Myelinated Axon Counts Research Articles

Transfer of the palmaris brevis motor branch to the thenar motor branch: A cadaveric preliminary study

ObjectiveHigh median nerve injuries can lead to poor distal recovery, especially poor functioning of median innervated thenar muscles involved in thumb opposition and palmar abduction. The palmaris brevis (PB) is a small subcutaneous muscle innervated by ulnar nerve. Innervation of the PB is in most of cases provided by the ulnar digital nerve to the little finger. The purpose of this study is to assess the feasibility of transferring the PB motor branch (PBMB) to the median nerve thenar motor branch (TMB), in order to allow for early restoration of thumb palmar abduction and opposition, through a preliminary cadaveric study. MethodsTwenty-five cadaver upper limbs were dissected under magnification. The length of the PBMB and TMB, and their origin were recorded. Nerve transfer from PBMB to TMB was conducted, and evaluated on 2 parameters: surgical feasibility, and distance from the coaptation site to the recipient nerve muscle entry point. The PBMB and TMB were harvested, fixated in formalin, then embedded in paraffin. They were sectioned transversely, and stained with a combination of hematoxylin–eosin and Luxol fast blue. Myelinated axons were counted in each specimen and the donor-to-recipient axon ratio was recorded. ResultsThe PBMB was constant and originated from the ulnar digital nerve of the little finger in all cases. The transfer from PBMB to TMB was feasible in all cases. Mean myelinated axon counts of PBMB and TMB were 253±142 and 356±198, respectively (p=0.06). The donor-to-recipient axon ratio was 1:1.41. The mean distance from coaptation of the PBMB to the recipient thenar muscles was 23.1±3.0mm. ConclusionsBased on our results, PBMB to TMB transfer is feasible. The PBMB has the advantage over other distal nerve transfer donors to be constant and superficial, allowing for an easier harvest. Moreover, this procedure does not sacrifice any intrinsic function of the hand, and the proximity of the PBMB with the carpal tunnel allows for a single incision procedure. Therefore, early restoration of the median innervated thenar muscles may be feasible by the PBMB to TMB transfer in cases of high median nerve lesions. Level of evidenceIV.

Evaluation of the effect of pitavastatin on motor deficit and functional recovery in sciatic nerve injury: A CatWalk study.

This study aims to investigate the electrophysiological, scintigraphic, and histopathological effects of pitavastatin and its impact on functional status in rats with sciatic nerve injury. A total of 30 Wistar albino rats were divided into three equal groups including 10 rats in each group: sham group (no injury), control group (nerve injury induced), and pitavastatin group (nerve injury induced and 2 mg/kg of pitavastatin administered orally once a day for 21 days). Before and at the end of intervention, quantitative gait analysis with the CatWalk system and sciatic nerve conduction studies were performed. After the intervention, the gastrocnemius muscle was scintigraphically evaluated, and the sciatic nerve was histopathologically examined. There was no significant difference in the sciatic nerve conduction before the intervention and Day 21 among the groups (p>0.05). According to the quantitative gait analysis, there were significant differences in the control group in terms of the individual, static, dynamic, and coordination parameters (p<0.05). The histopathological examination revealed a significant difference in the total myelinated axon count and mean axon diameter among the groups (p<0.001). Pitavastatin is effective in nerve regeneration and motor function recovery in rats with sciatic nerve injury.

(001) How many Nerve Fibers Innervate the Human Clitoris? A Histomorphometric Evaluation of the Dorsal Nerve of the Clitoris

Abstract Introduction It is frequently quoted in mainstream media that the clitoris has “8000 nerve endings”. However, no study has yet quantified the number of nerve fibers (axons) innervating the human clitoris. The dorsal nerve of the clitoris (DNC) is the primary source of somatic clitoral innervation. Reporting the number of axons in the DNC is an important step in our understanding of clitoral innervation and sexual response with implications for many fields of medical practice. Reconstruction following iatrogenic injuries to the dorsal clitoral nerve and restoration following cases of genital mutilation will benefit from a better understanding of the DNC. This knowledge will also be an important factor contributing to the optimization of nerve selection and sensory outcomes in gender-affirming genital surgery. Objective The purpose of this study is to quantify the number of nerve fibers in the human dorsal nerve of the clitoris (DNC) and report the approximate number of nerve fibers that innervate the human clitoris. Methods Following institutional approval and informed consent, dorsal clitoral nerve samples were obtained from 7 transmasculine patients undergoing gender-affirming phalloplasty surgery. In phalloplasty one of the two dorsal clitoral nerves is used to coapt to a sensory nerve in the phallus in order to provide for erogenous phallic sensation. At the time of nerve coaptation, a small excess of dorsal clitoral nerve (5 mm) was collected for analysis at the proximal level of the clitoral body, just after appearance of the dorsal clitoral nerve from underneath the pubic symphysis. The samples were placed into 3% glutaraldehyde fixative, post-fixed in 1% osmium tetroxide and serially dehydrate in ethanol and toluene. They were then embedded in araldite, sectioned on ultramicrotome into 1 μm cross sections and counter-stained with 1% toluidine blue. Histomorphometric evaluation was performed at 1000x magnification using a Leitz Laborlux S microscope and image analysis software (Clemex Vision Professional, Longueuil, Quebec) in order to obtain a nerve fiber count from each dorsal clitoral nerve specimen. Descriptive statistics were performed to yield a mean and range of the number of nerve fibers in the DNC. Assuming anatomic symmetry between bilateral DNC, the total number of somatic nerve fibers innervating the human clitoris was obtained by doubling the mean count of the DNC. Results 7 samples of the DNC were collected. The mean number of nerve fibers in the human dorsal clitoral nerve was 5140 with a range of 4926 to 5543. The mean number of myelinated nerve fibers innervating the human clitoris was 10281 (range 9852 to 11086). Conclusions This study is the first to report the number of myelinated nerve fibers in the human dorsal clitoral nerve and approximate the number of nerve fibers innervating the human clitoris. Findings demonstrate high and consistent myelinated axon counts and density. When taking into account the presence of uncounted unmyelinated axons, it is clear that far more than 8000 fibers innervate the human clitoris. Disclosure No

Drop-off in axonal regeneration along the length of a cross-face nerve graft: An experimental study in rats.

The average nerve graft length utilized in cross-face nerve grafting for reconstruction of facial nerve palsy is 20-22 cm. While the graft length is thought to be one of the greatest determinants of muscle strength, the mechanism through which this happens remains unknown. We studied changes in axonal regeneration along the length of a 2 cm cross-face nerve graft in a rat model. The hypothesis was that axon count would decrease along the length of the graft. A 2 cm nerve graft (sciatic nerve) was used as a cross-face nerve graft in 16 adult female, 210-250 g, Sprague Dawley rats. Thirteen weeks later, 5 mm nerve biopsies were taken at four sites: the facial nerve trunk (control), proximal graft, midpoint of graft (1 cm distal to coaptation) and distal graft (2 cm distal to coaptation). Retrograde nerve labeling with FluoroGold was performed at the biopsied nerve site and the facial motor nucleus was taken 1 week later. Microscopic imaging and manual counting of axons and labeled motor nuclei was performed. Retrograde-labeled motor neuron counts were decreased at the midway point of the graft compared to the facial trunk (1517 ± 335 axons, Δ% = 92.5, p = .01) and even further decreased at the distal end of the graft (269 ± 293 axons, Δ% = 175.5, p = .006). Analysis of the nerve biopsies demonstrated no significant differences in myelinated axon count between the nerve trunk and over the length of the nerve graft (range 6207-7179 axons, Δ% = 14.5, p = .07). In a rat model, the number of regenerating motor neurons drops off along the length of the graft and axon count is preserved due to axon sprouting. How this pattern correlates to ultimate muscle strength remains unknown, but this study provides insight into why shorter grafts may afford better outcomes.

71. Evaluation of Axonal Regeneration Across a Long Cross-face Nerve Graft in Rats

Purpose: Cross-face nerve grafting is a preferred technique in facial reanimation due to its capability in providing spontaneous action and symmetry to a patient following regeneration. The length of graft used is thought to be involved in differing functional outcomes. The present study evaluates changes in axonal regeneration at different lengths along a cross-face nerve graft. Methods: The sciatic nerve of sixteen adult Sprague Dawley rats was harvested as a 2-cm graft and coapted end-to-end to the proximal main trunk of the facial nerve. Thirteen weeks later, the graft site was re-exposed for sectioning. 5mm segments were taken from the native facial nerve trunk (Group 1), in the nerve graft, just distal to coaptation (Group 2), midway (Group 3), and just proximal to the distal end of the graft (Group 4). The nerve segments were preserved and embedded in paraffin, cut in 1um cross-sections, and stained with 1% toluidine blue for axon counting. After segmentation, the nerve stump was immersed in 4% Fluoro-Gold (FG) for one hour, returned to the head for retrograde labeling over one week. The brains were preserved in 4% PFA/30% sucrose, frozen in OCT, and sectioned in 40um cross-sections. Every third section was taken to avoid double-counting cells, and counter-stained with Fluoroshield PI (PI). Images were taken with a fluorescent microscope using a combined filter, and cells with double-positive stain were counted as regenerated motoneurons. Statistical analysis was performed using unpaired t-tests in GraphPad Prism7. Results: Analysis of the retrograde-labeled regenerated motoneuron counts showed no significant difference between the proximal portion of the graft (3,538 ± 1,969) and the facial trunk (4,128 ± 731) (p=0.67). However, the motoneuron count at the midway point of the graft (1,517 ± 335) and at the distal end (269 ± 293) were significantly lower than in the facial trunk (p=0.01 and 0.006, respectively). Analysis of the nerve biopsies along the length of the graft demonstrated no significant differences in myelinated axon count across the graft with a mean count of 6,207 ± 310 at the nerve trunk, and 6,606 ± 2399 (p=0.76), 6,405 ±2340 (p=0.88), and 7,179 ± 744 (p=0.07) moving farther down the graft segment.

QS1: Pre-implanted Nerve Grafts (PING) to Improve Functional Outcomes in VCA

Purpose:Improved graft reinnervation and functional recovery would greatly enhance the utility of vascularized composite allotransplantation (VCA). Pre-implantation of nerve grafts in a limb transplant recipient prior to transplantation to increase the length of the severed nerves in the amputation stump offers a novel, readily translatable approach to this problem. At the time of transplantation a pre-implanted nerve graft (PING) would be elevated from its wound bed in the amputated stump and unfurled distally into the transplanted limb, allowing for a more distal nerve coaptation.Methods:We used a reversed sciatic-tibial nerve isograft coapted end-to-end to a median nerve in a rat to: (1) determine viability of distal axons after elevating PINGs; and (2) assess for ischemic injury to axons in the distal PING following elevation from the wound bed. For axonal viability, grafts of varying lengths (2cm,3cm,4cm, n≥5/group) were left to regenerate for 8 weeks. The distal ends were then biopsied prior to elevation, and again one week after elevation. For ischemia assessment, blood flow along 4cm long PINGs was measured with laser doppler at multiple timepoints: baseline (at time of grafting), 8 weeks after grafting (before elevation), immediately after elevation, and 3 and 7 days post-elevation (n=8/timepoint). Means were compared with t-tests and ANOVA. A pig forelimb model was used to assess the functional impact of PINGs. A 6cm long reversed ulnar-PING was coapted to the proximally transected ulnar nerve. After 12 weeks of regeneration, the distal PING was transferred end-to-end to the median nerve just proximal to the elbow. A control group underwent ulnar-to-median nerve transfer 6cm proximal to the elbow (n=4 limbs/group). Hoof flexion force was measured every three weeks.Results Axonal viability:after 8 weeks, axons had regenerated into the distal grafts in all lengths: mean myelinated axon counts (±SD): 7043 (±790), 6066 (±1838), 6491 (±983) in 2cm,3cm,4cm grafts, with no differences between graft lengths (p=0.48). One week after elevation and unfurling, the number of myelinated axons was significantly decreased in all groups (p<0.006): 3821 (±567), 2953 (±1107), 2894 (±1761) in 2cm,3cm,4cm grafts, with no differences between lengths (p=0.91). Ischemic injury: immediately after elevating a PING, blood flow within the distal half of the graft fell to baseline levels (i.e. no blood flow). 3 days after elevation, blood flow throughout the graft was significantly above baseline (p≤0.04) and matched/exceeded flow measured immediately prior to elevation. Functional impact: preliminary results suggest PINGs can deliver rapid functional recovery.Conclusions:Axons remain present in clinically relevant numbers at the distal ends of PINGs after elevation and unfurling. A decrease in axon counts after elevation may relate to ischemia in the distal PING immediately after elevation. Preliminary functional results show PINGs are capable of delivering early functional recovery.

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

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. 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. 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. A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery. 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.

Temporal Progression of Acute Spinal Cord Injury Mechanisms in a Rat Model: Contusion, Dislocation, and Distraction.

Traumatic spinal cord injuries (SCIs) occur due to different spinal column injury patterns, including burst fracture, dislocation, and flexion-distraction. Pre-clinical studies modeling different SCI mechanisms have shown distinct histological differences between these injuries both acutely (3 h and less) and chronically (8 weeks), but there remains a temporal gap. Different rates of injury progression at specific regions of the spinal cord may provide insight into the pathologies that are initiated by specific SCI mechanisms. Therefore, the objective of this study was to evaluate the temporal progression of injury at specific tracts within the white matter, for time-points of 3 h, 24 h, and 7 days, for three distinct SCI mechanisms. In this study, 96 male Sprague Dawley rats underwent one of three SCI mechanisms: contusion, dislocation, or distraction. Animals were sacrificed at one of three times post-injury: 3 h, 24 h, or 7 days. Histological analysis using eriochrome cyanide and immunostaining for MBP, SMI-312, neurofilament-H (NF-H), and β-III tubulin were used to characterize white matter sparing and axon and myelinated axon counts. The regions analyzed were the gracile fasciculus, cuneate fasciculus, dorsal corticospinal tract, and ventrolateral white matter. Contusion, dislocation, and distraction SCIs demonstrated distinct damage patterns that progressed differently over time. Myelinated axon counts were significantly reduced after dislocation and contusion injuries in most locations and time-points analyzed (compared with sham). This indicates early myelin damage often within 3 h. Myelinated axon counts after distraction dropped early and did not demonstrate any significant progression over the next 7 days. Important differences in white matter degeneration were identified between injury types, with distraction injuries showing the least variability across time-points These findings and the observation that white matter injury occurs early, and in many cases, without much dynamic change, highlight the importance of injury type in SCI research-both clinically and pre-clinically.

A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting: Correction.

A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting: Correction.

Abstract 119: Novel Application Of Common Pharmacological Agents Nimodipine And Botulinum Toxin A On Traumatic Nerve Regeneration Following Microsurgical Repair: A Pilot Study

Purpose: Peripheral nerve damage is a frequent problem in civilian and military populations; an estimated 2.8-5% of trauma admissions contain a peripheral nerve injury. Currently, end-to-end, tension-free microsurgical nerve repair (neurorrhaphy) is the gold standard treatment. However, neurorrhaphy is not neuroprotective and does not address the complex molecular environment of a regenerating nerve required for complete functional restoration. Anecdotal clinical evidence indicates that widely used pharmacological agents botulinum toxin A (BTX, a neurotoxic protein) and nimodipine (NDP, a L-type calcium channel blocker) may improve functional recovery of injured nerves, but the mechanisms remain unknown. This research investigates BTX and NDP, independently and in combination, for their novel capacity to improve neural regeneration and functional recovery following neurorrhaphy. Methods: 32 Lewis rats underwent surgical tibial nerve transection and neurorrhaphy. Post-op pharmaceutical treatment groups included: 1) Sham surgery (n=4); 2) Sham surgery+BTX (n=4); 3) NDP+saline injection (control for BTX, N=6); 4) BTX+NDP (n=6); 5) Saline+placebo pill (control for NDP, n=6); 6) BTX+placebo pill (n=6). Outcomes were assessed using behavioral (rotarod, horizontal ladder walk), electrophysiological (nerve conduction velocity [NCV], compound nerve action potential [CNAP], duration of response), and stereological means (myelinated axon count estimation, epineurial cross sectional area). Statistical significance was determined by one-way ANOVA. Results: The NDP+saline group outperformed other treatment groups in the ladder walk, resulting in the fewest deep slips (15.07% vs 30.77% in BTX+NDP, p=0.117), fewest misses (3.54% vs 4.21% in BTX+NDP, p=0.809), and most correct steps (70.53% vs 55.58% in BTX+NDP, p=0.143). Rotarod testing resulted in no clear differences between treatment groups with all groups performing worse than sham controls. There was an observed sex bias between groups in which females tended to outperform males within groups in both behavioral modalities. Electrophysiological testing portrayed similar outcomes to ladder walk testing in which NDP+saline resulted in the fastest NCV (0.81m/s vs 0.59m/s in BTX+NDP, p=0.126), and shortest duration of response (22.68µs vs 65.96µs in BTX+NDP, p=0.171) among the treatment groups. Our unbiased stereological analyses resulted in the BTX+NDP group having the highest myelinated axon count (9,249.54 vs 7,334.94 in NDP+saline, p=0.036), but when epineurial area is controlled for, this difference diminishes (0.005/um2 vs 0.0043/um2 in NDP+saline, p=0.201). When separated by sex, males tended to have higher axon counts than females (8,615.03 vs 6,052.84 in NDP+saline, p=0.036). Conclusions: This pilot study represents the first approach to test NDP with BTX in a multimodal assessment of nerve recovery and regeneration following transection and neurorrhaphy. While an additive or synergistic effect between BTX and NDP was expected, NDP alone tended to outperform the combined treatment group in behavioral and electrophysiological assessments. However, and in accordance with past studies by our group and others, histologic axon count was inversely related to nerve function recovery, portraying a disadvantageous compensatory effect. Our findings of a sex bias parallel observations present in the current literature. Future work will expand on these studies focusing on nimodipine in males and females in an effort to improve nerve recovery in trauma patients.

Assessing pathological changes within the nucleus ambiguus of horses with recurrent laryngeal neuropathy: An extreme, length-dependent axonopathy.

Equine recurrent laryngeal neuropathy (RLN) is a naturally occurring model of length-dependent axonopathy characterized by asymmetrical degeneration of recurrent laryngeal nerve axons (RLn). Distal RLn degeneration is marked, but it is unclear whether degeneration extends to include cell bodies (consistent with a neuronopathy). With examiners blinded to RLN severity, brainstem location, and side, we examined correlations between RLN severity (assessed using left distal RLn myelinated axon count) and histopathological features (including chromatolysis and glial responses) in the nucleus ambiguus cell bodies, and myelinated axon count of the right distal RLn of 16 horses. RLN severity was not associated with RLn cell body number (P > .05), or degeneration. A positive correlation between the left and right distal RLn myelinated axon counts was identified (R2 = 0.57, P < .05). We confirm that RLN, a length-dependent distal axonopathy, occurs in the absence of detectable neuronopathy.

Neurofilament-histomorphometry comparison in the evaluation of unmyelinated axon regeneration following peripheral nerve injury: An alternative to electron microscopy

BackgroundCurrently, assessment of unmyelinated axon regeneration is limited to electron microscopy (EM), which is expensive, time consuming and not universally available. This study presents a protocol to estimate the number of unmyelinated axons in a regenerating peripheral nerve without the need for electron microscopy. New methodThe common peroneal nerve of Sprague-Dawley rats was transected, repaired and regenerated for 4 weeks. Two distal adjacent segments of the regenerating nerve were then processed for either conventional histomorphometry using toluidine blue or immunolabeling of neurofilament protein. Myelinated axon and total axon counts were obtained, respectively, to generate estimates of unmyelinated axon numbers, which were then compared to unmyelinated axon counts using EM from the same specimens. For comparison, unmyelinated axons were counted in an uninjured rat laryngeal nerve. ResultsAfter 4 weeks of regeneration, the estimated number of regenerating unmyelinated axons was 4044 ± 232 using this technique, representing 81.3% of the total axonal population. By comparison, the proportion of unmyelinated axons in the uninjured laryngeal nerve was 55% of the total axonal population. Comparison with existing methodThese estimates correlate with electron microscopy measurements, both in terms of the proportion of unmyelinated axons and also by linear regression analysis. ConclusionsThe neurofilament staining method correlates with electron microscopy estimates of the same nerve sections. It is useful for the efficient counting of unmyelinated axons in the regenerating peripheral nerve and can be used by laboratories that do not have access to EM facilities.

A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting.

Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures; however, there exists no reliable means for their intraoperative quantification. In this article, the authors report a novel protocol for rapid quantification of myelinated axons from frozen sections, and demonstrate its applicability to surgical practice. The impact of various fixation and FluoroMyelin Red staining strategies on resolved myelin sheath morphology from cryosections of rat and rabbit femoral and sciatic nerves was assessed. A protocol comprising fresh cryosection and rapid staining was developed, and histomorphometric results were compared against conventional osmium-postfixed, resin-embedded, toluidine blue-stained sections of rat sciatic nerve. The rapid protocol was applied for intraoperative quantification of donor nerve myelinated axon count in a cross-facial nerve grafting procedure. Resolution of myelinated axon morphology suitable for counting was realized within 10 minutes of tissue harvest. Although mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± SD; rapid, 9.25 ± 0.62 μm; conventional, 6.05 ± 0.71 μm; p < 0.001), no difference in axon counts was observed on high-power fields (rapid, 429.42 ± 49.32; conventional, 460.32 ± 69.96; p = 0.277). Whole nerve myelinated axon counts using the rapid protocol herein (8435.12 ± 1329.72) were similar to prior reports using conventional osmium processing of rat sciatic nerve. A rapid protocol for quantification of myelinated axon counts from peripheral nerves using widely available equipment and techniques has been described, rendering possible intraoperative assessment of donor nerve suitability for reanimation.

Double Motor Nerve Transfer for All Finger Flexion in Cervical Spinal Cord Injury: An Anatomical Study and a Clinical Report.

To explore the feasibility of restoring all finger flexion after a cervical spinal cord injury. Double nerve transfer was conducted in 22 cadaver upper extremities. Donor nerves were the brachialis branch of the musculocutaneous nerve and the extensor carpi radialis brevis (ECRB) branches of the radial nerve. Recipient nerves were the anterior interosseous nerve (AIN) and the flexor digitorum profundus (FDP) branch of ulnar nerve (ulnar-FDP). Nerve transfers were evaluated on 3 parameters: surgical feasibility, donor-to-recipient axon count ratio, and distance from the coaptation site to the muscle entry of recipient nerve. A complete C6 spinal cord injury reconstruction was accomplished in a patient using a double nerve transfer of ECRB to ulnar-FDP and brachialis to AIN. In the cadaver study, nerve transfers from ECRB to AIN, brachialis to AIN, and ECRB to ulnar-FDP were all feasible. The transfer from the brachialis to ulnar-FDP was not possible. Mean myelinated axon counts of AIN, brachialis, ulnar-FDP, and ECRB were 2,903 ± 1049, 1,497 ± 606, 753 ± 364, and 567 ± 175, respectively. The donor-to-recipient axon count ratios of ECRB to AIN, brachialis to AIN, and ECRB to ulnar-FDP were 0.24 ± 0.15, 0.55 ± 0.38, and 0.98 ± 0.60, respectively. The distance from coaptation of the ECRB to the ulnar-FDP muscle entry was shorter than for the other nerve transfers (54 ± 14.29 mm). At 18 months, there was restoration of flexion in all fingers and functional improvement from double nerve transfer of the brachialis to the AIN and the ECRB to theulnar-FDP. Restoration of all finger flexion may be feasible by the ECRB to ulnar-FDP and brachialis to AIN double nerve transfer. Double nerve transfer can be used in C6-C7 spinal cord injury and patients with lower arm-type brachial plexus injury who have no finger flexion but have good brachialis and ECRB.

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

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. 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. 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). 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.

Is it necessary to use the entire root as a donor when transferring contralateral C7nerve to repair median nerve?

If a partial contralateral C7 nerve is transferred to a recipient injured nerve, results are not satisfactory. However, if an entire contralateral C7 nerve is used to repair two nerves, both recipient nerves show good recovery. These findings seem contradictory, as the above two methods use the same donor nerve, only the cutting method of the contralateral C7 nerve is different. To verify whether this can actually result in different repair effects, we divided rats with right total brachial plexus injury into three groups. In the entire root group, the entire contralateral C7 root was transected and transferred to the median nerve of the affected limb. In the posterior division group, only the posterior division of the contralateral C7 root was transected and transferred to the median nerve. In the entire root + posterior division group, the entire contralateral C7 root was transected but only the posterior division was transferred to the median nerve. After neurectomy, the median nerve was repaired on the affected side in the three groups. At 8, 12, and 16 weeks postoperatively, electrophysiological examination showed that maximum amplitude, latency, muscle tetanic contraction force, and muscle fiber cross-sectional area of the flexor digitorum superficialis muscle were significantly better in the entire root and entire root + posterior division groups than in the posterior division group. No significant difference was found between the entire root and entire root + posterior division groups. Counts of myelinated axons in the median nerve were greater in the entire root group than in the entire root + posterior division group, which were greater than the posterior division group. We conclude that for the same recipient nerve, harvesting of the entire contralateral C7 root achieved significantly better recovery than partial harvesting, even if only part of the entire root was used for transfer. This result indicates that the entire root should be used as a donor when transferring contralateral C7 nerve.

Low-frequency pulsed electromagnetic field pretreated bone marrow-derived mesenchymal stem cells promote the regeneration of crush-injured rat mental nerve.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to promote the regeneration of injured peripheral nerves. Pulsed electromagnetic field (PEMF) reportedly promotes the proliferation and neuronal differentiation of BMSCs. Low-frequency PEMF can induce the neuronal differentiation of BMSCs in the absence of nerve growth factors. This study was designed to investigate the effects of low-frequency PEMF pretreatment on the proliferation and function of BMSCs and the effects of low-frequency PEMF pre-treated BMSCs on the regeneration of injured peripheral nerve using in vitro and in vivo experiments. In in vitro experiments, quantitative DNA analysis was performed to determine the proliferation of BMSCs, and reverse transcription-polymerase chain reaction was performed to detect S100 (Schwann cell marker), glial fibrillary acidic protein (astrocyte marker), and brain-derived neurotrophic factor and nerve growth factor (neurotrophic factors) mRNA expression. In the in vivo experiments, rat models of crush-injured mental nerve established using clamp method were randomly injected with low-frequency PEMF pretreated BMSCs, unpretreated BMSCs or PBS at the injury site (1 × 106 cells). DiI-labeled BMSCs injected at the injury site were counted under the fluorescence microscope to determine cell survival. One or two weeks after cell injection, functional recovery of the injured nerve was assessed using the sensory test with von Frey filaments. Two weeks after cell injection, axonal regeneration was evaluated using histomorphometric analysis and retrograde labeling of trigeminal ganglion neurons. In vitro experiment results revealed that low-frequency PEMF pretreated BMSCs proliferated faster and had greater mRNA expression of growth factors than unpretreated BMSCs. In vivo experiment results revealed that compared with injection of unpretreated BMSCs, injection of low-frequency PEMF pretreated BMSCs led to higher myelinated axon count and axon density and more DiI-labeled neurons in the trigeminal ganglia, contributing to rapider functional recovery of injured mental nerve. These findings suggest that low-frequency PEMF pretreatment is a promising approach to enhance the efficacy of cell therapy for peripheral nerve injury repair.

Axon and Myelin Morphology in Animal and Human Spinal Cord.

Characterizing precisely the microstructure of axons, their density, size and myelination is of interest for the neuroscientific community, for example to help maximize the outcome of studies on white matter (WM) pathologies of the spinal cord (SC). The existence of a comprehensive and structured database of axonal measurements in healthy and disease models could help the validation of results obtained by different researchers. The purpose of this article is to provide such a database of healthy SC WM, to discuss the potential sources of variability and to suggest avenues for robust and accurate quantification of axon morphometry based on novel acquisition and processing techniques. The article is organized in three sections. The first section reviews morphometric results across species according to range of densities and counts of myelinated axons, axon diameter and myelin thickness, and characteristics of unmyelinated axons in different regions. The second section discusses the sources of variability across studies, such as age, sex, spinal pathways, spinal levels, statistical power and terminology in regard to tracts and protocols. The third section presents new techniques and perspectives that could benefit histology studies. For example, coherent anti-stokes Raman spectroscopy (CARS) imaging can provide sub-micrometric resolution without the need for fixation and staining, while slide scanners and stitching algorithms can provide full cross-sectional area of SC. In combination with these acquisition techniques, automatic segmentation algorithms for delineating axons and myelin sheath can help provide large-scale statistics on axon morphometry.

Distal sensory to distal motor nerve anastomosis can protect lower extremity muscle atrophy in a murine model

Delayed reinnervation of denervated motor neuron has irreversible consequences. We introduced distal motor to distal sensory anastomosis (DDSA) as a practical, time-saving method to protect injured motor neurons and its target tissues. Two experimental groups of Wistar rats were studied. In DDSA group, the distal end of the tibial sensory nerve of the left leg was anastomosed to the distal common peroneal nerve. The same nerves were dissected without anastomosis in the control group. Four months later, visual functional assessment of sciatic nerves was performed, and histological structures of the nerves and muscles and ultra-structure of nerves were evaluated. Significant enhancement was seen in intermediate toe spread factor in DDSA group (P < 0.05), but toe spread factor and subsequently sciatic statistic index demonstrated no significant improvement. The surgical procedures resulted in an ipsilateral rehabilitation in DDSA group with statistically significant (P < 0.05) improvement in muscle weight and myelinated axon count. Light and electron microscopy evaluations of the histological specimen showed obvious prevention of nerve and muscle tissues degeneration following anastomosis. Overall, DDSA showed a peripheral nerve could repair, survive, and protect target tissues from degeneration without connection to their cell bodies and central nervous system. Some possible explanations for these positive results could be the restorative role of electrochemical signaling directly from the skin sensory nerve receptors and stimulation of Schwann cell to convert to its regenerative phenotype. Level of Evidence: Not ratable.

Selective Nerve Root Transection in the Rat Produces Permanent, Partial Nerve Injury Models with Variable Levels of Functional Deficit.

The goal of this study was to develop a partial, nonregenerative nerve injury model in a rat that results in permanently reduced motoneuron numbers and function. This model could serve as a platform for the study of therapeutics, such as a reverse end-to-side nerve transfer (i.e., supercharge). The authors hypothesized that transection of one or more of the L4 to L6 nerve roots supplying the sciatic nerve would cause a permanent reduction in muscle force. Rats were randomized into five groups that underwent variations of nerve root transections or sham injury. The L4 to L6 nerve roots were selectively transected and capped to prevent regeneration. Tibial and common peroneal nerves were harvested for quantitative histology and retrograde-labeled to assess the number of motoneurons projecting axons. Muscle force and relative muscle mass were assessed as metrics of postinjury motor function. At 6 months, the number of motoneurons projecting axons and myelinated axon counts were reduced in both the tibial and common peroneal nerves after injury in all groups. Transecting both L4 and L5 or both L4 and L6 reduced motoneuron numbers sufficiently below sham numbers to reduce muscle force and mass in major muscles of the hindlimb innervated by both nerves. Transecting L4 reduced muscle force and mass in common peroneal-innervated muscles, whereas transecting L5 reduced muscle force and mass in tibial-innervated muscles. These findings were stable over time. Transection of nerve roots produces stable (time-independent) partial nerve injury models with a selective decrease in motor function.