Distal Fascicles Research Articles

Heterogeneous fascicle behavior within the biceps femoris long head at different muscle activation levels

Magnetic resonance and ultrasound imaging have shown hamstring strain injuries occur most often in the biceps femoris long head (BFLH), and particularly in the proximal vs. distal region of this muscle. Animal research and musculoskeletal modeling (MSK) have detected heterogeneous fascicle behavior within muscle regions, and within fascicles. Understanding architectural behavior differences during muscle contractions may help to discern possible mechanisms behind proximal BFLH injuries. The purpose of our study was to assess the magnitude of shortening of the proximal and distal fascicles of the BFLH under a range of muscle activation levels under isometric conditions using ultrasound imaging (US). Thirteen healthy adults performed targeted sustained isometric contractions while US were taken of the entire BFLH. Measurements of fascicle lengths in both muscle regions were compared at 20%, 30%, 50%, and 67% MVIC. The results showed that while both regions shortened significantly with activation, the proximal fascicles were significantly longer, regardless of activation level (~38%), and shortened significantly more than the distal fascicles overall (~40%), and cumulatively at higher activation levels (30% and above). No significant strain differences were found between the two regions. These data suggest heterogeneous fascicle behavior exists in an absolute sense; however, differences in behavior are eliminated when normalized (strain). Coupled with MSK literature, the absence of regional fascicle strain differences in this study may indicate strain heterogeneity is not detectable at the whole fascicle level. Further knowledge of this commonly strained muscle׳s regional behavior during dynamic movements could provide evidence of proximal hamstring strain predisposition.

An anatomic study of the structure and innervation of the pronator quadratus muscle.

The pronator quadratus muscle is composed of the superficial and deep heads. However, to date there is no consensus on the variations of each head. To add to this enigma, the innervation patterns of each head have not been thoroughly studied. The present study was conducted to clarify the structure and innervation of the pronator quadratus muscle by minute dissection of 46 forearms from 26 cadavers. The origin, insertion, shape, and direction of the muscle fascicles in each head were observed. The intramuscular distribution and the innervation patterns to each head were investigated. The attachment of the deep head was examined using Masson's trichrome staining technique. Each head consisted of various muscle fascicles which differed in shape and direction. The most distal muscle fascicle extended toward the head of the ulna. On microscopic study, this most distal fascicle was observed to reach the base of the ulnar styloid process. The nerves innervating the superficial head penetrated through the deep head and ran on the anterior surface of the radius from medial to lateral. This tendency was common to all of the forearms studied. We confirmed that each head of the pronator quadratus muscle consisted of various muscle fascicles. The attachment to the base of the ulnar styloid process is considered to be an important structure that prevents the head of the ulna from impacting against the carpal bones. Knowledge of the innervation pattern to each head is critical for preserving the function of the pronator quadratus muscle during surgery for distal radial fracture.

Ankle Impingement Syndromes

Ankle injuries are common occurrences in athletics involving and requiring extreme ranges of motion. Ankle sprains specifically occur with a 1 in 10,000 person rate in active individuals each day. If trauma is repetitive, the ankle structures have potential to experience secondary injury and dysfunction. Included in this category of dysfunction are both anterior and posterior ankle impingement syndromes where disruption of the bony structures, joint capsule, ligaments, and tendons typically occurs. Ankle impingement is described as ankle pain that occurs during athletic activity, with recurrent, extreme dorsiflexion or plantar flexion with the joint under a load. Ankle impingements can be classified according to what structures become involved both anteriorly and posteriorly. Osseous impingement, soft tissue impingement, impingement of the distal fascicle of anterior inferior tibiofibular ligament, and meniscoid lesions are all documented causes of ankle impingement. These changes tend to be brought about and exacerbated by extreme ranges of motion. Understanding various impingement types will better enable the clinician to prevent, identify, treat, and rehabilitate affected ankles. Acknowledging activities that predispose to ankle impingement syndrome will enhance prevention and recovery processes. Description of ankle impingement etiology and pathology is the objective of the current review.

Arthroscopic Treatment of Anterolateral Impingement of the Ankle

Anterolateral ankle impingement is due to entrapment of pathological tissues in the anterolateral aspect of the joint. The aim of this study was to evaluate the results of arthroscopic diagnosis and treatment of anterolateral ankle impingement. Twenty patients with anterolateral ankle impingement who had arthroscopic debridement were included in this prospective study, including 16 men and 4 women with a mean age of 35.8 years. Synovial hypertrophy was found in 13 patients, impinging distal fascicle of the anterior inferior tibiofibular ligament in 2 patients, torn anterior inferior tibiofibular ligament in 3 patients, and fibrofatty scar tissue in 2 patients. Seven patients had associated talar cartilage damage. Four patients had additional inversion injuries after the arthroscopic procedures. Final results were excellent in 11 patients, good in 6 patients, fair in 2 patients, and poor in 1 patient. One of the 7 patients with cartilage damage had excellent results, 5 patients had good results, and the remaining patient had fair results. Two of the 3 patients with complete tear of the anterior inferior tibiofibular ligament had fair results, and the other had a poor result. One of the 4 patients with additional inversion injuries had good results, 2 had fair results, and 1 had a poor result. In conclusion, ankle arthroscopy is useful in the diagnosis and treatment of anterolateral ankle impingement. Factors that negatively affect final outcome are associated chondral lesions of the talus, associated syndesmotic lesions, and new inversion injuries after the arthroscopic procedure.

The distal fascicle of the anterior inferior tibiofibular ligament as a cause of tibiotalar impingement syndrome: a current concepts review

Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior, anterolateral, or posterior. Ankle impingement syndromes are painful conditions caused by the friction of joint tissues, which are both the cause and the effect of altered joint biomechanics. The distal fascicle of the anterior inferior tibiofibular ligament (AITFL) is possible cause of anterior impingement. The objective of this article was to review the literature concerning the anatomy, pathogenesis, symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations. The AITFL starts from the distal tibia, 5 mm in average above the articular surface, and descends obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus. The incidence of the accessory fascicle differs very widely in the several studies. The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding. It may become pathological, due to anatomical variations and/or anterolateral instability of the ankle resulting from an anterior talofibular ligament injury. When observed during an ankle arthroscopy, the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle. The presence of the AITFL and the contact with the talus is a normal finding. An impingement of the AITFL can result from an anatomical variant or anteroposterior instability of the ankle. The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion injury and have a stable ankle, normal plain radiographs, and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament. The impingement syndrome can be treated arthroscopically.

Immunohistochemical profile of transforming growth factor-β1 and basic fibroblast growth factor in sciatic nerve anastomosis following pinealectomy and exogenous melatonin administration in rats

Collagen scar formation at the cut end of a peripheral nerve, an important problem in clinical practice for neurosurgeons, obstructs sprouting of axons into appropriate distal fascicles, and thereby limits the regeneration process. Researchers have attempted to control collagen accumulation and neuroma formation with various physical and chemical methods, but with limited functional success. Recently, it has been demonstrated that transforming growth factor (TGF)-β and basic fibroblast growth factor (bFGF) play an important role in collagen production by fibroblasts and in Schwann cell activity. In our study, rats were divided into a control group, a melatonin-treated group, a surgical pinealectomy group, and a group treated with melatonin following pinealectomy. They then underwent a surgical sciatic nerve transection and primary suture anastomosis. At 2 months after anastomosis, the animals were sacrificed and unilateral sciatic nerve specimens, including the anastomotic region, were removed and processed for immunohistochemical study from two animals in each group. For each antibody, immunoreactivity was assessed using a semiquantitative scoring system. Strong TGF-β1 and/or bFGF expression was observed in the epineurium of animals that underwent pinealectomy, but no or weak staining was observed in animals in the control and melatonin treatment groups. Based on these data, we suggest that both TGF-β1 and bFGF have important roles in control of collagen accumulation and neuroma formation at the anastomotic site, and that the pineal neurohormone melatonin has a beneficial effect on nerve regeneration.

Muscle fascicle and series elastic element length changes along the length of the human gastrocnemius during walking and running

Ultrasound imaging has recently been used to distinguish the length changes of muscle fascicles from those of the whole muscle tendon complex during real life movements. The complicated three-dimensional architecture of pennate muscles can however cause heterogeneity in the length changes along the length of a muscle. Here we use ultrasonography to examine muscle fascicle length and pennation angle changes at proximal, distal and midbelly sites of the human gastrocnemius medialis (GM) muscle during walking (4.5 km/h) and running (7.5 km/h) on a treadmill. The results of this study have shown that muscle fascicles perform the same actions along the length of the human GM muscle during locomotion. However the distal fascicles tend to shorten more and act at greater pennation angles than the more proximal fascicles. Muscle fascicles acted relatively isometrically during the stance phase during walking, however during running the fascicles shortened throughout the stance phase, which corresponded to an increase in the strain of the series elastic elements (SEEs) (consisting of the Achilles tendon and aponeurosis). Measurement of the fascicle length changes at the midbelly level provided a good approximation of the average fascicle length changes across the length of the muscle. The compliance of the SEE allows the muscle fascicles to shorten at a much slower speed, more concomitant with their optimal speed for maximal power output and efficiency, with high velocity shortening during take off in both walking and running achieved by recoil of the SEE.

Assessment of effects of pinealectomy and exogenous melatonin administration on rat sciatic nerve suture repair: an electrophysiological, electron microscopic, and immunohistochemical study

Collagen scar formation at the cut end of a nerve, an important problem in clinical practice for neurosurgeons in peripheral nerve surgery, obstructs sprouting of axons into appropriate distal fascicles, and thereby limits nerve regeneration. Researchers attempt to control collagen accumulation in the formation of neuroma by various physical and chemical methods, but these have yielded only limited functional success. This is the first experimental study investigating the effects of melatonin (MLT) on nerve repair and neuronal regeneration in rat sciatic nerve suture repair. The hypothesis that exogenous MLT administration may inhibit the formation of neuroma in peripheral nerve surgery was investigated in rat sciatic nerve model. In this study, a total of 80 rats were used for control groups (Groups Ia, Ib, IIa, and IId), MLT group (Group Ic), surgical pinealectomy (Px) groups (Groups IIb and IIc), and group of MLT treatment following Px procedure (Group IIe). All animals underwent a surgical intervention consisting of bilateral sciatic nerve section and primary suture repair. At 8 weeks after repair, the animals were killed following completion of recording of nerve action potentials (NAPs). Then, unilateral sciatic nerve specimens including the suture repair region were carefully removed and the excised segments were processed for electron microscopy examination. Afterwards, contralateral sciatic nerve specimens from two animals from each group were removed and stained for immunohistochemical analysis. Results of morphometric analysis revealed that Px procedure caused an elevation of collagen content of the sciatic nerve and macroscopic neuroma formation, and that there was a statistically significant reduction in collagen content of the same region in pinealectomized animals treated with MLT (p<0.001). Accordingly, electrophysiological findings demonstrated that the stimulus intensities required to excite a NAP response were increased in surgical Px group, but the presence of a reduced threshold response was found in the group treated with MLT following Px procedure (p<0.01). Immunohistochemical staining for Type I collagen and Type III collagen was markedly more intense in the epineurium of animals after Px. Virtually no or only weak staining was observed in animals in control groups and the MLT treatment group. Results of immunohistochemical analysis revealed that surgical Px procedure caused a strong immunoreactivity for Type I collagen and Type III collagen in all connective tissue planes of the nerve, especially in the epineurium, and there was a statistically significant reduction in immunoreactivity of the repair region in animals receiving MLT treatment after Px procedure (p<0.001). This study demonstrates that exogenous MLT administration significantly inhibits collagen accumulation in the formation of neuroma in the suture repair site and thereby improves nerve regeneration. From a clinical standpoint, the positive effect of MLT administration on neuroma formation and nerve regeneration seems a particularly attractive treatment option. Therefore, we believe that nerve repair with addition of MLT may be a worthwhile option in addition to other treatment modalities in case of MLT deficiency, such as aging. However, further experimental and clinical studies using functional analysis warranted to confirm this result in future.

The Accessory Anteroinferior Tibiofibular Ligament as a Cause of Talar Impingement

Background A distal fascicle of the anteroinferior tibiofibular ligament has been reported as a possible cause of anterolateral ankle impingement. Hypothesis This fascicle represents an independent accessory ligament in the anterior aspect of the inferior tibiofibular syndesmosis. Study Design Descriptive anatomical study. Methods The inferior tibiofibular syndesmosis was exposed in 24 fresh-frozen cadaveric ankles. Morphological characteristics of the inferior tibiofibular ligaments and interactions between the anterior ligaments and the talus were investigated in different degrees of ankle motion. Results A separate accessory anterior ligament was identified in 22 specimens. Its orientation was parallel and distal to the anterior tibiofibular ligament, from which it was separated by a fibrofatty septum. Its length ranged from 17 to 22 mm, its thickness from 1 to 2 mm, and its width from 3 to 5 mm. Impingement of this ligament against the talar dome was documented in all specimens in neutral ankle position, with bending of the ligamentous bands in dorsiflexion. Distraction maneuvers reproducing traction during arthroscopic approaches relieved contact. Conclusions An accessory anteroinferior tibiofibular ligament with distinct anatomical features was recognized. Clinical Relevance Based on its increased incidence in the cadaver ankles, the accessory ligament reflects a normal anatomical finding. Nevertheless, it could lead to anterolateral impingement in cases with coexistent ankle instability.

Mixed Nerve Suture Facilitated by Enzyme-Staining Techniques

Identification of motor and sensory fascicles to correctly match the motor and sensory fascicles helps surgeons to correctly align nerve stumps. Motor fibers, with their high cholinesterase activity, can be differentiated from sensory fibers. I developed a new modification that requires less than 1 hour and shows clearer differentiation compared with other histochemical techniques. In my clinical research, proximal stumps can be stained as long as 16 months after injury, however, distal stumps cannot be stained after 5 days. After 5 days, this staining could provide useful proximal information and distal fascicles may be identified by anatomic or topographical knowledge. I think this staining is especially useful in proximal nerve lesions and nerve injuries that require nerve graft within 4 days after injury. Also, this technique is useful in functioning muscle transfer to find motor fascicles and, in a sensate flap, to identify the sensory fascicle.

The anterior inferior tibiofibular ligament and talar impingement: a cadaveric study.

Impingement by the distal fascicle of the anterior inferior tibiofibular ligament (AITFL) is a relatively new entity among the known causes of anterolateral impingement syndromes of the ankle. This study investigated the anatomy of the anterior inferior tibiofibular ligament and its possible role in talar impingement in 47 ankles of 27 cadavers. The length, width, insertion point to the fibula and the interactions with talus were noted, as was the relationship of the fascicle and talus during different ankle movements before and after incision of the lateral ligaments. A distal fascicle of the AITFL was found in 39 of the 47 ankles (83%) and appeared as a single-complete ligament in the remaining 8 ankles (17%). The fascicle averaged 16.1+/-2.94 mm in length (range 10-21) and 4.2+/-1.00 mm in width (range, 3-7). The insertion point of the fascicle on the fibula averaged 10.3+/-2.27 mm (5-13) distal to the joint level. Contact between the ligament and the lateral dome of the talus was observed in 42 specimens (89.3%). Bending of the fascicle was observed in 8 of these 42 ankles with forced dorsiflexion. These 8 specimens were significantly wider and longer than the specimens without bending of the fascicle. Incision of the anterior talofibular ligament led to bending in dorsiflexion in additional 11 ankles. The total 19 fascicles with bending after incision of the anterior talofibular ligament were significantly longer and inserted more distally than the remaining 20 fascisles without bending. Manual traction simulating distraction during arthroscopic procedures relieved the contact. These findings show that the presence of the distal fascicle of the AITFL and its contact with the talus is a normal finding. However, it may become pathological due to anatomical variations and/or instability of the ankle resulting from torn lateral ligaments. When observed during an ankle arthroscopy, the surgeon should look for the criteria described in the present study to decide whether it is pathological and needs to be resected.

Motor pudendal nerve characterization in the female rat.

The aim of our study was to provide quantitative data on pudendal motor neuron cell bodies and axons in the female rat. To confirm earlier studies, fluorescent retrograde tracers were used to label the motor neurons for correlation with myelinated axon counts along the length of the motor pudendal nerve. The external urethral sphincter of female rats was injected with diamidino yellow and the external anal sphincter with fast blue. The L(6) spinal cord revealed labeled motor neurons. Those in the dorsolateral column (60.8 +/- 10.6) had nuclei labeled yellow from the external urethral sphincter and those in the dorsomedial column (31.7 +/- 8.5) had cytoplasm labeled blue from the external anal sphincter. Double labeling was not present, suggesting that pudendal motor neurons in each column innervate separate sphincters. The motor pudendal nerve in the ischiorectal fossa was also characterized by light microscopy. The mean myelinated axon count (151.4 +/- 17.0) was highly correlated (r = 0.995) in the proximal fascicles and the sum of distal fascicles. This indicated that myelinated axons do not branch at the point where the main motor pudendal nerve branches into separate fascicles. Axon counts between sides were not as well correlated (r = 0.883). The ratio of motor neurons to myelinated axons is 56%, suggesting that some myelinated axons either innervate other muscles or are sensory. This reproducible characterization of the normal pudendal nerve anatomy provides an excellent basis for experimental studies associated with pudendal nerve denervation as a model for neurogenic incontinence.

The distal fascicle of the anterior inferior tibio-fibular ligament as a cause of anterolateral ankle impingement: results of arthroscopic resection.

We arthroscopically resected the impinged distal fascicle of the anterior inferior tibiofibular ligament (AIT-FL) in 21 patients (mean age 31 (11-68) years, 14 women) with chronic ankle pain after an ankle sprain. Clinical tests revealed moderate laxity in 2 and severe laxity in another 2, the remaining 17 ankles showing only mild laxity. During arthroscopy, an impinging distal fascicle of the AITFL was found in all cases. Following anterolateral synovectomy, the fascicle was excised. At the follow-up after mean 3 (2-4) years, good-to-excellent results were obtained in 17 patients. 19 patients were satisfied with the procedure and 17 patients returned to their previous level of activity. 2 patients who had mild laxity were graded as poor because of neuromas of the terminal branches of the superficial peroneal nerve. These patients became asymptomatic after an injection of steroids.

Functional results of low median and ulnar nerve repair with intraneural fascicular dissection and electrical fascicular orientation

Twenty-eight low median nerve injuries and 23 low ulnar nerve injuries were repaired using intraneural fascicular dissection and electrical fascicular orientation. Eleven freshly lacerated nerves were seen within 48 hours after injury; 40 nerve lacerations were chronic. Fascicular orientation between sensory and motor fascicles at the proximal nerve end could be accurately differentiated in 47 nerves (92%) independent of whether it was acute or chronic. At the distal nerve end in fresh lacerations, the motor fascicles could be determined conclusively by muscle contraction with sequential electrical stimulation of the fascicles. In chronic nerve lacerations, the distal fascicles could be estimated anatomically after internal neurolysis. After fascicular orientation, nerves were repaired with end-to-end group fascicular suture or interfascicular sural nerve grafting. Twenty-four nerves repaired with end-to-end suture and 13 nerves repaired with nerve grafting were monitored more than 25 months. Satisfactory sensory results (i.e., S3+ or S4 functions) were obtained in 29 nerves (78%) and M4 or M5 motor functions were achieved in 29 nerves (78%). There were no patients who needed additional tendon transfers to reconstruct thumb opposition or to correct claw finger deformity. These results suggest that low median or ulnar nerve lacerations, whether acute or chronic, partial or complete, may be successfully repaired with the aid of electrical fascicular orientation with or without intraneural fascicular dissection.

Eccentric training does not increase sarcomere number in rabbit dorsiflexor muscles

The hypothesis of this study was that eccentric muscle training induces serial sarcomere addition. Eccentric training of rabbit dorsiflexor muscles (50 maximal contractions, 2 times per week, for 12 weeks) increased sarcomere number by only 3% in superficial fascicles of the tibialis anterior (TA) and did not change sarcomere number in deep fascicles of the TA nor in proximal or distal fascicles of extensor digitorum longus. Thus eccentric training had little or no effect on sarcomere number. These results do not support previous speculation that eccentric training produces increases in sarcomere number.

Isolated Fascicular Abducens Nerve Palsy and Lyme Disease

A patient with Lyme disease developed an isolated sixth nerve palsy at the end of a 1 month course of oral antibiotics. Magnetic resonance imaging disclosed high-signal abnormality at the left pontomedullary junction, implicating involvement of the distal sixth nerve fascicle. Although facial numbness ensued during a subsequent course of intravenous antibiotics, corticosteroid therapy was associated with prompt improvement of neurologic signs, suggesting an immunologic mechanism for the central nervous system dysfunction.

Axonal projections and functional recovery following fascicular repair of the rat sciatic nerve with Y-tunnelled silicone chambers

The projection of regenerating axons and the specificity of motor reinnervation were studied after repair of the transected rat sciatic nerve with Y-tunnelled silicone chambers. This geometry was used experimentally to face either the proximal tibial or peroneal fascicle with two distal fascicular options usually the distal peroneal and tibial fascicle. A 4 mm gap separated the proximal and distal fascicles. Four weeks after the repair, preferential motor reinnervation could be demonstrated and there were always more axons projecting towards the distal homonymous fascicle. In contrast, if the distal stumps were disconnected from the target no fascicle specific projection of axons was observed. This was true even if segments from the median and ulnar nerve were used to replace either the distal tibial or peroneal segments. It appeared as though the size and not the type of fascicle determined the number of attracted axons. The results suggest that there is no fascicle specific guidance of regenerating nerve fibers.

Reinnervation of muscles in rats after repair of transsected sciatic nerves with Y-shaped and X-shaped silicone tubes. Muscle reinnervation after nerve repair.

Reinnervation of the gastrocnemius and anterior tibial muscles was assessed by measurements of tetanic force after repair of sciatic nerves with Y-shaped or X-shaped silicone tubes in rats. The transsected proximal stump of either the tibial or the peroneal fascicle was introduced into the opening of a Y-shaped silicone tube, or both fascicles were introduced into an X-shaped tube. The distal tibial and peroneal fascicles were inserted into the distal outlets of the tubes leaving a gap of 4 mm between proximal and distal stumps. In the X-shaped tubes the proximal inserts were placed opposite or adjacent to their respective distal parts. Sixteen weeks later reinnervation was evaluated by measurements of tetanic force of the gastrocnemius and anterior tibial muscles after electrical stimulation of the fascicles. There was preferential reinnervation in both types of tubes. In Y-shaped tubes about 90% of the tetanic force could be recorded from both muscles after stimulation of the peroneal and tibial fascicles, respectively. Recovery was lower in the X-shaped tubes, amounting to about 75%. Contractions evoked by misrouted fibres were similar (roughly 40%) in both models. We conclude that motor axons preferentially, but not exclusively, selected a path to reinnervate their original target muscle.

Talar impingement by the anteroinferior tibiofibular ligament. A cause of chronic pain in the ankle after inversion sprain.

Seven patients who had pain in the anterior aspect of the ankle were found to have a thickened distal fascicle of the anteroinferior tibiofibular ligament. Each patient had a history of an inversion sprain of the ankle followed by chronic pain in the anterior aspect of the ankle. The thickened distal fascicle was resected without loss of stability of the ankle. Five patients needed débridement of an area of abraded hyaline cartilage on the anterolateral aspect of the talus. Six patients were followed for a mean of thirty-nine months (range, twenty-four to fifty-nine months). Four of them had no pain in the ankle or limitation in activities, and two reported marked improvement, with only occasional pain in the ankle related to overuse. A separate distal fascicle of the anteroinferior tibiofibular ligament is present in most human ankles and can be a cause of talar impingement, abrasion of the articular cartilage, and pain in the anterior aspect of the ankle. Resection of this ligament usually will alleviate the pain caused by the impingement.

Cytochemistry and morphometry of rat sciatic nerve following laser and suture treatments

The success of nerve repair treatment can be measured by examining the factors (Wallerian degeneration) that prevent functional recovery, and determining the extent of regeneration of proximal axon sprouts in the endoneurial environment of the distal fascicles. In the present study, the neurosurgical alignment of fascicles was attempted by laser as well as suture microsurgery of transected rat sciatic nerve and the relative efficacy of the two procedures was evaluated after 6 and 12 weeks by Neurophysiological Study, Morphological Study with light microscopy, ultrastructure, and the permeability of blood-nerve barrier using horseradish peroxidase (HRP) tracer technique, and Morphometry of the nerve fibers.