PACAP facilitate the nerve regeneration factors in the facial nerve injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported as a strong neurotrophic factor in the various sites of nervous system. The facial nerve injury is one of the common problems in patients at the Otolaryngology since the nerve damage could occur easily due to the anatomical characteristics. Once it happens, the regeneration is little observed and functional recovery is poor. Thus, we investigated that PACAP might have some influence for regeneration after the facial nerve transaction in the guinea pig. PACAP treatment accelerated time for the appearance of compound muscle action potentials (CMAP) after the nerve transaction (first appeared at 1 versus 2 weeks in control) and shortened the latency at 4 weeks. The number of myelinated fibers increased at 4 weeks. Histochemical demonstration of GAP-43, a growth cone protein, was observed at the injury area at 2-4 days. PACAP increased the level of glial cell line-derived neurotrophic factor (GDNF), a neurotrophin, in facial target muscles at 1 day-4 weeks. These data indicated that PACAP promotes the regeneration factors and increases the possibility of functional recovery following the facial nerve injury.     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.     

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up-regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over-expression of P311 induces p21(WAF1/Cip1) expression, leading PC12 cells to differentiate and to have neuron-like morphologies. Adenovirus-mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.     

Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF-2) is expressed in the peripheral nervous system and is up-regulated after nerve lesion. It has been demonstrated that administration of FGF-2 protects neurons from injury-induced cell death and promotes axonal regrowth. Using transgenic mice over-expressing FGF-2 (TgFGF-2), we addressed the importance of endogenously generated FGF-2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild-type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF-2. Morphometric evaluation of intact nerves from TgFGF-2 mice revealed no difference in number and size of myelinated fibers compared to wild-type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF-2 over-expression on Schwann cell proliferation during the early regeneration process, we used BrdU-labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild-types. We propose that endogenously synthesized FGF-2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.     

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet–rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line–derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2′-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Regulation and function of neuronal GTP-Ras in facial motor nerve regeneration

Activation of Ras into the GTP-binding, 'ON' state is a key switch in the neurotrophin-mediated neuronal survival and neurite outgrowth, in vitro as well as in vivo . In the current study we explored changes in GTP-Ras levels following facial nerve injury and the ensuing regeneration and the effects of perturbing these changes in vivo using synapsin-promoter mediated neuronal expression of constitutively active Val12H-Ras (synRas). Quantification of GTP-Ras and total Ras revealed a precipitous drop in the relative GTP-Ras levels in the axotomized facial motor nucleus, to 40% of normal levels at 2 days after cut, followed by a partial recovery to 50–65% at 4–28 days. On western blots, control and axotomized nuclei from synRas mutants showed a 2.2- and 2.5-fold elevation in GTP-Ras, respectively, compared with their wild type littermate controls ( p  < 5%, anova , TUKEY post-hoc ), with the levels in the axotomized synRas nucleus slightly but not significantly above that in the uninjured littermate control ( p  = 9.9%). Similar increase was also observed in the pERK but not pAKT targets of the Ras cascade. This moderate elevation of GTP-Ras strongly curtailed post-traumatic neuronal cell death (−65%), the influx of T-cells (−48%) as well as other parameters of neuroinflammatory response. Although synRas did not affect the speed of axonal regeneration or functional recovery it caused a very pronounced increase in central axonal sprouting. These current data emphasize the role of reduced active Ras, and by extension, the reduced overall level of retrograde neurotrophin signalling after axotomy, in mediating post-traumatic cell death and inflammation and in restricting the sprouting response. Moreover, the neuroprotective and central sprouting-enhancing effects of neuronal Val12H-Ras could help promote recovery in CNS injury.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury

Over a half a century of research has confirmed that neurotrophic factors promote the survival and process outgrowth of isolated neurons in vitro. The mechanisms by which neurotrophic factors mediate these survival-promoting effects have also been well characterized. In vivo, peripheral neurons are critically dependent on limited amounts of neurotrophic factors during development. After peripheral nerve injury, the adult mammalian peripheral nervous system responds by making neurotrophic factors once again available, either by autocrine or paracrine sources. Three families of neurotrophic factors were compared, the neurotrophins, the GDNF family of neurotrophic factors, and the neuropoetic cytokines. Following a general overview of the mechanisms by which these neurotrophic factors mediate their effects, we reviewed the temporal pattern of expression of the neurotrophic factors and their receptors by axotomized motoneurons as well as in the distal nerve stump after peripheral nerve injury. We discussed recent experiments from our lab and others which have examined the role of neurotrophic factors in peripheral nerve injury. Although our understanding of the mechanisms by which neurotrophic factors mediate their effects in vivo are poorly understood, evidence is beginning to emerge that similar phenomena observed in vitro also apply to nerve regeneration in vivo.     

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.     

ERK/MAPK and PI3K/AKT signal channels simultaneously activated in nerve cell and axon after facial nerve injury

BackgroundThe in-vitro study indicated that ERK/MAPK and PI3K/AKT signal channels may play an important role in reparative regeneration process after peripheral nerve injury. But, relevant in-vivo study was infrequent. In particular, there has been no report on simultaneous activation of ERK/MAPK and PI3K/AKT signal channels in facial nerve cell and axon after facial nerve injury.ResultsThe expression of P-ERK enhanced in nerve cells at the injury side on the 1 d after the rat facial nerve was cut and kept on a higher level until 14 d, but decreased on 28 d. The expression of P-AKT enhanced in nerve cells at the injury side on 1 d after injury, and kept on a higher level until 28 d. The expression of P-ERK enhanced at the near and far sections of the injured axon on 1 d, then increased gradually and reached the maximum on 7 d, but decreased on 14 d, until down to the level before the injury on 28 d. The expression of P-AKT obviously enhanced in the injured axon on 1 d, especially in the axon of the rear section, but decreased in the axon of the rear section on 7 d, while the expression of axon in the far section increased to the maximum and kept on till 14 d. On 28 d, the expression of P-AKT decreased in both rear and far sections of the axon.ConclusionThe facial nerve simultaneously activated ERK/MAPK and PI3K/AKT signal channels in facial nerve cells and axons after the cut injury, but the expression levels of P-ERK and P-AKT varied as the function of the time. In particular, they were quite different in axon of the far section. It has been speculated that two signal channels might have different functions after nerve injury. However, their specific regulating effects should still be testified by further studies in regenerative process of peripheral nerve injury.     

Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep

Background

Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/Principal Findings

The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/Significance

This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.     

Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4⁺ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4⁺ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3^−/− mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2^−/− (recombination activating gene-2-deficient) mice adoptively transferred CD4⁺ T-cells isolated from CCR3^−/− mice, but not in CCR3^−/− mice adoptively transferred CD4⁺ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4⁺ T-cell- and CCR3-mediated neuroprotection after FMN injury.     

Gonadal steroid enhancement of facial nerve regeneration: Role of heat shock protein 70

Over the past decade, our laboratory has been investigating the now well-established neurotrophic capabilities of gonadal steroids in the context of peripheral nerve injury and repair. The focus of our work has been on the hamster facial motoneuron (FMN) system (Kujawa & Jones, 1995), although we have recently begun to explore two additional motoneuron injury paradigms, the rat sciatic and hamster rubrospinal systems (Kujawa et al., 1993). In this brief review, we will discuss the effects of androgens and estrogens on the regenerative properties and the molecular programming of injured hamster FMN. This will be followed by a discussion of the effects of androgens on the glial response to injury in the facial nucleus. Finally, a working model of the mechanism by which gonadal steroids enhance neuronal reparative processes will be advanced. Our proposed mechanism involves a neuroprotective role for gonadal steroids, in that we hypothesize that exposure to these agents at the time of injury reduces the need for the injured neuron to mount a classical “stress response” involving the production of heat shock protein 70. Finally, a series of future directions for our work will be presented.