An ultrasound-guided percutaneous electrical nerve stimulation regimen devised using finite element modeling promotes functional recovery after median nerve transection

Percutaneous electrical nerve stimulation of an injured nerve can promote and accelerate peripheral nerve regeneration and improve function.When performing acupuncture and moxibustion,locating the injured nerve using ultrasound before percutaneous nerve stimulation can help prevent further injury to an already injured nerve.However,stimulation parameters have not been standardized.In this study,we constructed a multi-layer human forearm model using finite element modeling.Taking current density and activated function as optimization indicators,the optimal percutaneous nerve stimulation parameters were established.The optimal parameters were parallel placement located 3 cm apart with the injury site at the midpoint between the needles.To validate the efficacy of this regimen,we performed a randomized controlled trial in 23 patients with median nerve transection who underwent neurorrhaphy.Patients who received conventional rehabilitation combined with percutaneous electrical nerve stimulation experienced greater improvement in sensory function,motor function,and grip strength than those who received conventional rehabilitation combined with transcutaneous electrical nerve stimulation.These findings suggest that the percutaneous electrical nerve stimulation regimen established in this study can improve global median nerve function in patients with median nerve transection.

Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice:effects of 4-aminopyridine

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine(4-AP),a potassium channel blocker,on functional recove ry and muscle atrophy after sciatic nerve crush injury in rodents.However,this effect of 4-AP is unknown in nerve transection,gap,and grafting models.To evaluate and compare the functional recovery,nerve morphology,and muscle atrophy,we used a novel stepwise nerve transection with gluing(STG),as well as 7-mm irreparable nerve gap(G-7/0)and 7-mm isografting in 5-mm gap(G-5/7)models in the absence and presence of 4-AP treatment.Following surgery,sciatic functional index was determined wee kly to evaluate the direct in vivo global motor functional recovery.After 12 weeks,nerves were processed for whole-mount immunofluorescence imaging,and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber crosssectional area and minimal Feret's diameter.Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model.4-AP did not affect the sciatic functional index recovery in any model.Compared to STG,nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting.While muscle weight,cross-sectional area,and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7,4-AP treatment significantly increased right TA muscle mass,cross-sectional area,and minimal Feret's diameter in G-7/0 model.These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap,and 4-AP exerts diffe rential effects on functional recove ry and muscle atrophy.

Expressions of nestin and glial fibrillary acidic protein in rat retina after optic nerve transection

AIM：To assess the expression of nestin and glial fibrillary acidic protein（GFAP）in rat retina after optic nerve transection.METHODS：Rats were randomly divided into normal control group,sham group and operation group,and used for establishing an animal model of optic nerve transection.Retinal specimen of each group was collected at 3,48h,7and 14d postoperative.Nestin and GFAP expressions on sagittal sections were analyzed by immunohistochemical staining,and protein extraction was analyzed by Western blot.RESULTS：Immunohistochemical analysis showed that nestin positive staining was rarely detected in normal control group and sham group,while sham group showed weak positive staining at 3h postoperative,the reaction gradually increased at 48h postoperative,and reached its maximum at 7d postoperative,and then decreased at 14d postoperative.Compared to the expression of GFAP,there was not statistically significant obvious difference among three groups（P〉0.05）.Result of Western blot method was consistent with that of immunohistochemical method.CONCLUSION：The expression of nestin increased in a time dependent fashion in Müller cells of retina following optic nerve transection,which was statistically significant,but there was no obvious difference in GFAP expression.The results indicate that an increase in colloid synthesis in retina following optic nerve transection can improve the retinal neurons’environment.

Dark rearing maintains tyrosine hydroxylase expression in retinal amacrine cells following optic nerve transection

The present study examined changes in retinal tyrosine hydroxylase （TH） expression in rats having undergone optic nerve transection and housed under a normal day/night cycle or in the dark. The aim was to investigate the effects of amacrine cells on axonal regeneration in retinal ganglion cells and on the synapses that transmit visual signals. The results revealed that retinal TH expression gradually decreased following optic nerve transection in rats housed under a normal day/night cycle reaching a minimum at 5 days. In contrast, retinal TH expression decreased to a minimum at 1 day following optic nerve transection in dark reared rats, gradually increasing afterward and reaching a normal level at 5 7 days. The number of TH-positive synaptic particles correlated with the TH levels indicating that dark rearing can help maintain TH expression during the synaptic degeneration stage （5 7 days after optic nerve injury） in retinal amacrine cells.

Spatiotemporal microRNA profile in peripheral nerve regeneration:miR-138 targets vimentin and inhibits Schwann cell migration and proliferation

While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.

The correlation between rat retinal nerve fiber layer thickness around optic disc by using optical coherence tomography and histological measurements

·AIM: To explore the correlation between the retinal nerve fiber layer (RNFL) thickness by using optical coherence tomography (OCT) and by histological measurements in normal adult rats and optic nerve transected rats. · METHODS: The RNFL thickness of 36 rats was scanned in a circle 3.46mm far from the optic disc by OCT. The two experimental groups were the normal group ( =20 rats) and the optic nerve transected group ( =16 rats). The latter group included 4 groups ( =4 /group) surviving for 1 day, 3, 5 and 7 days. Then the RNFL thickness of the same retina area was also measured by NF -200 immunohistochemical staining method. Linear regression was used to analyze the correlation between the data obtained from these two methods. ·RESULTS: The RNFL thickness of normal right eyes around optic disc by OCT was 72.35 ±5.71μm and that of the left eyes was 72.65 ±5.88μm ( =0.074). The RNFL thickness of the corresponding histological section by immunohistochemistry was 37.54 ±4.05μm (right eyes) and 37.38 ±4.23μm (left eyes) ( =0.059). There was a good correlation between the RNFL thickness measured by OCT and that measured by histology (R 2 =0.8131). After optic nerve transection, the trend of the RNFL thickness was thinner with the prolonged survival time. The correlation of the thickness detected by the above two methods was approximately (R 2 =0.8265). Value of the RNFL thickness in rats around optic disc measured by OCT was obviously higher than that measured by common histological measurement in normal adult rats and optic nerve transected rats. ·CONCLUSION: The RNFL thickness measured by OCT has a strong correlation with that measured by histological method. Through OCT scanning, we found that the thickness of RNFL gradually becomes thinner in a time-dependent manner.

The proteome of distal nerves: implication in delayed repair and poor functional recovery

Chronic denervation is one of the key factors that affect nerve regeneration.Chronic axotomy deteriorates the distal nerve stump,causes protein changes,and renders the microenvironment less permissive for regeneration.Some of these factors/proteins have been individually studied.To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect,we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model.Four rats that received immediate repair after sciatic nerve transection served as control,whereas four rats in the experimental group(chronic denervation)had their sciatic nerve repaired after a 12-week delay.All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins.Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels.NanoLC-MS/MS analysis was applied to the gels.Protein expression levels of nerves on the surgery side were compared to those on the contralateral side.Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed.All the differentially expressed proteins in both groups were further stratified according to the biological processes.A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration.Ingenuity Pathway Analysis(IPA)software was used for pathway analysis.The results showed that 709 differentially expressed proteins were identified in the delayed repair group,with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified.The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated.IPA revealed that protective pathways such as LXR/RXR,acute phase response,RAC,ERK/MAPK,CNTF,IL-6,and FGF signaling were inhibited in the delayed repair group,whereas three detrimental pathways,including the complement system,PTEN,and apoptosis signaling,were activated.An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types.The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins.The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.

EZH2-dependent myelination following sciatic nerve injury

Demyelination and remyelination have been major focal points in the study of peripheral nerve regeneration following peripheral nerve injury.Notably,the gene regulatory network of regenerated myelin differs from that of native myelin.Silencing of enhancer of zeste homolog 2(EZH2)hinders the differentiation,maturation,and myelination of Schwann cells in vitro.To further determine the role of EZH2 in myelination and recovery post-peripheral nerve injury,conditional knockout mice lacking Ezh2 in Schwann cells(Ezh2^(fl/fl);Dhh-Cre and Ezh2^(fl/fl);Mpz-Cre)were generated.Our results show that a significant proportion of axons in the sciatic nerve of Ezh2-depleted mice remain unmyelinated.This highlights the crucial role of Ezh2 in initiating Schwann cell myelination.Furthermore,we observed that 21 days after inducing a sciatic nerve crush injury in these mice,most axons had remyelinated at the injury site in the control nerve,while Ezh2^(fl/fl);Mpz-Cre mice had significantly fewer remyelinated axons compared with their wild-type littermates.This suggests that the absence of Ezh2 in Schwann cells impairs myelin formation and remyelination.In conclusion,EZH2 has emerged as a pivotal regulatory factor in the process of demyelination and myelin regeneration following peripheral nerve injury.Modulating EZH2 activity during these processes may offer a promising therapeutic target for the treatment of peripheral nerve injuries.

Special function of nestin^+ neurons in the medial septum-diagonal band of Broca in adult rats

Nestin＋ neurons have been shown to express choline acetyltransferase （CHAT） in the medial septum-diagonal band of Broca in adult rats. This study explored the projection of nestin＋ neu-rons to the olfactory bulb and the time course of nestin＋ neurons in the medial septum-diagonal band of Broca in adult rats during injury recovery after olfactory nerve transection. This study observed that all nestin＋ neurons were double-labeled with ChAT in the medial septum-diagonal band of Broca. Approximately 53.6% of nestin~ neurons were projected to the olfactory bulb and co-labeled with fast blue. A large number of nestin~ neurons were not present in each region of the medial septum-diagonal band of Broca. Nestin＋ neurons in the medial septum and vertical limb of the diagonal band of Broca showed obvious compensatory function. The number of nestin＋ neurons decreased to a minimum later than nestin/CHAT＋ neurons in the medial sep- turn-diagonal band of Broca. The results suggest that nestin＋ cholinergic neurons may have a closer connection to olfactory bulb neurons. Nestin＋ cholinergic neurons may have a stronger tolerance to injury than Nestin/CHAT＋ neurons. The difference between nestin＋ and nestin-/ ChAT＋ neurons during the recovery process requires further investigations.

The HMGB1 signaling pathway activates the inflammatory response in Schwann cells

Schwann cells are not only myelinating cells, but also function as immune cells and express numerous innate pattern recognition receptors, including the Toll-like receptors. Injury to peripheral nerves activates an inflammatory response in Schwann cells. However, it is unclear whether specific endogenous damage-associated molecular pattern molecules are involved in the inflammatory response following nerve injury. In the present study, we demonstrate that a key damage-associated molecular pattern molecule, high mobility group box 1（HMGB1）, is upregulated following rat sciatic nerve axotomy, and we show colocalization of the protein with Schwann cells. HMGB1 alone could not enhance expression of Toll-like receptors or the receptor for advanced glycation end products（RAGE）, but was able to facilitate migration of Schwann cells. When Schwann cells were treated with HMGB1 together with lipopolysaccharide, the expression levels of Toll-like receptors and RAGE, as well as inflammatory cytokines were upregulated. Our novel findings demonstrate that the HMGB1 pathway activates the inflammatory response in Schwann cells following peripheral nerve injury.

A novel technique using hydrophilic polymers to promote axonal fusion

The management of traumatic peripheral nerve injury remains a considerable concern for clinicians.With minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury,outcomes of surgical intervention have been unpredictable.The inability to manipulate the pathophysiology of nerve injury（i.e.,Wallerian degeneration） has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration（-1 mm/day）.When axons are severed,the endings undergo calcium-mediated plasmalemmal sealing,which limits the ability of the axon to be primarily repaired.Polythethylene glycol（PEG） in combination with a bioengineered process overcomes the inability to fuse axons.The mechanism for PEG axonal fusion is not clearly understood,but multiple studies have shown that a providing a calcium-free environment is essential to the process known as PEG fusion.The proposed mechanism is PEG-induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur.This review highlights PEG fusion,its past and current studies,and future directions in PEG fusion.

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine （L-NA） on the survival of retinal ganglion cells （RGCs） at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 （1.0, 3.0 and 4.5 mg/kg） and L-NA （1.5, 3.0, 4.5 and 6.0 mg/kg） increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.

Biopolymer-nanotube nerve guidance conduit drug delivery for peripheral nerve regeneration:In vivo structural and functional assessment

Peripheral nerve injuries account for roughly 3%of all trauma patients with over 900,000 repair procedures annually in the US.Of all extremity peripheral nerve injuries,51%require nerve repair with a transected gap.The current gold-standard treatment for peripheral nerve injuries,autograft repair,has several shortcomings.Engineered constructs are currently only suitable for short gaps or small diameter nerves.Here,we investigate novel nerve guidance conduits with aligned microchannel porosity that deliver sustained-release of neurogenic 4-aminopyridine(4-AP)for peripheral nerve regeneration in a critical-size(15 mm)rat sciatic nerve transection model.The results of functional walking track analysis,morphometric evaluations of myelin development,and histological assessments of various markers confirmed the equivalency of our drug-conduit with autograft controls.Repaired nerves showed formation of thick myelin,presence of S100 and neurofilament markers,and promising functional recovery.The conduit’s aligned microchannel architecture may play a vital role in physically guiding axons for distal target reinnervation,while the sustained release of 4-AP may increase nerve conduction,and in turn synaptic neurotransmitter release and upregulation of critical Schwann cell neurotrophic factors.Overall,our nerve construct design facilitates efficient and efficacious peripheral nerve regeneration via a drug delivery system that is feasible for clinical applications.