Chronic recording of hand prosthesis control signals via a regenerative peripheral nerve interface in a rhesus macaque.

OBJECTIVE: Loss of even part of the upper limb is a devastating injury. In order to fully restore natural function when lacking sufficient residual musculature, it is necessary to record directly from peripheral nerves. However, current approaches must make trade-offs between signal quality and longevity which limit their clinical potential. To address this issue, we have developed the regenerative peripheral nerve interface (RPNI) and tested its use in non-human primates. APPROACH: The RPNI consists of a small, autologous partial muscle graft reinnervated by a transected peripheral nerve branch. After reinnervation, the graft acts as a bioamplifier for descending motor commands in the nerve, enabling long-term recording of high signal-to-noise ratio (SNR), functionally-specific electromyographic (EMG) signals. We implanted nine RPNIs on separate branches of the median and radial nerves in two rhesus macaques who were trained to perform cued finger movements. MAIN RESULTS: No adverse events were noted in either monkey, and we recorded normal EMG with high SNR (>8) from the RPNIs for up to 20 months post-implantation. Using RPNI signals recorded during the behavioral task, we were able to classify each monkey's finger movements as flexion, extension, or rest with >96% accuracy. RPNI signals also enabled functional prosthetic control, allowing the monkeys to perform the same behavioral task equally well with either physical finger movements or RPNI-based movement classifications. SIGNIFICANCE: The RPNI signal strength, stability, and longevity demonstrated here represents a promising method for controlling advanced prosthetic limbs and fully restoring natural movement.

Conduction Properties Of Decellularized Nerve Biomaterials.

The purpose of this study is to optimize poly(3,4,-ethylenedioxythiophene) (PEDOT) polymerization into decellular nerve scaffolding for interfacing to peripheral nerves. Our ultimate aim is to permanently implant highly conductive peripheral nerve interfaces between amputee, stump, nerve fascicles and prosthetic electronics. Decellular nerve (DN) scaffolds are an FDA approved biomaterial (Axogen ) with the flexible tensile properties needed for successful permanent coaptation to peripheral nerves. Biocompatible, electroconductive, PEDOT facilitates electrical conduction through PEDOT coated acellular muscle. New electrochemical methods were used to polymerize various PEDOT concentrations into DN scaffolds without the need for a final dehydration step. DN scaffolds were then tested for electrical impedance and charge density. PEDOT coated DN scaffold materials were also implanted as 15-20mm peripheral nerve grafts. Measurement of in-situ nerve conduction immediately followed grafting. DN showed significant improvements in impedance for dehydrated and hydrated, DN, polymerized with moderate and low PEDOT concentrations when they were compared with DN alone (a ≤ 0.05). These measurements were equivalent to those for DN with maximal PEDOT concentrations. In-situ, nerve conduction measurements demonstrated that DN alone is a poor electro-conductor while the addition of PEDOT allows DN scaffold grafts to compare favorably with the "gold standard", autograft (Table 1). Surgical handling characteristics for conductive hydrated PEDOT DN scaffolds were rated 3 (pliable) while the dehydrated models were rated 1 (very stiff) when compared with autograft ratings of 4 (normal). Low concentrations of PEDOT on DN scaffolds provided significant increases in electro active properties which were comparable to the densest PEDOT coatings. DN pliability was closely maintained by continued hydration during PEDOT electrochemical polymerization without compromising electroconductivity.

Motion-specific laryngeal reinnervation using muscle-nerve-muscle neurotization.

There is no current treatment method that can reliably restore physiologic movement to a paralyzed vocal fold. The purposes of this study were to test the hypotheses that 1) muscle-nerve-muscle (M-N-M) neurotization can be induced in feline laryngeal muscles and 2) M-N-M neurotization can restore movement to a paralyzed vocal fold. Muscle-nerve-muscle neurotization can be defined as the reinnervation of a denervated muscle via axons that are induced to sprout from nerves within an innervated muscle and that then traverse a nerve graft interposed between it and the target denervated muscle. A paralyzed laryngeal muscle could be reinnervated by axons from its contralateral paired muscle, thus achieving motion-specific reinnervation. Eighteen adult cats were divided into sham, hemilaryngeal-denervated, and M-N-M-reinnervated thyroarytenoid muscle groups. Five of the 6 reinnervated animals had histologic evidence of axons in the nerve graft, 4 of the 6 had evoked electromyographic evidence of crossed reinnervation, and 1 of the 6 had a return of appropriately phased adduction. This technique has great potential and should be further investigated.

Range of motion physiotherapy reduces the force deficit in antagonists to denervated rat muscles.

BACKGROUND: We used a rat hindlimb model of tibial nerve transection to determine if a loss of mechanical function exists in innervated antagonists compared with denervated muscles. We tested two hypotheses: (1) denervation of the rat ankle plantar flexors results in decreased force production of the ankle dorsiflexors, and (2) daily passive ankle range of motion (ROM) physiotherapy prevents or reduces the force deficit. METHODS: Adult Lewis rats were assigned to one of three groups: (1) a sham (S) group, in which the tibial nerve was exposed but not transected; (2) a no rehabilitation (NR) group, in which a 2-cm segment of tibial nerve was excised at midthigh to denervate the ankle plantar flexors; or (3) a rehabilitation (R) group, in which a 2-cm segment of tibial nerve was excised and the animals were subjected to ankle passive ROM physiotherapy for two 5-min sessions each day. After 14 days, maximum isometric tetanic force (F(0)) and specific force (sF(0)) were measured in the extensor digitorum longus (EDL) muscle, an ankle dorsiflexor. RESULTS: Compared with those from animals in the S group, EDL muscles from animals in the NR group demonstrated a 22% decrease in both F(0) and sF(0). In the EDL from animals in the R group, daily passive ROM physiotherapy diminished the deficit in F(0) but not in sF(0). CONCLUSIONS: These data support the hypotheses that nerve injuries result in impaired mechanical function in the innervated antagonists to denervated muscles and that passive ROM physiotherapy can improve force production in these muscles.

"Donor" muscle structure and function after end-to-side neurorrhaphy.

End-to-end nerve coaptation is the preferred surgical technique for peripheral nerve reconstruction after injury or tumor extirpation. However, if the proximal nerve stump is not available for primary repair, then end-to-side neurorrhaphy may be a reasonable alternative. Numerous studies have demonstrated the effectiveness of this technique for muscle reinnervation. However, very little information is available regarding the potential adverse sequelae of end-to-side neurorrhaphy on the innervation and function of muscles innervated by the "donor" nerve. End-to-side neurorrhaphy is hypothesized to (1) acutely produce partial donor muscle denervation and (2) chronically produce no structural or functional deficits in muscles innervated by the donor nerve. Adult Lewis rats were allocated to one of two studies to determine the acute (2 weeks) and chronic (6 months) effects of end-to-side neurorrhaphy on donor muscle structure and function. In the acute study, animals underwent either sham exposure of the peroneal nerve (n = 13) or end-to-side neurorrhaphy between the end of the tibial nerve and the side of the peroneal nerve (n = 7). After a 2-week recovery period, isometric force (F(0) was measured, and specific force (sF(0) was calculated for the extensor digitorum longus muscle ("donor" muscle) for each animal. Immunohistochemical staining for neural cell adhesion molecule (NCAM) was performed to identify populations of denervated muscle fibers. In the chronic study, animals underwent either end-to-side neurorrhaphy between the end of the peroneal nerve and the side of the tibial nerve (n = 6) or sham exposure of the tibial nerve with performance of a peroneal nerve end-to-end nerve coaptation approximately 6), to match the period of anterior compartment muscle denervation in the end-to-side neurorrhaphy group. After a 6-month recovery period, contractile properties of the medial gastrocnemius muscle ("donor" muscle) were measured. Acutely, a fivefold increase in the percentage of denervated muscle fibers (1 +/0 0.7 percent to 5.4 +/-2.7 percent) was identified in the donor muscles of the animals with end-to-side neurorrhaphy (p < 0.001). However, no skeletal muscle force deficits were identified in these donor muscles. Chronically, the contractile properties of the medial gastrocnemius muscles were identical in the sham and end-to-side neurorrhaphy groups. These data support our two hypotheses that end-to-side neurorrhaphy causes acute donor muscle denervation, suggesting that there is physical disruption of axons at the time of nerve coaptation. However, end-to-side neurorrhaphy does not affect the long-term structure or function of muscles innervated by the donor nerve.

Specific force deficit in skeletal muscles of old rats is partially explained by the existence of denervated muscle fibers.

We tested the hypothesis that denervated muscle fibers account for part of the specific force (sF(o)) deficit observed in muscles from old adult (OA) mammals. Whole muscle force (F(o)) was quantified for extensor digitorum longus (EDL) muscles of OA and young adult (YA) rats. EDL muscle sF(o) was calculated by dividing F(o) by either total muscle fiber cross-sectional area (CSA) or by innervated fiber CSA. Innervated fiber CSA was estimated from EDL muscle cross sections labeled for neural cell adhesion molecules, whose presence is a marker for muscle fiber denervation. EDL muscles from OA rats contained significantly more denervated fibers than muscles from YA rats (5.6% vs 1.1% of total CSA). When compared with YA muscle, OA muscle demonstrated deficits of 34.1% for F(o), 28.3% for sF(o), and 24.9% for sF(o) calculated by using innervated CSA as the denominator. Denervated muscle fibers accounted for 11.3% of the specific force difference between normal YA and OA skeletal muscle. Other mechanisms in addition to denervation account for the majority of the sF(o) deficit with aging.

Thyroarytenoid muscle maintains normal contractile force in chronic vocal fold immobility.

BACKGROUND: Denervation of skeletal muscle typically results in irreversible denervation atrophy over time. This finding has generated controversy as to the efficacy of reinnervation procedures for chronic vocal fold immobility related to recurrent laryngeal nerve injury. OBJECTIVE: To test the hypothesis that chronic vocal fold immobility after recurrent laryngeal nerve injury does not result in diminished maximal isometric force generation in the thyroarytenoid muscle. STUDY DESIGN: Adult random-bred cats underwent either unilateral laryngeal denervation (n = 6) or sham surgery (n = 6). After 6 months, videolaryngoscopy was performed followed by in vitro measurement of maximal isometric tetanic force produced by the thyroarytenoid muscle. RESULTS: All animals in the denervation group showed right vocal fold paralysis after the initial denervation operation; none of these animals had return of appropriately phased movement with respiration. Four had intermittent disorganized twitching movements. One had these movements plus an occasional weak adduction, and one had no movement. Normal vocal fold mobility was observed in 6 of 6 animals undergoing sham surgery. The maximal isometric tetanic force measured from the thyroarytenoid muscle in the sham group was 438 mN (+/-92 mN standard deviation [SD]). The maximal isometric tetanic force measured from the thyroarytenoid muscle in the chronically immobile group was 405 mN (+/-107 mN SD). Differences were not statistically significant. CONCLUSION: Maximal isometric force in the thyroarytenoid muscle is not diminished in chronic vocal fold immobility after recurrent laryngeal nerve injury. We conclude that the possibility for restoration of contractile force to the chronically immobile thyroarytenoid muscle exists. This finding supports the pursuit of reinnervation procedures in the treatment of chronic vocal fold immobility.

Skeletal muscle reinnervation by reduced axonal numbers results in whole muscle force deficits.

Patients sustaining a peripheral nerve injury will frequently experience residual muscle weakness after muscle reinnervation, even if the nerve repair is performed under optimal circumstances to allow rapid muscle reinnervation. The mechanisms responsible for this contractile dysfunction remain unclear. It is hypothesized that after peripheral nerve injury and repair, a reduced number of axons are available for skeletal muscle reinnervation that results in whole muscle force and specific force deficits. A rat model of peroneal nerve injury and repair was designed so that the number of axons available for reinnervation could be systematically reduced. In adult rats, the peroneal nerve to the extensor digitorum longus muscle was either left intact (sham group, n = 8) or divided and repaired with either 50 percent (R50 group, n = 7) or 100 percent (R100 group, n = 8) of the axons in the proximal stump included in the repair. Four months after surgery, maximal tetanic isometric force was measured and specific force was calculated for each animal. Mean tetanic isometric force for extensor digitorum longus muscles from R50 rats (2765.7 +/- 767.6 mN) was significantly lower than sham (4082.8 +/- 196.5 mN) and R100 (3729.0 +/-370.2 mN) rats (p < 0.003). Mean specific force calculations revealed significant deficits in both the R100 (242.1 +/- 30 kN/m2) and R50 (190.6 +/- 51.8 kN/m2) rats compared with the sham animals (295.9 +/- 14 kN/m2) (p < 0.0005). These data support our hypothesis that after peripheral nerve injury and repair, reinnervation of skeletal muscle by a reduced number of axons results in a reduction in tetanic isometric force and specific force. The greater relative reduction in specific force compared with absolute force production after partial nerve repair may indicate that a population of residual denervated muscle fibers is responsible for this deficit.

Termino-lateral neurorrhaphy: the functional axonal anatomy.

The goal of this study was to determine the functional axonal anatomy of a termino-lateral neurorrhaphy (TLN). We hypothesize that axons populating a TLN must relinquish functional connections with their original targets prior to establishing new connections via the TLN. Two-month-old F344 rats underwent a TLN between the left peroneal nerve and a nerve graft tunneled to the contralateral hindlimb. Three months postoperatively, an end-to-end neurorrhaphy was performed between the nerve graft and the right peroneal nerve. Four months after the second operation, contractile properties and electromyographic (EMG) signals were measured in the bilateral hindlimbs. Left peroneal nerve stimulation proximal to the TLN site resulted in bilateral extensor digitorum longus (EDL) and tibialis anterior (TA) muscle contractions, with significantly lower forces on the side reinnervated by TLN. Evoked EMGs demonstrated that the right and left hindlimb musculature were electrically discontinuous following TLN. These data support our hypothesis that axons can form functional connections via a TLN, but they must first relinquish functional connections with their original targets.

Rat walking tracks do not reflect maximal muscle force capacity.

The relationship between walking-track measurements and maximum force generation in reinnervated rat hindlimb muscles was assessed. A rat model was designed to result in a broad range of recoveries of both muscle force and walking-track measurements after unilateral sciatic nerve injury and reconstruction. Three months following sciatic nerve injury, maximal force in the extensor digitorum longus (EDL) muscle ranged from 1325 to 3666 mN, and maximal specific forces ranged from 137.5 to 359.4 kNm(-2). In the same animals, functional intermediate toe spread factor (FIS) ranged from -0.03 to -0.78. Neither the correlation coefficient between EDL muscle maximal force and FIS (r = 0.4) nor that between EDL maximal specific force and FIS (r = -0.2) were statistically significant. The lack of correlation between muscle maximal force values and walking-track measurements suggests that these neuromuscular tests are assessing different factors.

The effect of reinnervation on force production and power output in skeletal muscle.

Failure to fully restore contractile function after denervation and reinnervation of skeletal muscle engenders significant disability in patients suffering peripheral nerve injuries. This work tested the hypothesis that skeletal muscle denervation and reinnervation result in a deficit in normalized power (W/kg), which exceeds the deficit in specific force (N/cm2), and that the mechanisms responsible for these deficits are independent. Adult Lewis rats underwent either transection and epineurial repair of the left peroneal nerve (denervation-reinnervation, n = 13) or SHAM exposure of the peroneal nerve (SHAM, n = 13). After a 4-month recovery period, isometric force, peak power, and maximum sustained power output were measured in the left extensor digitorum longus (EDL) muscle from each animal. Isometric force measurements revealed a specific force deficit of 14.3% in the reinnervated muscles. Power measurements during isovelocity shortening contractions demonstrated a normalized peak power deficit of 25.8% in the reinnervated muscles, which is accounted for by decreases in both optimal velocity (10.5%) and average force during shortening (13.7%). Maximum sustained power was similar in both groups. These data support our working hypothesis that both whole muscle force production and power output can be impaired in reinnervated muscle and that the relative deficits in power output exceed the deficits in force production. The mechanisms responsible for the deficits in force production appear to be independent of those that result in changes in peak power output. The measurement of muscle power output may represent a clinically relevant variable for studies of the recovery of mechanical function after motor nerve injury and repair.

Deletion of individual muscles alters rat walking-track parameters.

Rat walking-track analysis has been employed extensively to quantify motor recovery in studies of hindlimb nerve injury and repair. In order to clarify the relationship between individual print measurements and the function of specific hindlimb muscles, 40 young adult rats were assigned to one of five groups (n = 8/group) in which specific deletions of motors were created as follows: Group 1--division of the tendon of insertion of the gastrocnemius, soleus, and plantaris muscles (GSP); Group 2--division of the extensor digitorum longus muscles tendons of insertion (EDL); Group 3--division of the extensor hallicus longus muscle tendon of insertion (EHL); Group 4--division of the tibialis anterior muscle tendon of insertion (TA); or Group 5--division of the tibial nerve at the ankle (TNA). Parameters for print length (PL), intermediate toe spread (IS), and total toe spread (TS) were calculated from walking tracks recorded before and again five days after deletion of the motors. Specific, predictable patterns of change in footprint parameters were observed for each group. It was concluded that triceps surae and tibialis anterior muscle function directly affects print length; EHL function directly influences total toe spread; and EDL function is directly related to intermediary toe spread. These data demonstrate a direct relationship between individual print measurements and the function of individual rat hindlimb muscles.

The seven deadly sins of statistical analysis.

In a pedantic but playful way, we discuss some common errors in the use of 'statistical analysis' that are regularly observed in our professional plastic surgical literature. The seven errors we discuss are (1) the use of parametric analysis of ordinal data; (2) the inappropriate use of parametric analysis in general; (3) the failure to consider the possibility of committing type II statistical error; (4) the use of unmodified t-tests for multiple comparisons; (5) the failure to employ analysis of covariance, multivariate regression, nonlinear regression, and logistical regression when indicated; (6) the habit of reporting standard error instead of standard deviation; and (7) the underuse or overuse of statistical consultation. Confidence and common sense are advocated as a means to balance statistical significance with clinical importance.