NF synthesis failed to return to normal levels. These data suggest that activation of the physiological stop pathway by DRTZ astrocytes regulates NF protein synthesis in the DRG.

Dorsal root axonal regeneration in the adult frog spinal cord. A model of vertebrate CNS regeneration.

The frog dorsal root provides a useful model for the study of axonal regeneration in an adult vertebrate CNS. We have used the model to compare the regeneration of two very different types of axons within the same CNS environment and have found that regenerating dorsal root, as well as rerouted motoneuron axons, display similar growth patterns in the spinal cord. Both sensory and motor axons grow preferentially in some regions and not in others. They both regenerate effectively longitudinally as well as radially within the dorsolateral fasciculus (DLF). By contrast, fewer sensory and motor axons regenerate longitudinally or radially in the dorsal funiculus (DF). This similar preferential growth of two very different populations of axons suggests that the growth patterns reflect regional differences in the cellular environment of the cord. The DLF has fascicles of unmyelinated axons separated by radial glial processes and, after dorsal root injury, is mildly gliotic. By contrast, DF has very large myelinated axons, which widely separate the radial glial processes that traverse the region. After dorsal root injury, this region is markedly gliotic and contains myelin, debris and oligodendroglia, and microglial macrophages. Our data suggest that unmyelinated axons and radial glial processes are more preferred substrates for axonal growth than myelin debris, oligodendroglia and macrophages. It is not surprising, then, that regions of the adult mammalian CNS that are characterized by large myelinated axons fail to support axonal growth. Moreover, there is some evidence that regions of the adult mammalian CNS that are characterized by unmyelinated axons support axonal growth.

Regional specialization of the radial glial cells of the adult frog spinal cord.

The amphibian spinal cord is characterized by the presence of radially oriented astrocytic glial cells. These cells have their somata located in the grey matter of the spinal cord and radial processes that extend from the soma through the grey and white matters to the pial surface of the cord. Here we show that these radial glial cells are the predominant cell type labelled by horseradish peroxidase (HRP) when the marker is applied to the surface of the cord. The morphology of the HRP-labelled processes of an individual cell is different as they pass through the grey and white matter regions of the cord. By indirect immunofluorescence on frozen sections we show that the binding of an antibody raised against mammalian glial fibrillary acidic protein (GFAP) is preferentially localized in those areas of the glial process that traverse the white matter of the spinal cord. By transmission electron microscopy we confirm that there are no astrocyte cell bodies either at the pial surface or throughout the white matter region of the cord. These results demonstrate that all the astrocytes in the adult frog spinal cord can be selectively labelled through the application of HRP to the surface of the cord, and that the processes of these labelled cells display regional morphological and biochemical specializations depending on their location in the cord. We propose that these astrocytes may play an important role in setting up the grey-white matter arrangement of the amphibian spinal cord and that a single astrocyte of the frog spinal cord may combine the properties and functions of both grey and white matter mammalian astrocytes.

Short-term estrogen replacement increases beta-preprotachykinin mRNA levels in uninjured dorsal root ganglion neurons, but not in axotomized neurons.

Dorsal root ganglion (DRG) neurons that mediate nociception express the high affinity NGF receptor (trkA) gene and the preprotachykinin (PPT) gene. NGF has been shown to regulate both of these DRG neuronal genes. Our laboratory has shown that these genes are also regulated by estrogen. Long-term daily estrogen replacement, in adult ovariectomized (OVX) rats, causes a coordinate decline in trkA and beta-PPT mRNA levels in lumbar DRG neurons, while short-term estrogen replacement increases trkA mRNA levels in uninjured as well as in axotomized lumbar DRG neurons. The purpose of the current study was to test the hypothesis that short-term estrogen replacement increases DRG beta-PPT mRNA levels in lumbar DRG neurons of OVX rats and that the increase is dependent on target-derived NGF. Sciatic nerve transection (SNT) was used to eliminate target-derived NGF in L4 and L5 DRGs in adult OVX rats. Seven days later, one-half of the SNT and one-half of the animals that had received sham sciatic nerve transactions (SHAM) received two daily injections of estradiol benzoate (EB). The remaining rats received two daily injections of vehicle alone. Quantitative in situ hybridization analyses of sections from L4 and L5 DRGs showed that two daily injections of EB significantly increased beta-PPT mRNA levels in DRGs of SHAM animals, but had no effect on beta-PPT mRNA levels in DRGs from SNT animals. These data coupled with our earlier observations of the effect of short-term estrogen replacement on DRG trkA mRNA levels, indicate that the regulation of DRG beta-PPT mRNA levels by estrogen requires target-derived NGF.