Peripheral Nervous System Regeneration Research Articles

Expression of NGF and GDNF family members and their receptors during peripheral nerve development and differentiation of Schwann cells in vitro

Ligands of NGF and GDNF families of neurotrophic factors have important functions in the development of the vertebrate peripheral nervous system (PNS). It has been established that they also play key roles in the regeneration of PNS. Expression patterns of NGF and GDNF family members and their receptors have mostly been analyzed during regeneration, and less during development of the PNS. We describe the expression of mRNAs encoding these neurotrophic factors and their receptors during development of rat sciatic nerve and in three modes of differentiation of cultured rat Schwann cells. Our results demonstrate specific expression patterns of NGF and GDNF family ligands and their receptors during differentiation of Schwann cells in vivo and in vitro.

The effect of laminin peptide gradient in enzymatically cross‐linked collagen scaffolds on neurite growth

Guided neurite growth is critical in both peripheral nervous system and central nervous system nerve regeneration. Scaffolds that provide structural and guidance cues for neuronal cells have a potential role in neural regeneration application. Type I collagen is suitable to be processed as an engineered scaffold for nerve regeneration because of its biological and structural properties. A few previous studies have shown that cross-linking of collagen scaffolds with microbial transglutaminase improves the mechanical strength and degradation properties of the scaffolds. It was shown that laminin 5 can regulate neurite outgrowth and extension. A motif (PPFLMLLKGSTR) in the human laminin 5 alpha 3 chain is crucial for both integrin alpha 3 beta 1 receptor binding and cell adhesion. In the present work, we studied the guidance effect of a laminin peptide (PPFLMLLKGSTR) gradient in collagen and cross-linked collagen scaffolds on neurite growth. Neurites of rat pheochromocytoma (PC12) cells showed a preferential growth toward the high laminin concentration level on the collagen scaffold, while the incorporation of laminin peptide in the scaffold did not influence neurite length of PC12 cells.

Differences in c-jun and nNOS expression levels in motoneurons following different kinds of axonal injury in adult rats

In the peripheral nervous system (PNS), root avulsion causes motoneuron degeneration, but the majority of motoneurons can survive axotomy. In order to study the mechanism of motoneuron degeneration, we compared the expression patterns of c-jun and neuronal nitric oxide synthase (nNOS), the well-known molecular players in PNS regeneration and degeneration, among adult rats having undergone axotomy (Ax), avulsion (Av), or pre-axotomy plus secondary avulsion (Ax + Av) of the brachial plexus. Our results showed that the highest and longest-lasting c-jun activation occurred in Ax, which was much stronger than those in Av and Ax + Av. The time course and intensity of c-jun expression in Ax + Av were similar to those in Av except on day 1, while the pre-axotomy condition resulted in a transient up-regulation of c-jun to a level comparable to that in Ax. Axotomy alone did not induce nNOS expression in motoneurons. Pre-axotomy left-shifted the time course of nNOS induction in Ax + Av compared to that in Av. Motoneuron loss was not evident in Ax, while it was 70% in Av and more than 85% in Ax + Av at 8 weeks postinjury. The survival of motoneurons was positively correlated with c-jun induction, but not with nNOS expression in motoneurons. Moreover, c-jun induction was negatively correlated with nNOS induction in injured motoneurons. Our results indicate that functional crosstalk between c-jun and nNOS might play an important role in avulsion-induced motoneuron degeneration, while c-jun might act as a prerequisite survival factor and nNOS might act as a predictor for the onset of motoneuron degeneration.

Modulation of extracellular matrix components by metalloproteinases and their tissue inhibitors during degeneration and regeneration of rat sural nerve

The success of peripheral nervous system regeneration has been associated with changes on the microenvironment, particularly on the extracellular matrix (ECM) components. In the present study we analyzed by indirect immunohistochemistry, electron microscopy and Western blotting, the distribution of ECM components, metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), during Wallerian degeneration (WD) and nerve regeneration (2nd, 7th and 21st days after injury) on crushed rat sural nerves. Our results showed that laminin α 3-chain and α 2-chain are over expressed during the early stages of degeneration and regeneration respectively, whereas type IV collagen expression increased progressively after crush. On the other hand, the expression of chondroitin sulfate was down regulated during the early stages of degeneration, returning progressively to normal values during nerve regeneration. The expression of MMP-3 was almost normal immediately after lesion, and then reduced progressively achieving the smallest expression at 21 days after crush; on the contrary, the expression of MMP-7 increased significantly immediately after crush (2nd day) returning to normal values afterwards. TIMP-1 and TIMP-2 were over expressed at the beginning of WD, returning progressively to normal values after that. These results indicate that the modifications of ECM components, which are favorable for nerve regeneration, are correlated with changes on the balance between MMPs and TIMPs.

Growth-associated gene expression after stroke: evidence for a growth-promoting region in peri-infarct cortex

Stroke induces axonal sprouting in peri-infarct cortex. A set of growth-associated genes important in axonal sprouting in peripheral nervous system regeneration and cortical development has recently been defined. The expression profiles of these growth-associated genes were defined during the post-stroke axonal sprouting response using a model of stroke in barrel field cortex. Stroke induces sequential waves of neuronal growth-promoting genes during the sprouting response: an early expression peak (SPRR1), a mid expression peak (p21, Ta1 tubulin, L1, MARCKS), a late peak (SCG10, SCLIP), and an early/sustained pattern (GAP43, CAP23, c-jun). These expression peaks correspond to specific time points in the sprouting response. The expression of the growth-inhibiting chondroitin sulfate proteoglycans aggrecan, brevican, versican, and phosphacan are induced late in the sprouting process; except neurocan, which is increased during the peak of the growth-promoting gene expression. The developmentally associated growth inhibitors ephrin-A5, ephB1, semaphorin IIIa, and neuropilin 1 are also induced in the early phases of the sprouting response. At the cellular level, chondroitin sulfate proteoglycans, in the form of peri-neuronal nets, are reduced in the region of axonal sprouting, during the peak of growth-promoting gene expression. These results identify a unique profile of growth-promoting gene expression in adult cortex after stroke, the inhibitory molecules that are present during the sprouting response, and a region in which growth-promoting genes are increased, growth-inhibitory proteins are diminished and axonal sprouting occurs. This region may be a growth-promoting zone after stroke.

Cellular and molecular interactions after peripheral and central nerve injury

Injuries to the central nervous system (CNS) result in virtually irreversible neurologic deficits compared to the peripheral nervous system (PNS) where injuries may be followed by some functional recovery. This sharp difference in the regenerative capacity of CNS and PNS is attributed to intrinsic properties of the injured neurons, and the glial cells, specifically the Schwann cell of PNS and the oligodendrocyte of CNS. The myriad of cellular and molecular changes that result following nerve injury are intimately related to whether a regenerative-permissive environment is provided to injured neurons. In this review, we highlight some of the key injury-related cell-molecular changes that are triggered in neurons and glia and how these changes may be related to the promotion of axonal regeneration of injured PNS compared to CNS. Biomedical Reviews 2003; 14: 51-62.

Central and peripheral nerve regeneration by transplantation of Schwann cells and transdifferentiated bone marrow stromal cells.

In contrast to the peripheral nervous system (PNS), little structural and functional regeneration of the central nervous system (CNS) occurs spontaneously following injury in adult mammals. The inability of the CNS to regenerate is mainly attributed to its own inhibitorial environment such as glial scar formation and the myelin sheath of oligodendrocytes. Therefore, one of the strategies to promote axonal regeneration of the CNS is to experimentally modify the environment to be similar to that of the PNS. Schwann cells are the myelinating glial cells in the PNS, and are known to play a key role in Wallerian degeneration and subsequent regeneration. Central nervous system regeneration can be elicited by Schwann cell transplantation, which provides a suitable environment for regeneration. The underlying cellular mechanism of regeneration is based upon the cooperative interactions between axons and Schwann cells involving the production of neurotrophic factors and other related molecules. Furthermore, tight and gap junctional contact between the axon and Schwann cell also mediates the molecular interaction and linking. In this review, the role of the Schwann cell during the regeneration of the sciatic (representing the PNS) and optic (representing the CNS) nerves is explained. In addition, the possibility of optic nerve reconstruction by an artificial graft of Schwann cells is also described. Finally, the application of cells not of neuronal lineage, such as bone marrow stromal cells (MSCs), in nerve regeneration is proposed. Marrow stromal cells are known as multipotential stem cells that, under specific conditions, differentiate into several kinds of cells. The strategy to transdifferentiate MSCs into the cells with a Schwann cell phenotype and the induction of sciatic and optic nerve regeneration are described.

Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells.

Bone marrow stromal cells (MSCs) are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. We now demonstrate that MSCs can be induced to differentiate into cells with Schwann cell characteristics, capable of eliciting peripheral nervous system regeneration in adult rats. MSCs treated with beta-mercaptoethanol followed by retinoic acid and cultured in the presence of forskolin, basic-FGF, PDGF and heregulin, changed morphologically into cells resembling primary cultured Schwann cells and expressing p75, S-100, GFAP and O4. The MSCs were genetically engineered by transduction with retrovirus encoding green fluorescent protein (GFP), and then differentiated by treatment with factors described above. They were transplanted into the cut ends of sciatic nerves, which then responded with vigorous nerve fibre regeneration within 3 weeks of the operation. Myelination of regenerated fibers by GFP-expressing MSCs was recognized using confocal and immunoelectron microscopy. The results suggest that MSCs are able to differentiate into myelinating cells, capable of supporting nerve fibre re-growth, and they can therefore be applied to induce nerve regeneration.

The role of basic fibroblast growth factor in peripheral nerve regeneration.

In the peripheral nervous system regeneration and gradual functional restoration occur following peripheral nerve injury. Growth of regenerating axons depends on the presence of diffusible neurotrophic factors, in addition to the substratum. Neurotrophic factors that are involved in peripheral nerve regeneration include nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, and interleukin-6. Recent functional and expression studies of basic fibroblast growth factor and its receptors have emphasized a physiological role of these molecules in the peripheral nervous system. Basic fibroblast growth factor and its receptors are constitutively expressed in dorsal root ganglia and the peripheral nerve. These molecules display an upregulation in dorsal root ganglia and in the proximal and distal nerve stumps following peripheral nerve injury. In the ganglia these molecules show a mainly neuronal expression, whereas at the lesion site of the nerve, Schwann cells and invading macrophages represent the main cellular sources of basic fibroblast growth factor and the receptors 1-3. Exogenously applied basic fibroblast growth factor mediates rescue effects on injured sensory neurons and supports neurite outgrowth of transectioned nerves. Regarding the expression patterm and the effects after exogenous administration of basic fibroblast growth factor, this molecule seems to play a physiological role during nerve regeneration. Thus, basic fibroblast growth factor could be a promising candidate to contribute to the development of new therapeutic strategies for the treatment of peripheral nerve injuries.

Different Signaling Pathways Mediate Regenerative versus Developmental Sensory Axon Growth

Recent advances in defining neurotrophin signaling mediators have provided insights into the signal transduction mechanisms that underlie axon growth. Evidence is accumulating that major Trk effectors regulate the morphological development of embryonic peripheral neurons. Less is known about signaling related to the robust axon extension that follows peripheral axotomy of adult neurons. Regenerative axon growth can be mimicked in vitro by a "conditioning" lesion performed 2 weeks before culture (Smith and Skene, 1997). Previous work has implicated both neurotrophins and cytokines in this response. Because signal transduction mediators of both of these families of growth factors are well characterized, we have compared the role of neurotrophin and cytokine signaling in developmental versus regenerative sensory axon growth. Chemical inhibitors were administrated to embryonic and axotomized sensory neurons in vitro to block the activation of Erk kinase (MEK)-extracellular signal-regulated kinase (ERK), phosphatidylinositol-3 kinase (PI3-K), and janus kinase (JAK) signaling. As expected, both MEK and PI3-K inhibition blocked axon growth from both naive and NGF-stimulated embryonic day 13 sensory neurons, whereas inhibition of JAK phosphorylation had no effect. In contrast, neither MEK nor PI3-K inhibitors blocked elongation of adult sensory neurons after a conditioning lesion. However, the addition of a JAK2 inhibitor prevented the regenerative axon response. Consistent with these pharmacological results, the percentage of neurons showing intense nuclear signal transducers and activators of transcription 3 phosphorylation after a conditioning lesion was markedly increased compared with controls. These observations demonstrate that the signaling mediators that underlie regenerative axon growth are distinct from those used during development and suggest that cytokine signaling may be critical to peripheral nervous system regeneration.

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma.

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate-resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented.

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate-resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented.

The Effects of Collagen-Based Implants on Early Healing of the Adult Rat Spinal Cord

In previous studies, we have examined the substrate preferences of peripheral nervous system (PNS) axons elongating within a 15-mm gap in the rat sciatic nerve. Gaps were bridged with tubes filled with one of several porous collagen-glycosaminoglycan (GAG) copolymers. In this study, we have attempted to determine if this method for PNS regeneration can be used to study regeneration of axons in the central nervous system (CNS). A 5-mm gap was created in the midthoracic spinal cord of adult rats. Animals were divided into groups in which the gap was bridged with a collagen tube filled with one of two experimental collagen-GAG copolymers or bridged with an unfilled collagen tube. Lesions that were not bridged by a tubular device were included as controls. Rats were sacrificed at 30 days, and spinal cords, including the lesion site, were removed and studied histologically. Large numbers of myelinated axons were observed regrowing within the gap in animals of all groups. The presence of collagen tubes modified the organization and orientation of the fibrous component of reparative tissue and the migration pattern of astrocytes in the wound site. The results suggest that entubulation of the transected spinal cord is a useful technique for studying the substrate preferences of regenerating CNS axons.

Tissue-Engineering Approaches for Central and Peripheral Nervous-System Regeneration

The nervous system of the human body can be divided into two parts: the central and the peripheral nervous system. While the central nervous system (CNS) includes the brain and the spinal cord, the peripheral nervous system (PNS) consists of all the nerve branches exiting the spinal cord or brain stem that process information from our environment to the CNS and vice versa. The main extension that passes all incoming information to the next neuron or a peripheral target is called an axon. Axons can be as long as 1 meter, as in the case of axons sending electrical signals to the toes. All incoming information is received by smaller structures called dendrites. Axons are wrapped with glial cells, Schwann cells in the PNS, and oligodendrocytes in the CNS. These cells are responsible for the deposition of myelin, a powerful insulator. Each glial cell is covering an axonal segment of about 1-2 mm. An unmyelinated gap separates two glial cells. Action potentials are generated at those gaps. This electrical signal jumps from one unmyelinated gap to the next, giving rise to conduction velocities as fast as 1 m/s. In the PNS, axons are grouped together into fascicles, consisting of a layer of connective tissue surrounding bundles of axons. Several of these fascicles form a peripheral nerve surrounded by another connective tissue layer named epineurium. The epineurium is responsible for the integrity of the overall structure of a peripheral nerve. While the PNS shows regenerative capabilities, this is in general not the case for the CNS. The mechanisms explaining the poor regeneration capabilities of the CNS have started to be elucidated. The expression of inhibitory molecules in the mammalian-adult nervous system seems to be an essential component in the inherent lack of CNS regeneration. Examples for possible applications of materials systems addressing certain issues of PNS and CNS regeneration are presented in the following sections.

Change in the molecular phenotype of Schwann cells upon transplantation into the central nervous system: down-regulation of c- jun

Activated Schwann cells such as those in the distal stump of a cut peripheral nerve, or those cultured in vitro, develop a molecular phenotype very different from that of quiescent Schwann cells, and express high levels of the transcription factor c- jun. We studied the expression of c- jun messenger RNA, by in situ hybridization, and Jun-like immunoreactivity of Schwann cells in segments of peripheral nerve, or in cell suspensions grafted into the adult rat brain. Schwann cells rapidly lost their Jun immunopositivity, and down-regulated expression of c- jun messenger RNA once implanted into the brain, and only the Schwann cells contained in the portion of peripheral nerve which remained outside the brain maintained Jun-like immunopositivity. c- jun messenger RNA was also down-regulated in the grafts, but more slowly than the protein; however, a proximodistal gradient in the level of expression of c- jun messenger RNA along the graft, comparable to that found for Jun immunoreactivity, was not detected. Schwann cells transplanted into the lesioned central nervous system promote regeneration of some injured central nervous system axons, but this regenerative response is always much more limited than peripheral nervous system regeneration. We suggest a correlation between the limited regeneration of central nervous system axons into peripheral nerve grafts and the loss of c- jun expression in Schwann cells following exposure to the central nervous system environment.

Impaired peripheral nerve regeneration in a mutant strain of mice (Enr) with a Schwann cell defect

Schwann cell-axon interactions in the development, maintenance, and regeneration of the normal peripheral nervous system are complex. A previously described transgene-induced insertional mutation (BPFD#36), now referred to as Enervated (Enr), results in disrupted Schwann cell-axon interactions. In this report, after a crush or transection injury to Enr peripheral nerves, we demonstrate impaired nerve regeneration. There are fewer myelinated fibers per mm2 and thinner myelin sheaths surrounding regenerating axons in the nerves of homozygous mutant mice compared to wild type mice at 28 d after crush injury to the sciatic nerve. Abnormal Schwann cell-axon interactions remain in Enr/Enr animals as evidenced by the relatively frequent ultrastructural finding of unmyelinated large diameter axons in the regenerating nerves. Additionally, nerve graft experiments indicate that the impairment in regeneration is due to a Schwann cell defect. Morphologic and morphometric findings in conjunction with molecular analysis of regenerating nerves suggest that the Enr defect causes a disruption in the ability of "early" Schwann cells to differentiate to a more mature phenotype. In mutant homozygous and wild type nerves at 7 d after crush injury there are similar levels of mRNA for the low-affinity nerve growth factor receptor, but in the mutant homozygous regenerating nerves there is 11-fold less mRNA for glial fibrillary acidic protein, a more mature phenotypic marker of Schwann cells. This Schwann cell differentiation defect likely accounts for both the peripheral neuropathy and impaired nerve regeneration observed in Enr mice.

Immunolocalization of vgr (BMP‐6, DVR‐6), a TGF‐β related cytokine, to Schwann cells of the rat peripheral nervous system: Expression patterns are not modulated by autoimmune disease

The transforming growth factors type beta (TGF-beta) have been implicated in regulation of peripheral nervous system inflammation and regeneration. Here we demonstrate expression of a TGF-beta-related bone morphogenetic protein, the vgr (BMP-6, DVR-6) in Schwann cells of the rat peripheral nervous system. The expression of vgr in the peripheral nervous system suggests that this factor and probably other TGF-beta-related bone morphogenetic proteins might participate in Schwann cell function during aspects of peripheral nervous system physiology and pathology. However, we did not observe changes in expression patterns in response to autoimmune inflammation (experimental autoimmune neuritis) of the peripheral nervous system.

The role of the Schwann cell in trophic support and regeneration.

Schwann cells show remarkable versatility in undertaking a broad repertoire of functions. Ensheathment and myelination are specifically regulated by contact with axons, and the Schwann cell is centrally involved in extracellular matrix production in the peripheral nerve trunk. Additional Schwann cell functions include the promotion of both peripheral and central nervous system regeneration, provision of a versatile source of trophic factors, the capacity to remyelinate central nervous system axons, and the restoration of electrophysiological conduction. Since it is now possible to isolate Schwann cells both from neonatal and adult human peripheral nerve, their ability to promote regenerative efforts by many central neurons suggests a role for Schwann cell autografts in influencing central nervous system repair.

Nerve growth factor and regeneration of peripheral nervous system

Nerve growth factor, a well-known neurotrophic factor, supports the survival, differentiation and maintenance of sensory and sympathetic neurons during embryonic development and in the adult. This paper summarises the data on its involvement in peripheral nerve regeneration.

Transforming growth factor beta as a neuronoglial signal during peripheral nervous system response to injury.

In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) displays an important regenerative ability which is dependent, at least in part, on Schwann cell properties. The mechanisms which stimulate Schwann cells to adapt their behavior after a lesion to generate adequate conditions for PNS regeneration remain unknown. In this work, we report that adult rat dorsal root ganglion (DRG) neurons are able, after a lesion performed in vivo or when they are dissociated and cultured in vitro, to synthesize transforming growth factor beta (TGF beta), a pleiotropic growth factor implicated in wound healing processes and in carcinogenesis. This TGF beta is tentatively identified as the beta-1 isoform. Adult rat DRG neurons release a biologically active form of TGF beta which is able to elicit multiple Schwann cell responses including a stimulation to proliferate. Moreover, purified TGF beta-1 produces a Schwann cell morphology alteration and decreases the secretion of tissue-type plasminogen activator (tPA) and enhances the secretion of plasminogen activator inhibitor (PAI) by Schwann cells. This generates conditions which are thought to favor a successful neuritic regrowth. Furthermore, purified TGF beta-1 stimulates type IV collagen mRNA expression in Schwann cells. This subtype of collagen is associated with the process of myelinization. Finally, TGF beta-1 decreases nerve growth factor (NGF) mRNA expression by Schwann cells, an effect which could participate in the maintenance of a distoproximal NGF gradient during nerve regeneration. We propose that neuronal TGF beta plays an essential role as a neuronoglial signal that modulates the response of Schwann cells to injury and participates in the successful regeneration processes observed in the PNS.