Electrical stimulation or tacrolimus (FK506) alone enhances nerve regeneration and recovery after nerve surgery, while dual use reduces variance and combines strengths of each in promoting enhanced outcomes.

INTRODUCTION/AIMS: Repaired nerve injuries can fail to achieve functional recovery. Therapeutic options beyond surgery, such as systemic tacrolimus (FK506) and electrical stimulation (E-stim), can improve recovery. We tested whether dual administration of FK506 and E-stim enhances regeneration and recovery more than either therapeutic alone. METHODS: Rats were randomized to four groups: E-stim, FK506, FK506 + E-stim, and repair alone. All groups underwent tibial nerve transection and repair. Two sets of animals were created to measure outcomes of early nerve regeneration using nerve histology (n = 36) and functional recovery (n = 42) (21- and 42-day endpoints, respectively). Functional recovery was measured by behavioral analyses (walking track and grid walk) and, at the endpoint, muscle mass and force. RESULTS: Dual E-stim and FK506 administration produced histomorphometric measurements of nerve regeneration no different than either therapeutic alone. All treatments were superior to repair alone (FK506, P < .0001; E-stim, P < .05; FK506 + E-stim, P < .05). The E-stim and FK506 + E-stim groups had improved behavioral recovery compared with repair alone (at 6 weeks: E-stim, P < .05; FK506 + E-stim, P < .01). The FK506 group had improved recovery based on walking-track analysis (at 6 weeks: P < .001) and muscle force and mass (P < .05). The concurrent use of both therapies ensured earlier functional recovery and decreased variability in functional outcomes compared with either therapy alone, suggesting a moderate benefit. DISCUSSION: Dual administration of FK506 and E-stim showed minimal additive effects to further improve regeneration or recovery compared with either therapy alone. The data suggest the combination of FK506 and E-stim appears to combine the relative strengths of each therapeutic.

Lidocaine Nerve Block Diminishes the Effects of Therapeutic Electrical Stimulation to Enhance Nerve Regeneration in Rats.

BACKGROUND: Although electrical stimulation (ES) can improve nerve regeneration, the impact of nerve block, such as lidocaine (Lido), on the therapeutic benefits of ES remains unclear. We used a rat tibial nerve transection-and-repair model to explore how either preoperative (PreOp) or postoperative (PostOp) nerve block affects ES-related improvement in regeneration. METHODS: Lewis rats were used in 1 of 2 studies. The first evaluated the effects of extraneural Lido on both healthy and injured nerves. In the second study, rats were randomized to 5 experimental groups: No ES (negative control), PreOp Lido, ES + PreOp Lido, PostOp + ES, and ES (positive control). All groups underwent tibial nerve transection and repair. In both studies, nerves were harvested for histological analysis of regeneration distal to the injury site. RESULTS: Application of extraneural Lido did not damage healthy or injured nerve based on qualitative histological observations. In the context of nerve transection and repair, the ES group exhibited improved axon regeneration at 21 days measured by the total number of myelinated fibers compared with No ES. Fiber density and percentage of neural tissue in the ES group were greater than those in both No ES and PreOp Lido + ES groups. ES + PostOp Lido was not different from No ES or ES group. CONCLUSIONS: Extraneural application of Lido did not damage nerves. Electrical stimulation augmented nerve regeneration, but Lido diminished the ES-related improvement in nerve regeneration. Clinical studies on the effects of ES to nerve regeneration may need to consider nerve block as a variable affecting ES outcome.

Acellular Nerve Allografts in Major Peripheral Nerve Repairs: An Analysis of Cases Presenting With Limited Recovery.

BACKGROUND: Acellular nerve allografts have been used successfully and with increasing frequency to reconstruct nerve injuries. As their use has been expanded to treat longer gap, larger diameter nerve injuries, some failed cases have been reported. We present the histomorphometry of 5 such cases illustrating these limitations and review the current literature of acellular nerve allografts. METHODS: Between 2014 and 2019, 5 patients with iatrogenic nerve injuries to the median or ulnar nerve reconstructed with an AxoGen AVANCE nerve allograft at an outside hospital were treated in our center with allograft excision and alternative reconstruction. These patients had no clinical or electrophysiological evidence of recovery, and allograft specimens at the time of surgery were sent for histomorphological examination. RESULTS: Three patients with a median and 2 with ulnar nerve injury were included. Histology demonstrated myelinated axons present in all proximal native nerve specimens. In 2 cases, axons failed to regenerate into the allograft and in 3 cases, axonal regeneration diminished or terminated within the allograft. CONCLUSIONS: The reported cases demonstrate the importance of evaluating the length and the function of nerves undergoing acellular nerve allograft repair. In long length, large-diameter nerves, the use of acellular nerve allografts should be carefully considered.

Effect of Veau Class on Levator Veli Palatini Muscle Composition.

OBJECTIVE: To examine levator veli palatini muscle composition in patients with nonsyndromic cleft palate and investigate the impact of Veau class. DESIGN: Prospective cohort study. SETTING: Tertiary care academic hospital. PATIENTS/PARTICIPANTS: Thirteen patients with nonsyndromic cleft palate were recruited. INTERVENTIONS: During primary palatoplasty, a sample of levator veli palatini muscle was excised and prepared for histological analysis. MAIN OUTCOME MEASURES: Fat and collagen content were determined utilizing Oil Red and Sirius red stains, respectively, while muscle fiber cross-sectional areas were calculated from H&E-stained samples, with analysis using histomorphometric methods. Immunofluorescent staining of myosin heavy chain isoforms was performed. RESULTS: Patients underwent repair at 10.8 months of age (interquartile range [IQR] 10.2-12.9). Fat content of the levator veli palatini muscle was low in both groups, ranging from 0% to 5.2%. Collagen content ranged from 8.5% to 39.8%; neither fat nor collagen content showed an association with Veau classes. Mean muscle fiber cross-sectional area decreased with increasing Veau class, from 808 µm2 (range 692-995 µm2) in Veau II to 651 µm2 (range 232-750 µm2) in Veau III (P = .02). There was also a nonsignificant decrease in proportion of type I muscle fibers with increasing Veau class (44.3% [range 31.4%-84.4%] in Veau II vs 35.3% [range 17.4%-61.3%] in Veau III). CONCLUSIONS: Muscle fiber area in levator veli palatini muscles decreases in Veau III clefts in comparison to Veau II. The impact of these differences in velopharyngeal dysfunction requires further analysis of a larger cohort.

Controlling axonal regeneration with acellular nerve allograft limits neuroma formation in peripheral nerve transection: An experimental study in a swine model.

BACKGROUND: Symptomatic neuromata are a common indication for revision surgery following amputation. Previously described treatments, including traction neurectomy, nerve transposition, targeted muscle re-innervation, and nerve capping, have provided inconsistent results or are technically challenging. Prior research using acellular nerve allografts (ANA) has shown controlled termination of axonal regrowth in long grafts. The purpose of this study was to determine the ability of a long ANA to prevent neuroma formation following transection of a peripheral nerve in a swine model. MATERIALS AND METHODS: Twenty-two adult female Yucatan miniature swine (Sus scrofa; 4-6 months, 15-25 kg) were assigned to control (ulnar nerve transection only, n = 10), treatment (ulnar transection and coaptation of 50 mm ANA, n = 10), or donor (n = 2) groups. Nerves harvested from donor group animals were treated to create the ANA. After 20 weeks, the transected nerves including any neuroma or graft were harvested. Both qualitative (nerve architecture, axonal sprouting) and quantitative histologic analyses (myelinated axon number, cross sectional area of nerve tissue) were performed. RESULTS: Qualitative histologic analysis of control specimens revealed robust axon growth into dense scar tissue. In contrast, the treatment group revealed dwindling axons in the terminal tissue, consistent with attenuated neuroma formation. Quantitative analysis revealed a significantly decreased number of myelinated axons in the treatment group (1232 ± 540) compared to the control group (44,380 ± 7204) (p < .0001). Cross sectional area of nerve tissue was significantly smaller in treatment group (2.83 ± 1.53 mm2 ) compared to the control group (9.14 ± 1.19 mm2 ) (p = .0012). CONCLUSIONS: Aberrant axonal growth is controlled to termination with coaptation of a 50 mm ANA in a swine model of nerve injury. These early results suggest further investigation of this technique to prevent and/or treat neuroma formation.

Assessing the Impact of Secondary Fluorescence on X-Ray Microanalysis Results from Semiconductor Thin Films.

The impact of secondary fluorescence on the material compositions measured by X-ray analysis for layered semiconductor thin films is assessed using simulations performed by the DTSA-II and CalcZAF software tools. Three technologically important examples are investigated: AlxGa1−xN layers on either GaN or AlN substrates, InxAl1−xN on GaN, and Si-doped (SnxGa1−x)2O3 on Si. Trends in the differences caused by secondary fluorescence are explained in terms of the propensity of different elements to reabsorb either characteristic or bremsstrahlung X-rays and then to re-emit the characteristic X-rays used to determine composition of the layer under investigation. Under typical beam conditions (7­12 keV), the quantification of dopants/trace elements is found to be susceptible to secondary fluorescence and care must be taken to prevent erroneous results. The overall impact on major constituents is shown to be very small with a change of approximately 0.07 molar cation percent for Al0.3Ga0.7N/AlN layers and a maximum change of 0.08 at% in the Si content of (SnxGa1−x)2O3/Si layers. This provides confidence that previously reported wavelength-dispersive X-ray compositions are not compromised by secondary fluorescence.

Short-Duration, Pulsatile, Electrical Stimulation Therapy Accelerates Axon Regeneration and Recovery following Tibial Nerve Injury and Repair in Rats.

BACKGROUND: Repair of nerve injuries can fail to achieve adequate functional recovery. Electrical stimulation applied at the time of nerve repair can accelerate axon regeneration, which may improve the likelihood of recovery. However, widespread use of electrical stimulation may be limited by treatment protocols that increase operative time and complexity. This study evaluated whether a short-duration electrical stimulation protocol (10 minutes) was efficacious to enhance regeneration following nerve repair using rat models. METHODS: Lewis and Thy1-green fluorescent protein rats were randomized to three groups: 0 minutes of electrical stimulation (no electrical stimulation; control), 10 minutes of electrical stimulation, and 60 minutes of electrical stimulation. All groups underwent tibial nerve transection and repair. In the intervention groups, electrical stimulation was delivered after nerve repair. Outcomes were assessed using immunohistochemistry, histology, and serial walking track analysis. RESULTS: Two weeks after nerve repair, Thy1-green fluorescent protein rats demonstrated increased green fluorescent protein-positive axon outgrowth from the repair site with electrical stimulation compared to no electrical stimulation. Serial measurement of walking tracks after nerve repair revealed recovery was achieved more rapidly in both electrical stimulation groups as compared to no electrical stimulation. Histologic analysis of nerve distal to the repair at 8 weeks revealed robust axon regeneration in all groups. CONCLUSIONS: As little as 10 minutes of intraoperative electrical stimulation therapy increased early axon regeneration and facilitated functional recovery following nerve transection with repair. Also, as early axon outgrowth increased following electrical stimulation with nerve repair, these findings suggest electrical stimulation facilitated recovery because of earlier axon growth across the suture-repaired site into the distal nerve to reach end-organ targets. CLINICAL RELEVANCE STATEMENT: Brief (10-minute) electrical stimulation therapy can provide similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair rat model and merit further studies, as they represent a translational advantage.

The Effects of Intraoperative Electrical Stimulation on Regeneration and Recovery After Nerve Isograft Repair in a Rat Model.

Background: Therapeutic electrical stimulation (ES) applied to repaired nerve is a promising treatment option to improve regeneration. However, few studies address the impact of ES following nerve graft reconstruction. The purpose of this study was to determine if ES applied to a nerve repair using nerve isograft in a rodent model could improve nerve regeneration and functional recovery. Methods: Adult rats were randomized to 2 groups: "ES" and "Control." Rats received a tibial nerve transection that was repaired using a tibial nerve isograft (1.0 cm length), where ES was applied immediately after repair in the applicable group. Nerve was harvested 2 weeks postrepair for immunohistochemical analysis of axon growth and macrophage accumulation. Independently, rats were assessed using walking track and grid-walk analysis for up to 21 weeks. Results: At 2 weeks, more robust axon regeneration and greater macrophage accumulation was observed within the isografts for the ES compared to Control groups. Both walking track and grid-walk analysis revealed that return of functional recovery was accelerated by ES. The ES group demonstrated improved functional recovery over time, as well as improved recovery compared to the Control group at 21 weeks. Conclusions: ES improved early axon regeneration into a nerve isograft and was associated with increased macrophage and beneficial M2 macrophage accumulation within the isograft. ES ultimately improved functional recovery compared to isograft repair alone. This study supports the clinical potential of ES to improve the management of nerve injuries requiring a nerve graft repair.

Brief Electrical Stimulation Accelerates Axon Regeneration and Promotes Recovery Following Nerve Transection and Repair in Mice.

BACKGROUND: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation (ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study was to determine if a 10-minute ES protocol could improve outcomes. METHODS: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated. RESULTS: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regeneration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to 19.0%], respectively). There was no difference between the ES groups in measured outcomes. CONCLUSIONS: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery. CLINICAL RELEVANCE: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair mice model and merits further studies.

Quantification of Trace-Level Silicon Doping in Al x Ga1-xN Films Using Wavelength-Dispersive X-Ray Microanalysis.

Wavelength-dispersive X-ray (WDX) spectroscopy was used to measure silicon atom concentrations in the range 35-100 ppm [corresponding to (3-9) × 1018 cm-3] in doped AlxGa1-xN films using an electron probe microanalyser also equipped with a cathodoluminescence (CL) spectrometer. Doping with Si is the usual way to produce the n-type conducting layers that are critical in GaN- and AlxGa1-xN-based devices such as LEDs and laser diodes. Previously, we have shown excellent agreement for Mg dopant concentrations in p-GaN measured by WDX with values from the more widely used technique of secondary ion mass spectrometry (SIMS). However, a discrepancy between these methods has been reported when quantifying the n-type dopant, silicon. We identify the cause of discrepancy as inherent sample contamination and propose a way to correct this using a calibration relation. This new approach, using a method combining data derived from SIMS measurements on both GaN and AlxGa1-xN samples, provides the means to measure the Si content in these samples with account taken of variations in the ZAF corrections. This method presents a cost-effective and time-saving way to measure the Si doping and can also benefit from simultaneously measuring other signals, such as CL and electron channeling contrast imaging.

Long Acellular Nerve Allografts Cap Transected Nerve to Arrest Axon Regeneration and Alter Upstream Gene Expression in a Rat Neuroma Model.

BACKGROUND: Treatments to manage painful neuroma are needed. An operative strategy that isolates and controls chaotic axonal growth could prevent neuroma. Using long acellular nerve allograft to "cap" damaged nerve could control axonal regeneration and, in turn, regulate upstream gene expression patterns. METHODS: Rat sciatic nerve was transected, and the distal nerve end was reversed and ligated to generate a model end-neuroma. Three groups were used to assess their effects immediately following this nerve injury: no treatment (control), traction neurectomy, or 5-cm acellular nerve allograft cap attached to the proximal nerve. Regeneration of axons from the injured nerve was assessed over 5 months and paired with concurrent measurements of gene expression from upstream affected dorsal root ganglia. RESULTS: Both control and traction neurectomy groups demonstrated uncontrolled axon regeneration revealed using Thy1-GFP rat axon imaging and histomorphometric measures of regenerated axons within the most terminal region of regenerated tissue. The acellular nerve allograft group arrested axons within the acellular nerve allograft, where no axons reached the most terminal region even after 5 months. At 5 months, gene expression associated with regeneration and pain sensitization, including Bdnf, cfos, and Gal, was decreased within dorsal root ganglia obtained from the acellular nerve allograft group compared to control or traction neurectomy group dorsal root ganglia. CONCLUSIONS: Long acellular nerve allografts to cap a severed nerve arrested axon regeneration within the acellular nerve allograft. This growth arrest corresponded with changes in regenerative and pain-related genes upstream. Acellular nerve allografts may be useful for surgical intervention of neuroma.

Neuroma Management: Capping Nerve Injuries With an Acellular Nerve Allograft Can Limit Axon Regeneration.

Background: Management of painful neuromas continues to challenge clinicians. Controlling axon growth to prevent neuroma has gained considerable traction. A logical extension of this idea is to therefore develop an approach to control and arrest axon growth. Given the limits in axonal regeneration across acellular nerve allografts (ANAs), these constructs could provide a means to reliably terminate axon regeneration from an injured nerve. The purpose of this study was to determine if attaching an ANA to an injured nerve could provide a means to control and limit axon regeneration in a predictable manner. Methods: Twenty (20) adult rats received a sciatic nerve transection, where only the proximal nerve was repaired using an ANA of variable length (0.5, 2.5, and 5.0 cm) or left unrepaired (control). The nerves were harvested 5 weeks post-operatively for gross and histomorphometric analysis. The extent of myelinated axons in regenerated tissue was quantified. Results: At 5 weeks, limited axon regeneration within the ANAs was observed. All lengths of ANAs lead to reduced myelinated axon numbers in the most terminal tissue region compared to untreated injured nerve (P = .002). Additionally, ANA length 2.5 cm or greater did not contain any axons at the most terminal tissue region. Conclusions: This study demonstrates a proof of concept that ANAs attached to the proximal end of an injured nerve can limit axon growth in a controlled manner. Furthermore, the extent of axon growth from the injured nerve into the ANA is dependent on the ANA length.

T cells modulate IL-4 expression by eosinophil recruitment within decellularized scaffolds to repair nerve defects.

Decellularized nerve, or acellular nerve allografts (ANAs), are an increasingly used alternative to nerve autografts to repair nerve gaps to facilitate regeneration. The adaptive immune system, specifically T cells, plays a role in promoting regeneration upon these ANA scaffolds. However, how T cells promote regeneration across ANAs is not clear. Here, we show that T cells accumulate within ANAs repairing nerve gaps resulting in regulation of cytokine expression within the ANA environment. This in turn ultimately leads to robust nerve regeneration and functional recovery. Nerve regeneration across ANAs and functional recovery in Rag1KO mice was limited compared to wild-type (WT) mice. Prior to appreciable nerve regeneration, ANAs from Rag1KO mice contained fewer eosinophils and reduced IL-4 expression compared to ANAs from WT mice. During this period, both T cells and eosinophils regulated IL-4 expression within ANAs. Eosinophils represented the majority of IL-4 expressing cells within ANAs, while T cells regulated IL-4 expression. Finally, an essential role for IL-4 during nerve regeneration across ANAs was confirmed as nerves repaired using ANAs had reduced regeneration in IL-4 KO mice compared to WT mice. Our data demonstrate T cells regulate the expression of IL-4 within the ANA environment via their effects on eosinophils. STATEMENT OF SIGNIFICANCE: The immune system has been emerging as a critical component for tissue regeneration, especially when regeneration is supported upon biomaterials. The role of T cells, and their roles in the regeneration of nerve repaired with biomaterials, is still unclear. We demonstrated that when nerves are repaired with decellularized nerve scaffolds, T cells contribute to regeneration by regulating cytokines. We focused on their regulation of cytokine IL-4. Unexpectedly, T cells do not produce IL-4, but instead regulate IL-4 by recruiting eosinophils, which are major cellular sources of IL-4 within these scaffolds. Thus, our work demonstrated how IL-4 is regulated in a model biomaterial, and has implications for improving the design of biomaterials and understanding immune responses to biomaterials.

Design-Based stereology and binary image histomorphometry in nerve assessment.

BACKGROUND: Stereology and histomorphometry are widely used by investigators to quantify nerve characteristics in normal and pathological states, including nerve injury and regeneration. While these methods of analysis are complementary, no study to date has systematically compared both approaches in peripheral nerve. This study investigated the reliability of design-based stereology versus semi-automated binary imaging histomorphometry for assessing healthy peripheral nerve characteristics. NEW METHOD: Stereological analysis was compared to histomorphometry with binary image analysis on uninjured sciatic nerves to determine nerve fiber number, nerve area, neural density, and fiber distribution. RESULTS: Sciatic nerves were harvested from 6 male Lewis rats, aged 8-12 weeks for comprehensive analysis of 6 nerve specimens. From each animal, sciatic nerve specimens were fixed, stained, and sectioned for analysis by light and electron microscopy. Both histomorphometry and stereological peripheral nerve analyses were performed on all specimens by two blinded and independent investigators who quantified nerve fiber count, fiber width, density, and related distribution parameters. COMPARISON WITH EXISTING METHODS: Histomorphometry and stereological analysis provided similar outcomes in nerve fiber number and total nerve area. However, the light microscopy, but not electron microscopy, stereological analysis yielded higher nerve fiber area compared to histomorphometry or manual measurement. CONCLUSION: Both methods measure similar fiber number and overall nerve fiber area; however, stereology with light microscopy quantified higher fiber area. Histomorphometry optimizes throughput and comprehensive analysis but requires user thresholding.

Evaluation for Late Nerve Transfer Surgery in Spinal Cord Injury: Predicting the Degree of Lower Motor Neuron Injury.

PURPOSE: Nerve transfer surgery is used to restore upper extremity function following cervical spinal cord injury (SCI) with substantial variation in outcomes. The injury pattern in SCI is complex and can include isolated upper motor neuron (UMN) and combined UMN/lower motor neuron (LMN) dysfunction. The purpose of the study was to determine the most effective diagnostic technique for determining suitable candidates for nerve transfer surgery in SCI. METHODS: Medical records were reviewed of patients who had nerve transfers to restore upper extremity function in SCI. Data collected included (1) preoperative clinical examination and electrodiagnostic testing; (2) intraoperative neuromuscular stimulation (NMS); and (3) nerve histopathology. Preoperative, intraoperative, and postoperative data were compared to identify predictors of isolated UMN versus combined UMN/LMN injury patterns. RESULTS: The study sample included 22 patients with 50 nerve transfer surgeries and included patients ranging from less than 1 year to over a decade post-SCI. Normal recipient nerve conduction studies (NCS) before surgery corresponded to the intraoperative presence of recipient NMS and postoperative histopathology that showed normal nerve architecture. Conversely, abnormal recipient NCS before surgery corresponded with the absence of recipient NMS during surgery and patterns of denervation on postoperative histopathology. Normal donor preoperative manual muscle testing corresponded with the presence of donor NMS during surgery and normal nerve architecture on postoperative histopathology. An EMG of corresponding musculature did not correspond with intraoperative donor or recipient NMS or histopathological findings. CONCLUSIONS: NCS better predict patterns of injury in SCI than EMG. This is important information for clinicians evaluating people for late nerve transfer surgery even years post-SCI. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic II.

Comparison of Myelin-Associated Glycoprotein With Vincristine for Facial Nerve Inhibition After Bilateral Axotomy in a Transgenic Thy1-Gfp Rat Model.

IMPORTANCE: Aberrant synkinetic movement after facial nerve injury can lead to prominent facial asymmetry and resultant psychological distress. The current practices of neuroinhibition to promote greater facial symmetry are often temporary in nature and require repeated procedures. OBJECTIVE: To determine whether myelin-associated glycoprotein (MAG), a specific neuroinhibitor, can prevent neuroregeneration with efficacy comparable with that of vincristine, a well-established neurotoxin. DESIGN, SETTING, AND PARTICIPANTS: Rats transgenic for Thy-1 cell surface antigen-green fluorescent protein (Thy1-Gfp) were randomized into 3 groups. Each rat received bilateral crush axotomy injuries to the buccal and marginal mandibular branches of the facial nerves. The animals received intraneural injection of saline, MAG, or vincristine. MAIN OUTCOMES AND MEASURES: The animals were imaged via fluorescent microscopy at weeks 1, 3, 4, and 5 after surgery. Quantitative fluorescent data were generated as mean intensities of nerve segments proximal and distal to the axotomy site. Electrophysiological analysis, via measurement of compound muscle action potentials, was performed at weeks 0, 3, 4, and 5 after surgery. RESULTS: A total of 12 rats were included in the study. Administration of MAG significantly reduced fluorescent intensity of the distal nerve in comparison with the control group at week 3 (mean [SD], MAG group: 94 [11] intensity units vs control group: 130 [11] intensity units; P < .001), week 4 (MAG group: 81 [19] intensity units vs control group: 103 [9] intensity units; P = .004), and week 5 (MAG group: 76 [10] intensity units vs control group: 94 [10] intensity units; P < .001). In addition, rats treated with MAG had greater fluorescent intensity than those treated with vincristine at week 3 (mean [SD], MAG group: 94 [11] intensity units vs vincristine group: 76 [6] intensity units; P = .03), although there was no significant difference for weeks 4 and 5. At week 5, both MAG and vincristine demonstrated lower distal nerve to proximal nerve intensity ratios than the control group (control group, 0.94; vs MAG group, 0.82; P = .01; vs vincristine group; 0.77; P < .001). There was no significant difference in amplitude between the experimental groups at week 5 of electrophysiological testing. CONCLUSIONS AND RELEVANCE: Lower facial asymmetry and synkinesis are common persistent concerns to patients after facial nerve injury. Using the Thy1-Gfp rat, this study demonstrates effective inhibition of neuroregeneration via intraneural application of MAG in a crush axotomy model, comparable with results with vincristine. By potentially avoiding systemic toxic effects of vincristine, MAG demonstrates potential as an inhibitor of neural regeneration for patients with synkinesis. LEVEL OF EVIDENCE: NA.

The accumulation of T cells within acellular nerve allografts is length-dependent and critical for nerve regeneration.

Repair of traumatic nerve injuries can require graft material to bridge the defect. The use of alternatives to bridge the defect, such as acellular nerve allografts (ANAs), is becoming more common and desired. Although ANAs support axon regeneration across short defects (<3 cm), axon regeneration across longer defects (>3 cm) is limited. It is unclear why alternatives, including ANAs, are functionally limited by length. After repairing Lewis rat nerve defects using short (2 cm) or long (4 cm) ANAs, we showed that long ANAs have severely reduced axon regeneration across the grafts and contain Schwann cells with a unique phenotype. But additionally, we found that long ANAs have disrupted angiogenesis and altered leukocyte infiltration compared to short ANAs as early as 2 weeks after repair. In particular, long ANAs contained fewer T cells compared to short ANAs. These outcomes were accompanied with reduced expression of select cytokines, including IFN-γ and IL-4, within long versus short ANAs. T cells within ANAs did not express elevated levels of IL-4, but expressed elevated levels of IFN-γ. We also directly assessed the contribution of T cells to regeneration across nerve grafts using athymic rats. Interestingly, T cell deficiency had minimal impact on axon regeneration across nerve defects repaired using isografts. Conversely, T cell deficiency reduced axon regeneration across nerve defects repaired using ANAs. Our data demonstrate that T cells contribute to nerve regeneration across ANAs and suggest that reduced T cells accumulation within long ANAs could contribute to limiting axon regeneration across these long ANAs.

Short-term evaluation of hepatic toxicity of titanium dioxide nanofiber (TDNF).

Various in vitro and in vivo studies have shown titanium dioxide nanoparticles (TDNPs) increase the production of reactive oxygen species and change the expression of genes and proteins involved in the inflammatory response and cell division. Although, the cytotoxicity of TDNPs has been shown to be largely dependent on the characteristics of the particles including shape and surface area. This present study investigates the effects of titanium dioxide nanofibers (TDNFs) with a diameter of 300-800 nm, on the histopathology of liver tissue, changes in feed efficiency and liver weights, changes in hepatic gene expression, and serum biochemical parameters in male Sprague-Dawley rats. Male Sprague-Dawley rats were fed concentrations of 0 ppm, 40 ppm, and 60 ppm TDNF by oral gavage for two weeks. Selected inflammatory response, oxidative stress, and regulatory cell cycle genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Differences in gene expression compared to the 0 ppm group were observed in genes Gnat3, IghA, IL-1ß, p21, p53, and TNF-α. Histopathology, body and liver weights, and feed efficiency showed no significant differences. Albumin levels in all groups were not significantly higher than the reference range while ALT levels for all groups were high compared to the reference value. Currently, the results suggest TDNF does not exhibit significant hepatic toxicity. This may be explained by the rutile crystalline structure of the nanofibers, the lower concentration or the short duration of exposure toxic used during experimentation.

Anatomic characteristics of supraorbital and supratrochlear nerves relevant to their use in corneal neurotization.

BACKGROUND: Corneal denervation can lead to opacification and blindness. A new treatment technique, surgical corneal neurotization, transfers healthy donor nerve, (most commonly contralateral supratrochlear or supraorbital) to the affected limbus to prevent corneal destruction and improve healing potential of the cornea following insult. We examine gross and histomorphometric anatomy of the supratrochlear and supraorbital nerves relevant to their use in corneal neurotization. METHODS: For each of nine adult cadaver heads, bilateral supraorbital and supratrochlear nerves were dissected from the supraorbital rim to the anterior hairline. The following data were recorded for each nerve: exit from the orbit through a notch versus foramen; horizontal distance from midline at the supraorbital rim; and distance from orbital exit to first branching point. Samples of all left supraorbital and supratrochlear nerves were obtained at the level of the supraorbital rim and at points 3 cm and 6 cm distally for histomorphometric analysis. Myelinated axon counts were determined for each sample. RESULTS: Four supraorbital foramina, 14 supraorbital notches, two supratrochlear foramina, and 15 supratrochlear notches were identified. Average supraorbital and supratrochlear distances to midline were 26.5 mm and 21 mm respectively. Average myelinated axon counts for both nerves were greater at the orbital rim (supraorbital: 6018, supratrochlear: 2533) than at 6 cm distally (supraorbital: 1621, supratrochlear: 1112). CONCLUSIONS: Anatomic dissection shows relative close approximation of the supraorbital and supratrochlear nerves, with a high proportion of both nerves exiting the orbit through foramina. The supraorbital nerve at the orbital rim contains the greatest number of myelinated axons.

Short-Term Effects of Titanium Dioxide Nanofiber on the Renal Function of Male Sprague Dawley Rats.

Titanium dioxide nanofiber (TDNF) is widely used in the manufacture of various household products, including cosmetics. As a result, the possibility exists for TDNFs to affect human health. Because the kidneys are responsible for filtering out waste from the blood, the goal of the present study was to investigate the short-term effects of TDNF on kidney function of male Sprague Dawley rats. To achieve study objectives, 6- to 7-wk-old male rats were exposed via oral gavage to a total of 0, 40, and 60 parts per million of TDNF for 2 wk. The TDNF was fabricated by electrospinning and then dissolved in water. We measured serum concentration of lactate dehydrogenase, renal histopathology, identification of TDNF in kidney tissue via scanning electron microscopy, and quantitative amounts of titanium-47 in kidney tissue. We also measured specific gene-expression analysis of transcripts involved in apoptosis, inflammation, cell-division regulation, cell structure, and motility. Results showed a slight dose-dependent reduction in renal weight. In contrast, a concentration-dependent elevation in titanium-47 amounts was noted in kidney tissue. We found no significant differences in histopathological patterns. Gnat3 and Hepacam3 were up-regulated in TDNF-treated groups. Up-regulation of NF-κB likely indicated the involvement of renal-tissue inflammation via an independent mechanism. Similarly, Gadd45-α was significantly overexpressed in kidney tissues. This transcript was previously increased following stressful growth-arrest conditions and treatment with DNA-damaging agents. Our overall results suggest marginal renal toxicity in Sprague Dawley rats after ingesting TDNF.