Spontaneous regeneration after resection of various lengths of hypoglossal nerve in rats.

OBJECTIVES: The objective of this study was to investigate spontaneous neural regeneration and functional recovery after resection of various lengths of the hypoglossal (XII) nerve in adult rats. METHODS: Twelve weeks after XII nerve resection at lengths ranging from 0.0-15.8 mm, the tongue deviation angle of rats was measured to evaluate the severity of paralysis; thereafter, the XII neurons in the XII nucleus were labeled with Fluoro-Gold (FG), which was injected into the tongue to visualize regenerated XII neurons re-innervating the tongue muscles. RESULTS: In the XII nerve-resected rats, the regenerative rates, that is, the percentage of the total number of FG-positive neurons on the injured side relative to that on the uninjured side, were divided into two groups; the regenerative rates were more than 77% and less than 6%, respectively. Upon comparing the two groups, the boundary resection length was approximately 10.0 mm. Moreover, the former and latter groups demonstrated tongue deviation angles less than or greater than 15°, respectively. CONCLUSIONS: The critical nerve gap length for spontaneous neural regeneration was approximately 10.0 mm in XII nerve-resected adult rats, and nerve regeneration occurred in both morphological and functional aspects after resection at less than the critical length.

The calcaneofibular ligament groove at the inferior fibula, an ultrasonographic anatomical landmark.

PURPOSE: Calcaneofibular ligament (CFL) injuries are harder to diagnose than anterior talofibular ligament (ATFL) ones. This study aimed to clarify the fibular attachment of the CFL and verify the bony landmark for evaluating the CFL on ultrasonography. METHODS: Fifty-nine ankles were used in this anatomical study. To confirm the control function of the CFL, we performed passive movement manually using cadaveric ankles and observed the ankle positions where the CFLs were tense. Histological observation of CFL attachment of the fibula was performed using Masson's trichrome stain. The ATFL and CFL were removed, and the bone morphology of the CFL attachment and inferior fibular end was imaged using a stereomicroscope and a 3D scanner. Using ultrasonography, we evaluated the bone morphology of the fibular attachment of the CFL in short-axis images of 27 healthy adult ankles. RESULTS: The CFL was tensed according to ankle motions: supination, maximum dorsi flexion, maximum plantar flexion, and mild plantar flexion-external rotation. Below the CFL attachment of the fibula was a slight groove between the inferior tip and the obscure tubercle of the fibula. This groove was observed in 81.5% of cases using short-axis ultrasonography. CONCLUSION: The CFL was tensed in various ankle positions to control the movements of the talocrural and subtalar joints. There was a slight groove at the inferior end of the fibula where the CFL coursed downward. We called it the CFL groove and proposed that it could serve as a landmark for the short-axis image of ultrasonography.

Difference in the fibular attachment structure between the superior and inferior fascicles of the anterior talofibular ligament using ultrasonography and histological examinations.

PURPOSE: The anterior talofibular ligament (ATFL) is divided into superior (SB) and inferior bands (IB). Although the differences in length and width are known, the structure of the fibular attachment had not been elucidated. The present study aimed to clarify the differences in the fibular attachment structure between ATFL's SB and IB using cross-sectional images along the ligament. METHODS: An anatomical study using 15 formalin-fixed ankles was performed. The lateral ankle ligament complex was collected after a longitudinal image of SB/IB was visualized by ultrasonography. The specimens were decalcified and sectioned longitudinally at the center of SB/IB using a microtome. Histological evaluation of the enthesis structure at the fibular attachment of SB/IB was performed using hematoxylin-eosin and Masson's trichrome stains. RESULTS: A fibrillar pattern could not be observed in the longitudinal image at the IB level by ultrasonography. The lengths of ATFL's SB and IB were 20.6 ± 1.6 and 15.3 ± 1.3 mm, respectively, with thicknesses of 1.8 ± 0.4 and 1.0 ± 0.4 mm, respectively. The ATFL's IB was significantly shorter and thinner than the ATFL's SB. The fibular attachment of ATFL's SB had distinct enthesis structure, whereas in the attachment structure of the ATFL's IB, there were several variations including a type with a narrower enthesis structure than the ATFL's SB and a type that merged with or wrapped around the calcaneofibular ligament. CONCLUSION: The fibular attachment structure between ATFL's SB and IB differs. Our results could be useful information when performing ultrasonography and MRI diagnosis.

Relationship between inferior fascicle of anterior talofibular ligament and articular capsule in lateral ankle ligament complex.

PURPOSE: The lateral ankle ligament complex (LALC) is composed of anterior talofibular (ATFL), calcaneofibular (CFL), and posterior talofibular (PTFL) ligaments, all of which have a connection/continuous fiber. However, the structural link between the LALC and the articular capsule remains unknown. The goal of our study was to determine the connection between ATFL's inferior fascicle and the articular capsule. METHODS: In this study, we utilized 84 formalin-fixed ankles to elucidate the structure of LALC. Between ATFL and CFL, the bundle number of ATFL and arciform fiber was investigated. The specimens were decalcified and sectioned coronally using a freezing microtome, in the case of double bundles of ATFL, to study the connection between the inferior fascicle of ATFL and the articular capsule. RESULTS: ATFL had a single (25%), double (74%), and triple (1%) bundle number, respectively. The arciform fiber connecting the ATFL and the CFL was found in the superficial layer of all ankles (100%). There were two types of relationships between the inferior fascicle of ATFL and the articular capsule: 36 ankles (58%) were extracapsular, and 26 of 62 ankles (42%) were integrated with the inferior-lateral articular capsule. There are two kinds of relationships between the inferior fascicle of the ATFL and the articular capsule: extracapsular and integrated-capsular. CONCLUSIONS: The inferior fascicle of ATFL has a variant and integrated-capsular type is reinforced inferior-lateral articular capsule and enters the joint to form continuous fibers with PTFL, making LALC. These anatomical findings are helpful in ultrasonography diagnosis and arthroscopic ankle surgery.

Continuous and Connective Fibers of the Lateral Ankle Ligament Complex.

The lateral ankle ligament complex (LALC) is an intricate structure; therefore precise anatomic knowledge is required by the surgeon. However, the structural relationship of the LALC remains unclear. Here, the features of the posterior talofibular ligament (PTFL) and the relationship to the LALC at the distal fibula were clarified in a cadaver study. The lengths of most of the anterior and posterior parts, and the widths of the anterior-posterior and superior-inferior parts, were measured with a digital caliper. In addition, the relationship between the anterior talofibular ligament (ATFL) and PTFL inside of the capsule is described. The small fiber bundles of the PTFL were manually divided, and the footprint of each bundle at the fibula and talus was clarified. The relationship between the ATFL and CFL, outside of the capsule, was examined on axial slices at the inferior fibula. The lengths of the most anterior and most posterior parts of the PTFL were 9.8 ± 1.7 and 29.4 ± 1.9 mm, respectively. The widths of the anterior-posterior and superior-inferior parts were 10.0 ± 0.9 and 5.8 ± 1.1 mm, respectively. Approximately 83% of the fibers between the ATFL and PTFL were continuous. The anterior-inferior fibers of the PTFL were continuous with the inferior fibers of the ATFL inside of the capsule. The ATFL and CFL converged with connective tissue from outside of the capsule at the distal fibula. The results of this study should prove useful to further clarify the relationships of the LALC both inside and outside of the capsule at the distal fibula.

Hypoglossal nerve injury with long nerve resection leading to slow motoneuron death.

Crush injury to peripheral nerves in adult animals is considered not to trigger retrograde neuronal cell death; however, several studies reported neuronal cell death following severe injuries including nerve transection, resection, or avulsion. However, the rate of neuronal cell death varied among studies. In this study, we evaluated the outcomes of very severe nerve injury by long nerve resection in adult rats. Right hypoglossal (XII) nerve was exposed, and a 9-mm section was resected. At 4, 8, and 12 weeks after the resection, the number of XII neurons were counted in from the rostral to caudal sections. The number of XII neurons in the injured right side was reduced after the XII nerve resection compared with the uninjured left side. The mean rates of surviving neurons at 4, 8, and 12 weeks after the nerve resection were 83.5 %, 73.9 %, and 61.1 %, respectively, which were significantly lower than those of the control. The number of XII neurons after extensive XII nerve resection declined gradually over a relatively long time period, revealing that extensive nerve resection led to slow cell death of the injured neurons.

Independent Attachment of Lateral Ankle Ligaments: Anterior Talofibular and Calcaneofibular Ligaments - A Cadaveric Study.

Anatomic knowledge of lateral ligaments around the lateral malleolus is important for repair or reconstruction of ankle instability. The detailed structure of the connective fibers between the anterior talofibular ligament (ATFL) and the calcaneofibular ligament (CFL) is unknown. To clarify the anatomic structure of ATFL and CFL and the connective fiber between the 2 ligaments, the lateral ligament was dissected in 60 ankles of formalin-fixed cadavers, and the distance was measured between bony landmarks and fibular attachment of ATFL and CFL using a digital caliper. All ankles had connective fibers between ATFL and CFL. The structure of connective fibers consisted of a thin fiber above the surface layer of ATFL and CFL; it comprised thin fibrils of the surface layer covering the lower part of ATFL and the front part of CFL. Both ATFL and CFL were independent fibers, and both attachments of the fibula were isolated. Single bands of ATFL were noted in 14 of 60 (23.3%) ankles, double bands that divided the superior and inferior bands were observed in 42 of 60 (70.0%) ankles, and multiple bands were observed in 4 of 60 (6.7%) ankles. A cord-like and a flat and fanning type of CFL was noted in 22 (36.7%) and 38 (63.3%) of the 60 ankles, respectively. Distances between ATFL/CFL and articular and inferior tips of the fibula were 4.3 ± 1.1 mm/7.6 ± 1.6 mm and 14.3 ± 1.9 mm/7.4 ± 1.7 mm, respectively (mean ± standard deviation). The results of this study suggest that knowledge of more anatomic structures of ATFL, CFL, and connective fiber will be beneficial for surgeons in the repair or reconstruction of the lateral ligament of the ankle.

Effect of graded nerve pressure injuries on motor function.

OBJECT: The purpose of this study was to determine the minimum amount of nerve fibers required to maintain normal motor function after nerve injury in rats. METHODS: The authors first confirmed that a common peroneal nerve injury caused more aggravating effects on lower limb motor function than tibial nerve injury, as assessed by the static sciatic index (SSI). Thereafter, rats were subjected to varying degrees of crush injury to the common peroneal nerve. At 48 hours after the injury, motor function was assessed using the SSI and slope-walking ability (with slope angles of 30° and 45°). The tibialis anterior muscle, a main muscle innervated by the common peroneal nerve, was removed. Muscle sections were co-labeled with neuronal class III ß-tubulin polyclonal antibody to identify the presence of axons and Alexa Fluor 488-conjugated α-bungarotoxin to identify the presence of motor endplates. RESULTS: The evaluation of neuromuscular innervation showed a correlation between SSI scores and ratios of residual axons (rs = 0.68, p < 0.01), and there was a statistically significant difference between slope-walking ability and ratios of residual axons (p < 0.01). Moreover, the ratios of residual axons in the nerve-crushed rats with normal motor function (SSI above -20) ranged from 36.5% to 88.7%, and those ratios in the success group with slope-walking angles of 30° and 45° ranged from 14.7% to 88.7% and from 39.8% to 88.7%, respectively. CONCLUSIONS: In this study of rodents, less than half of the motor axons were sufficient to maintain normal motor function of the lower limb.

Correlation between motor function and axonal morphology in neonatally sciatic nerve-injured rats.

The present study was conducted to investigate the correlation between motor function and axonal morphology in neonatally sciatic nerve-injured rats. The left sciatic nerve of newborn rats was transected or crushed, and functionality of the sciatic nerve was assessed by the static sciatic index after 8 weeks. After functional assessment, the common peroneal nerves in the control, nerve-transected, and nerve-crushed rats were removed and prepared for morphometric examinations. The cross-sectional area of the nerve, total number of myelinated axons, and size of each myelinated axon were analyzed for each group. The control rats showed normal motor function, whereas the nerve-transected rats showed severe motor dysfunction. The cross-sectional area of the nerve and total number of myelinated axons were reduced after nerve transection. Moreover, the percentage per size class of myelinated axons was almost uniform in the control rats, while the distribution was shifted to the left in the nerve-transected rats. Furthermore, no large myelinated axons were observed in the nerve-transected rats. The nerve-crushed rats showed various gait functions with various distribution patterns of axonal size, and the rats were divided into two groups with and without uninjured residual large axons. The results showed that the importance of regenerated medium-sized axons in cases without large axons and of residual large axons in cases with large axons in motor function. It was revealed that motor function was related closely to axonal size in neonatally nerve-injured rats.

Bony Landmarks of the Anterior Cruciate Ligament Tibial Footprint: A Detailed Analysis Comparing 3-Dimensional Computed Tomography Images to Visual and Histological Evaluations.

BACKGROUND: Although the importance of tibial tunnel position for achieving stability after anterior cruciate ligament (ACL) reconstruction was recently recognized, there are fewer detailed reports of the anatomy of the tibial topographic footprint compared with the femoral side. HYPOTHESIS: The ACL tibial footprint has a relationship to bony prominences and surrounding bony landmarks. STUDY DESIGN: Descriptive laboratory study. METHODS: This study consisted of 2 anatomic procedures for the identification of bony prominences that correspond to the ACL tibial footprint and 3 surrounding landmarks: the anterior ridge, lateral groove, and intertubercular fossa. In the first procedure, after computed tomography (CT) was performed on 12 paired, embalmed cadaveric knees, 12 knees were visually observed, while their contralateral knees were histologically observed. Comparisons were made between macroscopic and microscopic findings and 3-dimensional (3D) CT images of these bony landmarks. In the second procedure, the shape of the bony prominence and incidence of their bony landmarks were evaluated from the preoperative CT data of 60 knee joints. RESULTS: In the first procedure, we were able to confirm a bony prominence and all 3 surrounding landmarks by CT in all cases. Visual evaluation confirmed a small bony eminence at the anterior boundary of the ACL. The lateral groove was not confirmed macroscopically. The ACL was not attached to the lateral intercondylar tubercle, ACL tibial ridge, and intertubercular space at the posterior boundary. Histological evaluation confirmed that the anterior ridge and lateral groove were positioned at the anterior and lateral boundaries, respectively. There was no ligament tissue on the intercondylar space corresponding to the intercondylar fossa. In the second investigation, the bony prominence showed 2 morphological patterns: an oval type (58.3%) and a triangular type (41.6%). The 3 bony landmarks, including the anterior ridge, lateral groove, and intertubercular fossa, existed in 96.6%, 100.0%, and 96.6% of the cases, respectively. CONCLUSION: There is a bony prominence corresponding to the ACL footprint and bony landmarks on the anterior, posterior, and lateral boundaries. CLINICAL RELEVANCE: The study results may help create an accurate and reproducible tunnel, which is essential for successful ACL reconstruction surgery.

Quantitative analysis of survival of hypoglossal neurons in neonatally nerve-injured rats: Correlation with milk intake.

INTRODUCTION: Tongue movement innervated by the hypoglossal (XII) nerve is essential for the survival of neonatal rats. The pups with bilateral XII nerve resection failed to suckle milk and did not survive, and the pups with unilateral XII nerve resection showed disturbed suckling capability and lower survival rates. The present study was performed to investigate the relation between neuronal population and milk intake of developing rats that had received various degrees of crush injuries to the unilateral XII nerve during the neonatal period. METHODS: The right XII nerve of postnatal day 1 (P1) pups was crushed and milk intake was estimated at 3 days and 6 days after the nerve injury. As nerve injury at the neonatal stage results in death of axotomized neurons, varying degrees of crushing was estimated by the number of survived motor neurons. RESULTS: In nerve-crushed rats, the populations of XII motor neurons and amounts of milk intake were reduced in a varied manner. Statistically significant positive correlations were observed between increasing XII neuron survival and increasing milk intake at 3 (r=0.62) and 6 (r=0.71) days after the nerve injury. CONCLUSION: The results indicate that there is a strong relationship between the number of XII motor neurons and the amount of milk intake in neonatally XII nerve-injured rats.

Effects of repeated crush injuries on motor functional recovery of the sciatic nerve.

OBJECTIVES: The present study was conducted to examine whether repeated crush injuries have significant effects on motor functional recovery of peripheral nerves. METHODS: Repeated crush injuries of the sciatic nerve were inflicted on adult rats at 1-week intervals, and functionality of the sciatic nerve was assessed by the static sciatic index each week for 8 weeks after the final injury. To determine the effects of repeated crush injuries on motor functional recovery of the sciatic nerve, tibialis anterior muscle fibers from single and triple crush injuries were examined, and fiber size and fiber reinnervation during the 2- to 4-week period after the final injury were measured. RESULTS: Compared to single crush injuries, which completely recovered by post-injury week 4, double crush injuries resulted in retarded, but complete recovery by post-injury week 6, whereas triple crush injuries resulted in marked retardation in the regenerative process with incomplete recovery during week 8 of the experimental period. Muscle fiber size for rats with triple crush did not recover to normal range at post-injury week 4, despite its normal size for rats with single crush. The rate of reinnervation increased prominently between post-injury weeks 2 and 3 in both injuries, but the rate with triple crush was lower than that with single crush at post-injury week 3. DISCUSSION: These results, which contradict those of a previous study that reported early functional recovery, indicate that repeated crush injuries inhibit motor functional recovery of the damaged sciatic nerve, as evidenced by delayed and incomplete regeneration, atrophied muscle fibers, and delayed reinnervation.

Re-evaluation of spontaneous regeneration of the lateral olfactory tract.

Spontaneous regeneration of the lateral olfactory tract (LOT) was re-evaluated in newborn rats using a fluorescent retrograde neuronal tracer as objective indicators of complete LOT transection. Complete LOT transection was evidenced by the loss of the white myelinated band characteristic for adult LOT and the total lack of retrograde neuronal labeling of mitral cells by Fast Blue that was injected during LOT transection. In completely LOT-transected young adult rats, mitral cells were retrogradely labeled consistently only by Fluoro-Gold that was injected into the olfactory cortex at the adult stage. Moreover, an anterograde neuronal tracer, biotinylated dextran amine (BDA), was demonstrated to pass from the neonatally LOT-transected bulb, through the transected retrobulbar site, towards the olfactory cortex, far caudally at a level near the optic chiasm. The regenerated structures lacked immunoreactivity for myelin basic protein and electron-dense myelinated axon bundles, and were also characterized by the thinness of the BDA+ terminal zone within the olfactory cortex and the lack of its caudal extension. Young adult rats subjected to unilateral bulbectomy contralateral to the neonatally LOT-transected side showed perfect ability to discriminate cycloheximide solution by olfaction. From these findings, we conclude that the spontaneously regenerated olfactory system is functional despite structural incompleteness.

Tyrosine hydroxylase-immunoreactive fibers in the human vagus nerve.

Sympathetic catecholaminergic fibers in the vagus nerve were immunohistochemically examined in formalin-fixed human cadavers using an antibody against the noradrenalin-synthetic enzyme tyrosine hydroxylase (TH). TH-positive fibers were extensively distributed in the vagal nerve components, including the superior and inferior ganglia, the main trunk and the branches (superior and recurrent laryngeal, superior and inferior cardiac, and pulmonary branches). The inferior ganglion and its continuous cervical main trunk contained numerous TH-positive fibers with focal or diffuse distribution patterns in each nerve bundle. From these findings, we conclude that sympathetic fibers are consistently included in the human vagus nerve, a main source of parasympathetic preganglionic fibers to the cervical, thoracic and abdominal visceral organs.

Neural plasticity of neonatal hypoglossal nerve for effective suckling.

The adaptive movement of the tongue after unilateral lesion of the hypoglossal (XII) nerve during the early postnatal days is essential for recovery of milk intake. The present study investigated the basic mechanisms underlying such adaptation, focusing on the neural plasticity that allows effective suckling. After resection of the ipsilateral XII nerve on P1, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlolate (DiI), a postmortem neuronal tracer, was applied to the contralateral uninjured XII nerve on P4 and P7. DiI-labeled fibers were traced successfully within the tongue and showed gradually increased extension over the XII nerve-injured side in the central core portion of the denervated tongue between P4 and P7. Systematic neuroanatomic experiments showed that contralateral axonal sprouting occurred as early as 1 day after nerve injury (P2), and that such axonal sprouting occurred exclusively from the medial branch of the XII nerve responsible for tongue protrusion, an essential movement for suckling. These findings provide direct evidence of functional neural plasticity that allows effective suckling in XII nerve-injured newborns with suckling disturbance.

Functional role of lingual nerve in breastfeeding.

Functional role of lingual nerve in breastfeeding was investigated in rat pups during the suckling period. DiI, a postmortem neuronal tracer, was used to confirm the immature lingual nerve (LN) responsible for tongue sensation and resulted in successful fiber labeling anterogradely to the tongue, which showed different distribution patterns from fiber labeling derived from the hypoglossal nerve. Unilaterally LN-injured pups did not show suckling disturbance with absence of any shortening (P11 pups: 559+/-16s; 105% of the control value) in nipple attachment time and the survival rate remained high (P11: 100%). Bilaterally LN-injured pups showed suckling disturbance with marked shortening (P11 pups: 220+/-54 s; 42% of the control value) in nipple attachment time and a low survival rate (P1: 33%; P11: 41%). Bilaterally infraorbital nerve-injured or bilaterally bulbectomized pups did not show any nipple attachment at all and there were no survivors, confirming the crucial roles of upper lip sensation and olfaction in suckling. Based on these findings, we conclude that tongue sensation is very important, but not essential for suckling.

Motor neurons essential for normal sciatic function in neonatally nerve-injured rats.

The present study was aimed to determine neuronal population essential for normal motor function in young adult rats receiving various degrees of crushing to the sciatic nerve at the neonatal stage. Motor function was estimated by the static sciatic index, and a neuronal tracer was applied to the common peroneal nerve. The total numbers of the tracer-labeled neurons of the nerve-crushed rats were 74-383 in the normal function group, 14-61 in the disordered function group, and 0-32 in the severely disordered function group. We conclude that normal motor function can be well preserved by a very small population of motor neurons (approximately 15% of the control value) in the neonatally sciatic nerve-injured rats.