Distal Nerve Stump Research Articles

Sensory nerve baby-sitting in reverse end-to-side fashion improves the recovery of denervated muscle in rats

Objective To delay the atrophy of denervated skeletal muscle by baby-sitting in reverse end-to-side fashion and explore the feasibility of this operation to improve the recovery of denervated muscle in rats. Methods From July, 2015 to March, 2016, 32 female SD rats were divided into 4 groups randomly and the tibial nerve of each rats was transected. In immediate repair group, the transected tibial nerve was sutured in situ. In unprotected group, the tibial nerve stump was putting-aside. The sural nerve was transected in other 2 groups. The proximal sural nerve was connected to the distal tibial nerve stump directly in end-to-end (ETE) protected group and to the side of the distal tibial nerve stump in end-to-side (ETS) protected group. After 3 months, connected the two ends of tibial nerve except immediate repair group. After another 3 months, the tibial nerve functional index, electrophysiological testing, histology and morphology were examined. Results The gastrocnemius muscle was atrophy obviously in unprotected group, while in other groups the muscle structure was well protected. The tibial nerve functional index (-39.54±24.32) , motor nerve conduction velocity[ (30.25±12.65) m/s], and muscle contraction [ (0.98 ± 0.38) N) ] indicated that the gastrocnemius muscle function in ETS protected group was statistically better recovery than in unprotected group [-75.65±32.13, (24.93±8.69) m/s and (0.64±0.20) N, respectively] and ETE protected group[-62.34±21.65, (16.90±7.92) m/s and (0.75±0.15) N, respectively] (P 0.05) . Conclusion Sensory nerve baby-sitting in reverse end-to-side fashion is a effective method to improve the recovery of denervated muscle in rats. Key words: Muscular atrophy; Denervated muscle; Nerve baby-sitting; End-to-side suture; Rat

Mechano- and thermosensitivity of injured muscle afferents 20 to 80 days after nerve injury.

Chronic injury of limb nerves leading to neuropathic pain affects deep somatic nerves. Here the functional properties of injured afferent fibers in the lateral gastrocnemius-soleus nerve were investigated 20 and 80 days after suturing the central stump of this muscle nerve to the distal stump of the sural nerve in anesthetized rats. Neurophysiological recordings were made from afferent axons identified in either the sciatic nerve (87 A-, 63 C-fibers) or the dorsal root L4/L5 (52 A-, 26 C-fibers) by electrical stimulation of the injured nerve. About 70% of the functionally identified A-fibers had regenerated into skin by 80 days after nerve suture; the remaining A-fibers could be activated only from the injured nerve. In contrast, 93% of the functionally identified C-fibers could only be activated from the injured sural nerve after 80 days. Nearly half of the injured A- (45%) and C-fibers (44%) exhibited ongoing and/or mechanically or thermally evoked activity. Because ~50% of the A- and C-fibers are somatomotor or sympathetic postganglionic axons, respectively, probably all injured muscle afferent A- and C-fibers developed ectopic activity. Ongoing activity was present in 17% of the A- and 46% of the C-fibers. Mechanosensitivity was present in most injured A- (99%) and C-fibers (85%), whereas thermosensitivity was more common in C-fibers (cold 46%, heat 47%) than in A-fibers (cold 18%, heat 12%). Practically all thermosensitive A-fibers and C-fibers were also mechanosensitive. Thus, unlike cutaneous axons, almost all A- and C-fibers afferents in injured muscle nerves demonstrate ectopic activity, even chronically after nerve injury. NEW & NOTEWORTHY After chronic injury of a muscle nerve, allowing the nerve fibers to regenerate to the target tissue, 1) most afferent A-fibers are mechanosensitive and regenerate to the target tissue; 2) ectopic ongoing activity, cold sensitivity, and heat sensitivity significantly decrease with time after injury in A-afferents; 3) most afferent C-fibers do not regenerate to the target tissue; and 4) injured C-afferents maintain the patterns of ectopic discharge properties they already show soon after nerve injury.

α6 and β1 Integrin Heterodimer Mediates Schwann Cell Interactions with Axons and Facilitates Axonal Regeneration after Peripheral Nerve Injury

Several isoforms of integrin subunits are expressed in Schwann cells and mediate Schwann cell interactions with axons. Here, we identify α6 and β1 integrins as heterodimeric proteins expressed in Schwann cells and define their functions in axonal regeneration. α6 and β1 integrins are induced in Schwann cells in the sciatic nerve after a crush injury, and the blocking of integrin activity by siRNA expression and by treatment with anti-integrin antibodies attenuates Schwann cell contact with cultured neurons and decreases neurite outgrowth. After nerve transection, the levels of α6 and β1 integrins in the distal nerve stump are lower than those in the corresponding nerve area after a crush injury. Schwann cells prepared from the distal nerves 7 days after transection are less supportive of neurite outgrowth in co-cultured neurons than those prepared from the nerves 7 days after a crush injury. When the transected nerves are reconnected after a delay of 1 to 2 weeks, the induced levels of α6 and β1 integrins in the reconnected distal nerves are significantly reduced compared to those in the nerves after a crush injury. These changes correlate with retarded axonal regeneration in animals that have experienced nerve transections and delayed coaptation, which implies an attenuated Schwann cell capacity to support axonal regeneration due to delayed Schwann cell contact with axons. The present data suggest that α6 and β1 integrins induced in Schwann cells after nerve injury may play a role in mediating Schwann cell interactions with axons and promote axonal regeneration.

Lysophosphatidic acid propagates post-injury Schwann cell dedifferentiation through LPA1 signaling.

Lysophosphatidic acid (LPA) is a pleiotropic signaling lipid that acts as ligand for at least six specific G-protein coupled receptors. Schwann cells (SC) are known to mainly express the LPA1 receptor subtype. An emerging body of evidence has linked LPA with injury-induced peripheral nerve demyelination as well as neuropathic pain. However, the molecular mechanisms underlying its demyelinating effect have not been conclusively elucidated. We aimed to decipher the demyelinating effect in vitro as well as in vivo by studying markers of SC differentiation and dedifferentiation: Myelinated dorsal root ganglia (DRG) cultures were treated either with LPA, LPA plus AM095 (LPA1 antagonist) or vehicle. Myelin content was subsequently investigated by Sudan Black staining and immunocytochemistry. In vivo, we performed sciatic nerve crush in C57BL/6 mice treated with AM095 at 10mg/kg. In DRG cultures, LPA caused a significant reduction of myelin as demonstrated by both Sudan Black staining and immunocytochemical analysis of myelin basic protein. Demyelination was paralleled by an upregulation of TNF-alpha as well as downregulation of Sox10, a marker for SC differentiation. LPA mediated effects were largely blocked by the addition of the LPA1 receptor antagonist AM095. In the in vivo model, AM095 treatment prior to crush injury increased Sox10 expression in SCs in the distal nerve stump while reducing the number of cells expressing the SC dedifferentiation marker Sox2. Additionally, TNF-alpha immunofluorescence was reduced in CD11b-positive cells. These data indicate that LPA may be a critical factor that shifts SCs towards a post-injury phenotype and contributes to the onset of Wallerian degeneration.

Functional and Molecular Characterization of a Novel Traumatic Peripheral Nerve-Muscle Injury Model.

Traumatic injuries to human peripheral nerves are frequently associated with damage to nerve surrounding tissues including muscles and blood vessels. Currently, most rodent models of peripheral nerve injuries (e.g., facial or sciatic nerve) employ surgical nerve transection with scissors or scalpels. However, such an isolated surgical nerve injury only mildly damages neighboring tissues and weakly activates an immune response. In order to provide a rodent nerve injury model accounting for such nerve-associated tissue damage and immune cell activation, we developed a drop tower-based facial nerve trauma model in mice. We compare nerve regeneration in this novel peripheral nerve trauma model with the established surgical nerve injury along several parameters. These include gene expression, histological and functional facial motoneuron (FMN) regeneration, facial nerve degeneration, immune cell activation and muscle damage. Regeneration-associated genes (RAGs; e.g., Atf3) were strongly induced in FMNs subjected to traumatic and surgical injury. Regeneration of FMNs and functional recovery of whisker movement were faster in traumatic versus complete surgical injury, thus cutting down experimentation time. Wallerian degeneration of distal nerve stumps was readily observed in this novel trauma injury model. Importantly, drop tower-inflicted facial nerve injury resulted in muscle damage, activation of muscle satellite cell markers (PAX7) and pronounced infiltration of immune cells to the injury site only in this model but not upon surgical nerve transection. Thus, we provide a novel rodent PNS trauma model that can be easily adopted to other PNS nerves such as the sciatic nerve. Since this nerve trauma model replicates multiple tissue damage frequently encountered in clinical routine, it will be well suited to identify molecular and cellular mechanisms of PNS nerve repair in wild-type and genetically modified rodents.

Abstract 51: Acellularized Nerve Allografts Guide Axon Regeneration to a Controlled Termination in a Rat Model

PURPOSE: The myriad of existing management approaches to neuroma highlight the difficulty in treating this clinical entity, and no satisfactory approach has been developed. Evaluation of “off-the-shelf” acellular nerve allografts (ANAs) as a means to bridge nerve gaps has shown an unintentional, controlled termination of axonal regrowth within long (>3cm) ANAs. We hypothesized that long ANAs can be beneficially utilized to “cap” injured nerve and guide regenerating axons to a gradual termination effectively neutralizing neuroma formation. METHODS: Thy1-GFP and Lewis rats were randomized to eight groups which received: 1) nerve transection alone, 2) traction neurectomy, 3) transection and 0.5 cm closed end silicone conduit, 4) transection and 0.5 cm ANA, 5) transection and 2.5 cm ANA, 6) transection and 5.0 cm ANA, 7) transection and proximal nerve crush, or 8) transection, proximal nerve crush and 5.0 cm ANA. In all groups, the distal nerve stump was ligated and the distal nerve turned from the proximal end to remove any trophic influence. The Thy1-GFP rat nerves were serially imaged at 4, 8, and 20 weeks to provide a visual history of regeneration. Lewis rats were sacrificed at 5 and 20 weeks for quantitative nerve histology and IHC. ANOVA with post hoc analysis were performed to evaluate significance (p<0.05). RESULTS: GFP animals that received transection alone, traction neurectomy, or transection and crush showed signs of neuroma with chaotic nerve regeneration (multidirectional axonal regrowth confirmed by histology) extending from the proximal stump as early as 4 weeks. At 5 weeks, axons grew through the entirety of the 0.5 cm ANAs, with neuroma formation extending beyond the grafts. In the 2.5 and 5.0 cm ANAs, robust axonal regeneration was demonstrated in the proximal portions of the grafts with a gradual tapering of regeneration as it moved distally, and axons failed to grow beyond the grafts. At 20 weeks, gross visualization of Thy1-GFP labeled axons demonstrates that regeneration dwindles and terminates within 5.0 cm ANAs without neuroma formation. Further histological analysis is ongoing, as are additional 20 week experiments to evaluate controlled termination with histology and IHC. CONCLUSION: Following nerve transection, long ANA “caps” can be used to control disorganized axonal regrowth, and therefore prevent the formation of a neuroma. As such, the “capping” of a transected nerve with a long ANA is a potential surgical tool in the future of neuroma management. Based upon these results, further studies are underway in a swine model to evaluate the use of ANAs in neuroma prevention in a neuroma model more similar to the human.

Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve.

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-WldS (WldS ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of WldS compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2+ Schwann cells located in the sciatic nerve distal stump of WT mice, but not WldS mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017 GLIA 2017;65:657-669.

Irreversible changes occurring in long-term denervated Schwann cells affect delayed nerve repair.

OBJECTIVE Multiple factors may affect functional recovery after peripheral nerve injury, among them the lesion site and the interval between the injury and the surgical repair. When the nerve segment distal to the lesion site undergoes chronic degeneration, the ensuing regeneration (when allowed) is often poor. The aims of the current study were as follows: 1) to examine the expression changes of the neuregulin 1/ErbB system during long-term nerve degeneration; and 2) to investigate whether a chronically denervated distal nerve stump can sustain nerve regeneration of freshly axotomized axons. METHODS This study used a rat surgical model of delayed nerve repair consisting of a cross suture between the chronically degenerated median nerve distal stump and the freshly axotomized ulnar proximal stump. Before the suture, a segment of long-term degenerated median nerve stump was harvested for analysis. Functional, morphological, morphometric, and biomolecular analyses were performed. RESULTS The results showed that neuregulin 1 is highly downregulated after chronic degeneration, as well as some Schwann cell markers, demonstrating that these cells undergo atrophy, which was also confirmed by ultrastructural analysis. After delayed nerve repair, it was observed that chronic degeneration of the distal nerve stump compromises nerve regeneration in terms of functional recovery, as well as the number and size of regenerated myelinated fibers. Moreover, neuregulin 1 is still downregulated after delayed regeneration. CONCLUSIONS The poor outcome after delayed nerve regeneration might be explained by Schwann cell impairment and the consequent ineffective support for nerve regeneration. Understanding the molecular and biological changes occurring both in the chronically degenerating nerve and in the delayed nerve repair may be useful to the development of new strategies to promote nerve regeneration. The results suggest that neuregulin 1 has an important role in Schwann cell activity after denervation, indicating that its manipulation might be a good strategy for improving outcome after delayed nerve repair.

Chronically denervated distal nerve stump inhibits peripheral nerve regeneration.

Chronically denervated distal nerve stump inhibits peripheral nerve regeneration.

Critical signaling pathways during Wallerian degeneration of peripheral nerve.

Wallerian degeneration is a critical biological process that occurs in distal nerve stumps after nerve injury. To systematically investigate molecular changes underlying Wallerian degeneration, we used a rat sciatic nerve transection model to examine microarray analysis outcomes and investigate significantly involved Kyoto Enrichment of Genes and Genomes (KEGG) pathways in injured distal nerve stumps at 0, 0.5, 1, 6, 12, and 24 hours, 4 days, 1, 2, 3, and 4 weeks after peripheral nerve injury. Bioinformatic analysis showed that only one KEGG pathway (cytokine-cytokine receptor interaction) was significantly enriched at an early time point (1 hour post-sciatic nerve transection). At later time points, the number of enriched KEGG pathways initially increased and then decreased. Three KEGG pathways were studied in further detail: cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and axon guidance. Moreover, temporal expression patterns of representative differentially expressed genes in these KEGG pathways were validated by real time-polymerase chain reaction. Taken together, the above three signaling pathways are important after sciatic nerve injury, and may increase our understanding of the molecular mechanisms underlying Wallerian degeneration.

Phenotypic changes of Schwann cells on the proximal stump of injured peripheral nerve during repair using small gap conduit tube.

Dedifferentiation of Schwann cells is an important feature of the response to peripheral nerve injury and specific negative myelination regulators are considered to have a major role in this process. However, most experiments have focused on the distal nerve stump, where the Notch signaling pathway is strongly associated with Schwann cell dedifferentiation and repair of the nerve. We observed the phenotypic changes of Schwann cells and changes of active Notch signaling on the proximal stump during peripheral nerve repair using small gap conduit tubulization. Eighty rats, with right sciatic nerve section of 4 mm, were randomly assigned to conduit bridging group and control group (epineurium suture). Glial fibrillary acidic protein expression, in myelinating Schwann cells on the proximal stump, began to up-regulate at 1 day after injury and was still evident at 5 days. Compared with the control group, Notch1 mRNA was expressed at a higher level in the conduit bridging group during the first week on the proximal stump. Hes1 mRNA levels in the conduit bridging group significantly increased compared with the control group at 3, 5, 7 and 14 days post-surgery. The change of the Notch intracellular domain shared a similar trend as Hes1 mRNA expression. Our results confirmed that phenotypic changes of Schwann cells occurred in the proximal stump. The differences in these changes between the conduit tubulization and epineurium suture groups correlate with changes in Notch signaling. This suggests that active Notch signaling might be a key mechanism during the early stage of neural regeneration in the proximal nerve stump.

Ingenuity Pathway Analysis of Gene Expression Profiles in Distal Nerve Stump following Nerve Injury: Insights into Wallerian Degeneration.

Nerve injury is a common and difficult clinical problem worldwide with a high disability rate. Different from the central nervous system, the peripheral nervous system is able to regenerate after injury. Wallerian degeneration in the distal nerve stump contributes to the construction of a permissible microenvironment for peripheral nerve regeneration. To gain new molecular insights into Wallerian degeneration, this study aimed to identify differentially expressed genes and elucidate significantly involved pathways and cellular functions in the distal nerve stump following nerve injury. Microarray analysis showed that a few genes were differentially expressed at 0.5 and 1 h post nerve injury and later on a relatively larger number of genes were up-regulated or down-regulated. Ingenuity pathway analysis indicated that inflammation and immune response, cytokine signaling, cellular growth and movement, as well as tissue development and function were significantly activated following sciatic nerve injury. Notably, a cellular function highly related to nerve regeneration, which is called Nervous System Development and Function, was continuously activated from 4 days until 4 weeks post injury. Our results may provide further understanding of Wallerian degeneration from a genetic perspective, thus aiding the development of potential therapies for peripheral nerve injury.

The Present and Future for Peripheral Nerve Regeneration.

Peripheral nerve injury can have a potentially devastating impact on a patient's quality of life, resulting in severe disability with substantial social and personal cost. Refined microsurgical techniques, advances in peripheral nerve topography, and a better understanding of the pathophysiology and molecular basis of nerve injury have all led to a decisive leap forward in the field of translational neurophysiology. Nerve repair, nerve grafting, and nerve transfers have improved significantly with consistently better functional outcomes. Direct nerve repair with epineural microsutures is still the surgical treatment of choice when a tension-free coaptation in a well-vascularized bed can be achieved. In the presence of a significant gap (>2-3 cm) between the proximal and distal nerve stumps, primary end-to-end nerve repair often is not possible; in these cases, nerve grafting is the treatment of choice. Indications for nerve transfer include brachial plexus injuries, especially avulsion type, with long distance from target motor end plates, delayed presentation, segmental loss of nerve function, and broad zone of injury with dense scarring. Current experimental research in peripheral nerve regeneration aims to accelerate the process of regeneration using pharmacologic agents, bioengineering of sophisticated nerve conduits, pluripotent stem cells, and gene therapy. Several small molecules, peptides, hormones, neurotoxins, and growth factors have been studied to improve and accelerate nerve repair and regeneration by reducing neuronal death and promoting axonal outgrowth. Targeting specific steps in molecular pathways also allows for purposeful pharmacologic intervention, potentially leading to a better functional recovery after nerve injury. This article summarizes the principles of nerve repair and the current concepts of peripheral nerve regeneration research, as well as future perspectives. [Orthopedics. 2017; 40(1):e141-e156.].

Clinical Study of Sensory Recovery of Artificial Nerve Grafts (Nerbridge®)

Purpose: To examine postoperative sensory recovery of patients who used Nerbridge® for sensory nerves, and the indications. Subjects and methods: We investigated length of the transplanted nerve, postoperative sensory evaluation, and Disabilities of Arm, Shoulder and Hand (DASH) score in patients who underwent surgery at our center and could be followed up for at least 6 months. The participants were 11 patients (8 men and 3 women) with 17 nerves for study whose mean age at the time of injury was 48 years (15-64 years) and mean follow-up period was 14.4 months (6-28 months). We used the Semmes Weinstein (SW) test for postoperative sensory evaluation. Results: Mean length of the transplanted nerve was 17.3 mm (3-41 mm); the sensory evaluation had 8 patients in the blue range, 6 in the purple range, and 3 in the red range; and the mean DASH score was 16.3 points (1.7-37.5 points). The results tended to be unfavorable for patients with severe injuries such as an amputated finger and those with relatively long transplanted nerves. The results of this study indicated that Nerbridge® worked well with digital nerve deficits on the order of 20 mm. Discussion: When the results following artificial nerve transplants were as good as or better than nerve autografts, the donor site was not problematic and the transplantations with artificial nerves were clearly useful. The product would be decomposed and absorbed in vivo in approximately 3 months. Although the product can be used for a maximum length of 50 mm, in a deficit over 30 mm, the regenerating nerve might be metabolized before reaching its distal nerve stump; thus, a nerve autograft would be preferred in that situation.

Chitosan crosslinked flat scaffolds for peripheral nerve regeneration

Chitosan (CS) has been widely used in a variety of biomedical applications, including peripheral nerve repair, due to its excellent biocompatibility, biodegradability, readily availability and antibacterial activity. In this study, CS flat membranes, crosslinked with dibasic sodium phosphate (DSP) alone (CS/DSP) or in association with the γ-glycidoxypropyltrimethoxysilane (CS/GPTMS_DSP), were fabricated with a solvent casting technique. The constituent ratio of crosslinking agents and CS were previously selected to obtain a composite material having both adequate mechanical properties and high biocompatibility.In vitro cytotoxicity tests showed that both CS membranes allowed cell survival and proliferation. Moreover, CS/GPTMS_DSP membranes promoted cell adhesion, induced Schwann cell-like morphology and supported neurite outgrowth from dorsal root ganglia explants.Preliminary in vivo tests carried out on both types of nerve scaffolds (CS/DSP and CS/GPTMS_DSP membranes) demonstrated their potential for: (i) protecting, as a membrane, the site of nerve crush or repair by end-to-end surgery and avoiding post-operative nerve adhesion; (ii) bridging, as a conduit, the two nerve stumps after a severe peripheral nerve lesion with substance loss.A 1 cm gap on rat median nerve was repaired using CS/DSP and CS/GPTMS_DSP conduits to further investigate their ability to induce nerve regeneration in vivo. CS/GPTMS_DSP tubes resulted to be more fragile during suturing and, along a 12 week post-operative lapse of time, they detached from the distal nerve stump. On the contrary CS/DSP conduits promoted nerve fiber regeneration and functional recovery, leading to an outcome comparable to median nerve repaired by autograft.

Electrical muscle stimulation elevates intramuscular BDNF and GDNF mRNA following peripheral nerve injury and repair in rats

Despite advances in surgery, patients with nerve injuries frequently have functional deficits. We previously demonstrated in a rat model that daily electrical muscle stimulation (EMS) following peripheral nerve injury and repair enhances reinnervation, detectable as early as two weeks post-injury. In this study, we explain the enhanced early reinnervation observed with electrical stimulation. In two groups of rats, the tibial nerve was transected and immediately repaired. Gastrocnemius muscles were implanted with intramuscular electrodes for sham or muscle stimulation. Muscles were stimulated daily, eliciting 600 contractions for one hour/day, repeated five days per week. Sixteen days following nerve injury, muscles were assessed for functional reinnervation by motor unit number estimation methods using electromyographic recording. In a separate cohort of rats, surgical and electrical stimulation procedures were identical but muscles and distal nerve stumps were harvested for molecular analysis. We observed that stimulated muscles had significantly higher motor unit number counts. Intramuscular levels of brain-derived and glial cell line-derived neurotrophic factor (BDNF and GDNF) mRNA were significantly upregulated in muscles that underwent daily electrical stimulation compared to those without stimulation. The corresponding levels of trophic factor mRNA within the distal stump were not different from one another, indicating that the intramuscular electrical stimulus does not modulate Schwann cell-derived trophic factor transcription. Stimulation over a three-month period maintained elevated muscle-derived GDNF but not BDNF mRNA. In conclusion, EMS elevates intramuscular trophic factor mRNA levels which may explain how EMS enhances neural regeneration following nerve injury.

Can mammalian vision be restored following optic nerve degeneration?

Can mammalian vision be restored following optic nerve degeneration? Damien P Kuffler Institute of Neurobiology, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico Abstract: For most adult vertebrates, glaucoma, trauma, and tumors close to retinal ganglion cells (RGCs) result in their neuron death and no possibility of vision reestablishment. For more distant traumas, RGCs survive, but their axons do not regenerate into the distal nerve stump due to regeneration-inhibiting factors and absence of regeneration-promoting factors. The annual clinical incidence of blindness in the United States is 1:28 (4%) for persons &gt;40 years, with the total number of blind people approaching 1.6 million. Thus, failure of optic nerves to regenerate is a significant problem. However, following transection of the optic nerve of adult amphibians and fish, the RGCs survive and their axons regenerate through the distal optic nerve stump and reestablish appropriate functional retinotopic connections and fully functional vision. This is because they lack factors that inhibit axon regeneration and possess factors that promote regeneration. The axon regeneration in lower vertebrates has led to extensive studies by using them as models in studies that attempt to understand the mechanisms by which axon regeneration is promoted, so that these mechanisms might be applied to higher vertebrates for restoring vision. Although many techniques have been tested, their successes have varied greatly from the recovery of light and dark perceptions to partial restoration of the optomotor response, depth perception, and circadian photoentrainment, thus demonstrating the feasibility of reconstructing central circuitry for vision after optic nerve damage in mature mammals. Thus, further research is required to induce the restoration of vision in higher vertebrates. This paper examines the causes of vision loss and techniques that promote transected optic nerve axons to regenerate and reestablish functional vision, with a focus on approaches that may have clinical applicability. Keywords: vision, RGC, axon regeneration, optic nerve trauma, restoration of vision

Can the Babysitter Procedure Improve Nerve Regeneration and Denervated Muscle Atrophy in the Treatment of Peripheral Nerve Injury?

The authors evaluated the long-term efficacy of the "babysitter" procedure in improving nerve regeneration and denervated muscle atrophy for peripheral nerve repair. Eighty Lewis rats were allocated equally into four groups. The peroneal nerves of all animals were divided. In group A, the peroneal nerve stumps were anchored into adjacent muscles. Rats in group B underwent end-to-end neurorrhaphy. Rats in group C underwent end-to-side neurorrhaphy of the distal peroneal nerve stump to an epineurial window on the tibial nerve. Rats in group D underwent end-to-side neurorrhaphy of the distal stump to the tibial nerve with 40 percent neurectomy. After 8 weeks, end-to-end neurorrhaphy of the peroneal nerve stumps was performed in group A, C, and D during a second-stage procedure. Electrophysiology, myelinated fiber counts, muscle force and weight, and muscle histomorphometry were analyzed at 4, 8, 12, and 24 weeks postoperatively. At 4 weeks, the end-to-end group showed predominant advantages in nerve regeneration and muscle preservation. No differences were observed in the latency delaying rate, tetanic tension, myelinated fiber number, or muscle weight between groups B and D by 24 weeks (p > 0.05). At 24 weeks, the results revealed superior latency delaying rate, myelinated axon regeneration, and size of muscle fibers in group D as compared with group C. Peripheral nerve repair with an initial motor nerve babysitter with 40 percent neurectomy of the donor nerve can achieve high efficacy in functional and structural recovery of the recipient system. Nerve babysitter by motor nerve with an epineural window was less effective.

ErbB2 blockade with Herceptin (trastuzumab) enhances peripheral nerve regeneration after repair of acute or chronic peripheral nerve injury.

Attenuation of the growth supportive environment within the distal nerve stump after delayed peripheral nerve repair profoundly limits nerve regeneration. Levels of the potent Schwann cell mitogen neuregulin and its receptor ErbB2 decline during this period, but the regenerative impact of this change is not completely understood. Herein, the ErbB2 receptor pathway is inhibited with the selective monoclonal antibody Herceptin (trastuzumab) to determine its significance in regulating acute and chronic regeneration in a rat hindlimb. The common peroneal nerve of Sprague-Dawley rats was transected and repaired immediately or after 4 months of chronic denervation, followed by administration of Herceptin or saline solution. Regenerated motor and sensory neurons were counted using a retrograde tracer 1, 2, or 4, weeks after repair. Distal myelinated axon outgrowth after 4 weeks was quantified using histomorphometry. Immunofluorescent imaging was used to evaluate Schwann cell proliferation and epidermal growth factor receptor (EGFR) activation in the regenerating nerves. Herceptin administration increased the rate of motor and sensory neuron regeneration and the number of proliferating Schwann cells in the distal stump after the first week. Herceptin also increased the number of myelinated axons that regenerated 4 weeks after immediate and delayed repair. Reduced EGFR activation was observed using immunofluorescent imaging. Inhibition of the ErbB2 receptor with Herceptin unexpectedly enhances nerve regeneration after acute and delayed nerve repair. This finding raises the possibility of using targeted molecular therapies to improve outcomes of peripheral nerve injuries. The mechanism may involve a novel inhibitory association between ErbB2 and EGFR. Ann Neurol 2016;80:112-126.

Impairment of toll-like receptors 2 and 4 leads to compensatory mechanisms after sciatic nerve axotomy.

BackgroundPeripheral nerve injury results in retrograde cell body-related changes in the spinal motoneurons that will contribute to the regenerative response of their axons. Successful functional recovery also depends on molecular events mediated by innate immune response during Wallerian degeneration in the nerve microenvironment. A previous study in our lab demonstrated that TLR 2 and 4 develop opposite effects on synaptic stability in the spinal cord after peripheral nerve injury. Therefore, we suggested that the better preservation of spinal cord microenvironment would positively influence distal axonal regrowth. In this context, the present work aimed to investigate the influence of TLR2 and TLR4 on regeneration and functional recovery after peripheral nerve injury.MethodsEighty-eight mice were anesthetized and subjected to unilateral sciatic nerve crush (C3H/HeJ, n = 22, C3H/HePas, n = 22; C57Bl6/J, n = 22 and TLR2−/−, n = 22). After the appropriate survival times (3, 7, 14 days, and 5 weeks), all mice were killed and the sciatic nerves and tibialis cranialis muscles were processed for immunohistochemistry and transmission electron microscopy (TEM). Gait analysis, after sciatic nerve crushing, was performed in another set of mice (minimum of n = 8 per group), by using the walking track test (CatWalk system).ResultsTLR4 mutant mice presented greater functional recovery as well as an enhanced p75NTR and neurofilament protein expression as compared to the wild-type strain. Moreover, the better functional recovery in mutant mice was correlated to a greater number of nerve terminal sprouts. Knockout mice for TLR2 exhibited 30 % greater number of degenerated axons in the distal stump of the sciatic nerve and a decreased p75NTR and neurofilament protein expression compared to the wild type. However, the absence of TLR2 receptor did not influence the overall functional recovery. End-point equivalent functional recovery in transgenic mice may be a result of enhanced axonal diameter found at 2 weeks after lesion.ConclusionsAltogether, the present results indicate that the lack of TLR2 or the absence of functional TLR4 does affect the nerve regeneration process; however, such changes are minimized through different compensatory mechanisms, resulting in similar motor function recovery, as compared to wild-type mice. These findings contribute to the concept that innate immune-related molecules influence peripheral nerve regeneration by concurrently participating in processes taking place both at the CNS and PNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0579-6) contains supplementary material, which is available to authorized users.