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.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

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.     

Neuroprotective effect of sonic hedgehog up-regulated in Schwann cells following sciatic nerve injury

The physiological roles of sonic hedgehog (Shh) have been intensively characterized in development of various organs. However, their functions in adult tissues have not been fully elucidated. We investigated the expression and the potential function of Shh in crush-injured adult rat sciatic nerves. The Shh expression was up-regulated in Schwann cells adjacent to the injured site. The time-course analyses of various neurotrophic factors revealed the up-regulation of Shh mRNA followed by that of brain-derived neurotrophic factor (BDNF) mRNA. The continuous administration of cyclopamine, a hedgehog signal inhibitor, to the injured site suppressed the increase of BDNF expression and deteriorated the survival of motor neurons in lumbar spinal cord. Treatment of exogenous Shh in cultured Schwann cells enhanced the BDNF expression. The BDNF promoter activity (exon I and II) was increased in IMS32 cells co-transfected with Shh and its receptor Smoothened. These findings imply that the up-regulated expression of Shh in Schwann cells may play an important role in injured motor neurons through the induction of BDNF.     

P0 and myelin basic protein-like immunoreactivities following ligation of the sciatic nerve in the rat

In this work we analyzed variations in the expression of MBPs and P₀ in ligated sciatic nerves of young and adult rats at 3, 7, and 14 days postligation (PL), by immunohistochemistry and SDS-PAGE of isolated myelin. A protein redistribution was seen in the distal stump of ligated nerves with the appearance of immunoreactive clusters. Using the KS400 image analyzer, immunostained area values were obtained from the different nerves dissected. In adult rats, there was an increase of the immunostained area for MBP from 3 to 7 days PL, coincident with a reorganization of the marker in clusters, followed by a marked decrease at 14 days. P₀ immunolabeling gave similar results without, however, a decrease of the immunostained area at the longer survival time tested. Young animals showed an acceleration in the process of protein redistribution and digestion within ligated nerves, which followed a similar pattern as that of adult animals. Analysis by electrophoresis showed a marked decrease in P₀ and MBP at 7 days PL in young rats and 14 days PL in adult rats. The functional significance of protein clustering within myelin in injured nerves deserves further analysis.     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

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.     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

Evaluation of combined effects of brief electrical stimulation and Schwann-like cells on sciatic nerve injury model

Severe nerve injuries can be treated with electrical stimulation and stem cell therapies, but little is known about the potential benefits of combining these two treatments. In an effort to investigate this combination, we conducted a study to evaluate the effectiveness of electrical stimulation and Schwann-like cell transplantation in female Wistar albino rats. Our study consisted of five groups of rats: a sham group, an injury group, an electrical stimulation group, a Schwann-like cell group, and a combination group. The experimental groups received electrical stimulation, Schwann-like cell transplantation, or both. The animals sciatic function index was evaluated during a 6-week recovery period, and nerve conduction velocity, wet muscle mass, and nerve tissues were also analyzed. The results of the study showed that all experimental groups had a faster functional recovery compared to the injury group, although the difference between groups was not statistically significant. Both the combination group and the Schwann-like cell transplantation group had a higher nerve conduction velocity compared to the other experimental groups. However, there was no significant difference between the combination and Schwann-like cell transplantation groups. Nonetheless, histological analysis showed a better axonal reorganization in the combination group. The study provides preliminary evidence of the potential benefits of combining electrical stimulation and Schwann-like cell transplantation in treating severe nerve injuries. However, further studies with larger sample sizes are needed to confirm these findings and optimize the treatment parameters.     

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.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by ∼30%. The death of motor neurons was confirmed using the terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl‐modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 185–201, 1999     

Salidroside protects retinal endothelial cells against hydrogen peroxide-induced injury via modulating oxidative status and apoptosis

Oxidative stress can cause injury in retinal endothelial cells. Salidroside is a strong antioxidative and cytoprotective supplement in Chinese traditional medicine. In this study, we investigated the effects of salidroside on H₂O₂-induced primary retinal endothelial cells injury. Salidroside decreased H₂O₂-induced cell death, and efficiently suppressed cellular ROS production, malondialdehyde generation, and cell apoptosis induced by H₂O₂ treatment. Salidroside induced the intracellular mRNA expression, protein expression, and enzymatic activities of catalase and Mn-SOD and increased the ratio of Bcl2/Bax. Our results demonstrated that salidroside protected retinal endothelial cells against oxidative injury through increasing the Bcl2/Bax signaling pathway and activation of endogenous antioxidant enzymes. This finding presents salidroside as an attractive agent with potential to attenuate retinopathic diseases.     

Byakangelicin protects against carbon tetrachloride–induced liver injury and fibrosis in mice

Liver fibrosis is a disease caused by long‐term damage that is related to a number of factors. The current research on the treatment of liver fibrosis mainly focuses on the activation of hepatic stellate cell, in addition to protecting liver cells. byakangelicin has certain anti‐inflammatory ability, but its effect on liver fibrosis is unclear. This study aims to explore whether byakangelicin plays a role in the development of liver fibrosis and to explore its mechanism. We determined that byakangelicin has a certain ability to resist fibrosis and reduce liver cell damage in a model of carbon tetrachloride–induced liver fibrosis in mice. Thereafter, we performed further verification in vitro. The signalling pathways of two important pro‐fibrotic cytokines, transforming growth factor‐β and platelet‐derived growth factor, were studied. Results showed that byakangelicin can inhibit related pathways. According to the hepatoprotective effect of byakangelicin observed in animal experiments, we studied the effect of byakangelicin on 4‐HNE–induced hepatocyte (HepG2) apoptosis and explored its related pathways. The results showed that byakangelicin could attenuate 4‐HNE–induced hepatocyte apoptosis via inhibiting ASK‐1/JNK signalling. In conclusion, byakangelicin could improve carbon tetrachloride–induced liver fibrosis and liver injury by inhibiting hepatic stellate cell proliferation and activation and suppressing hepatocyte apoptosis.     

Adoptive transfer of Th1-conditioned lymphocytes promotes axonal remodeling and functional recovery after spinal cord injury

The role of T lymphocytes in central nervous system (CNS) injuries is controversial, with inconsistent results reported concerning the effects of T-lymphocyte transfer on spinal cord injury (SCI). Here, we demonstrate that a specific T-lymphocyte subset enhances functional recovery after contusion SCI in mice. Intraperitoneal adoptive transfer of type 1 helper T (Th1)-conditioned cells 4 days after SCI promoted recovery of locomotor activity and tactile sensation and concomitantly induced regrowth of corticospinal tract and serotonergic fibers. However, neither type 2 helper T (Th2)- nor IL-17-producing helper T (Th17)-conditioned cells had such effects. Activation of microglia and macrophages were observed in the spinal cords of Th1-transfered mice after SCI. Specifically, M2 subtype of microglia/macrophages was upregulated after Th1 cell transfer. Neutralization of interleukin 10 secreted by Th1-conditioned cells significantly attenuated the beneficial effects by Th1-conditioned lymphocytes after SCI. We also found that Th1-conditioned lymphocytes secreted significantly higher levels of neurotrophic factor, neurotrophin 3 (NT-3), than Th2- or Th17-conditioned cells. Thus, adoptive transfer of pro-inflammatory Th1-conditioned cells has neuroprotective effects after SCI, with prospective implications in immunomodulatory treatment of CNS injury.     

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of H     

Loss and recovery of the blood–nerve barrier in the rat sciatic nerve after crush injury are associated with expression of intercellular junctional proteins

The blood–nerve barrier in peripheral nerves is important for maintaining the environment for axons. Breakdown of the barrier by nerve injury causes various pathologies. We hypothesized that the breakdown and recovery of the blood–nerve barrier after injury are associated with the changes in the expression of intercellular junctional proteins. To test this hypothesis, we induced crush injuries in the rat sciatic nerve by ligation and analyzed spatiotemporal changes of claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 by immunoconfocal microscopy and morphometry and compared them with changes in the permeability of the blood–nerve barrier by intravenous and local administration of Evans blue–albumin (EBA). On day 1 after removal of the ligature EBA leaked into the connective tissue in the endoneurium and then the leakage gradually decreased and disappeared on day 7. On day 1 claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 had totally disappeared from the perineurium and endoneurium. Thereafter, claudin-1, claudin-5, occludin, and VE-cadherin recovered from day 2, whereas connexin43 was redetected on day 5. These results indicate that the breakdown and following recovery of the blood–nerve barrier are closely associated with changes in the expression of claudins, occludin, VE-cadherin, and connexin43 and that the recovery time course is similar but nonidentical.     

Changes in lectin binding of lumbar dorsal root ganglia neurons and peripheral axons after sciatic and spinal nerve injury in the rat

Summary The effects of chronic lesions of rat lumbar spinal or sciatic nerves on the binding of Glycine max (soybean) agglutinin to galacto-conjugates, in small-and medium-size primary sensory neurons of the L₄ and L₅ dorsal root ganglia, were examined over a 580-day period. Spinal nerve section resulted in a marked decrease in the population of stained neurons within 7 days. However, despite some retrograde morphological changes triggered by axonal injury, the proportion of stained nerve cells was normalized 180 days postoperatively. This temporary decrease in perikaryal lectin reactivity was initially associated with a marked accumulation of stained material in the nerve, proximal and distal to the site of section, with similar accumulations also being noticeable at each level of injury in sciatic nerves subjected to double ligature. This may reflect the presence of glycocompounds linked to the autolysis of nerve fibers during the phase of retrograde dying-back and Wallerian degeneration. At later stages, stained deposits could be seen scattered along central and peripheral axonal processes of the dorsal root ganglion neurons in the vicinity of the cell body. They may indicate a disturbance in the peripheral turnover of glycoproteins in chronically-transected nerves, with piling up of neuronal products. Sciatic nerve injury caused similar but less severe effects which, except for the L₄ ganglion cells, were rapidly reversible.     

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.