Intravenous mesenchymal stem cell therapy after recurrent laryngeal nerve injury: a preliminary study.

OBJECTIVES/HYPOTHESIS: Intravenous administration of mesenchymal stem cells (MSCs) has been recently shown to enhance functional recovery after stroke and spinal cord injury. The therapeutic properties of MSCs are attributed to their secretion of a variety of potent antiinflammatory and neurotrophic factors. We hypothesize that intravenous administration of MSCs after recurrent laryngeal nerve (RLN) injury in the rat may enhance functional recovery. STUDY DESIGN: Animal Research. METHODS: Twelve 250-gram Sprague-Dawley rats underwent a controlled crush injury to the left RLN. After confirming postoperative vocal fold immobility, each rat was intravenously infused with either green fluorescent protein-expressing MSCs or control media in a randomized and blinded fashion. Videolaryngoscopy was performed weekly. The laryngoscopy video recordings were reviewed and rated by a fellowship-trained laryngologist who remained blinded to the intervention using a 0 to 3 scale. RESULTS: At 1 week postinjury, the MSC-infused group showed a trend for higher average functional recovery scores compared to the control group (2.2 vs 1.3), but it did not reach statistical significance (P value of 0.06). By 2 weeks, however, both groups exhibited complete return of function. CONCLUSIONS: These pilot data indicate that with complete nerve transection by crush injury of the RLN in rat, there is complete recovery of vocal fold mobility at 2 weeks. At 1 week postinjury, animals receiving intravenous infusion of MSCs showed a trend for greater functional recovery, suggesting a potential beneficial effect of MSCs; however, this did not reach statistical significance. Therefore, no definite conclusions can be drawn from these data and further study is required. LEVEL OF EVIDENCE: N/A.

Functional regeneration of recurrent laryngeal nerve injury during thyroid surgery using an asymmetrically porous nerve guide conduit in an animal model.

BACKGROUND: Vocal cord paralysis (VCP) caused by recurrent laryngeal nerve (RLN) damage during thyroidectomy commonly results in serious medico-legal problems. The purpose of this study was to evaluate the usefulness of an asymmetrically porous polycaprolactone (PCL)/Pluronic F127 nerve guide conduit (NGC) for functional regeneration in a RLN injury animal model. METHODS: A biodegradable, asymmetrically porous PCL/F127 NGC with selective permeability was fabricated for use in this study. A 10-mm segment of left RLN was resected in 28 New Zealand white rabbits, and then an asymmetrically porous NGC or a nonporous silicone tube was interposed between both stumps and securely fixed. Vocal cord mobility was endoscopically evaluated at one, four, and eight weeks postoperatively. Nerve growth through NGCs was assessed by toluidine blue staining, and thyroarytenoid (TA) muscle atrophy was evaluated by hematoxylin and eosin staining. Immunohistochemical stainings for acetylcholinesterase (AchE), anti-neurofilament (NF), and anti-S100 protein were also conducted, and transmission electron microscopy (TEM) was used to evaluate functional nerve regeneration. RESULTS: At eight weeks postoperatively, endoscopic evaluations showed significantly better recovery from VCP in the asymmetrically porous PCL/F127 NGC group (6 of 10 rabbits) than in the silicone tube group (1 of 10 rabbits). Continued nerve growth on the damaged nerve endings was observed with time in the asymmetrically porous PCL/F127 NGC-interposed RLNs. TA muscle dimensions and AchE expressions in TA muscle were significantly greater in the asymmetrically porous PCL/F127 NGC group than in the silicone tube group. Furthermore, immunohistochemical staining revealed the expression of NF and S100 protein in the regenerated nerves in the asymmetrically porous PCL/F127 NGC group at eight weeks postoperatively, and at this time, TEM imaging showed myelinated axons in the regenerated RLNs. CONCLUSION: The study shows that asymmetrically porous PCL/F127 NGC provides a favorable environment for RLN regeneration and that it has therapeutic potential for the regeneration of RLN damage.

Histological and ultrastructural evidence that recurrent laryngeal neuropathy is a bilateral mononeuropathy limited to recurrent laryngeal nerves.

REASONS FOR PERFORMING STUDY: Recurrent laryngeal neuropathy (RLN) is a common and debilitating peripheral nerve disease of horses, but it remains unclear if this disease is a mono- or polyneuropathy. An understanding of the distribution of the neuropathological lesions in RLN affected horses is fundamental to studying the aetiology of this very significant disease of tall horses. OBJECTIVE: To determine whether RLN should be classified as a mono- or polyneuropathy. METHODS: Multiple long peripheral nerves and their innervated muscles were examined systematically in 3 clinically affected RLN horses RESULTS: Severe lesions were evident in the left as well as right recurrent laryngeal nerves in all horses, both distally and, in one case, also proximally. No primary axonal lesions were evident in other nerves nor were changes found in their innervated muscles. CONCLUSIONS: RLN is not a polyneuropathy but should be classified as a bilateral mononeuropathy. POTENTIAL RELEVANCE: Genetic and local factors specifically affecting the recurrent laryngeal nerves in RLN-affected horses should now be investigated further.

Axons enter the human posterior cricoarytenoid muscle from the superior direction.

OBJECTIVE: To examine the possibility that axons from the superior laryngeal nerve travel inferiorly through connections between the recurrent and the superior laryngeal nerves within the interarytenoid muscle to innervate intrinsic laryngeal muscles. MATERIALS: The branch point from the recurrent laryngeal nerve to the posterior cricoarytenoid muscle was excised from five human larynges. METHODS: The section of nerve was first microdissected to remove the epineurium. Then, the specimen was incubated at 37 degrees C in a 1% collagenase solution, which was alternated with 0.4% chymopapain or 0.25% trypsin. Finally, the specimen was stained with 1% toluidine blue. RESULTS: In all five specimens, axons could be seen entering the nerve branch to the posterior cricoarytenoid muscle from the superior direction. In one specimen, about one third of the axons entering the muscle could be identified as arising from the superior direction. In two cases, the superior axons could be traced back to the recurrent laryngeal nerve branch to the interarytenoid muscle. CONCLUSIONS: These results suggest that axons from the superior laryngeal nerve connect with the recurrent laryngeal nerve in the area of the interarytenoid muscle. Furthermore, these axons travel inferiorly to innervate the posterior cricoarytenoid muscle.

Misdirected reinnervation in the feline intrinsic laryngeal muscles after long-term denervation.

Nerve terminals which resembled the autonomic nerve varicosities were found at all neuromuscular junctions of the feline intrinsic laryngeal muscles after 21 months of unilateral laryngeal paralysis. The structure of muscle fibers was preserved throughout this period, although there was no evidence of regeneration of transected recurrent laryngeal nerve. In the cricothyroid muscle, misdirected reinnervation was recognized in the different pathways from the other intrinsic laryngeal muscles.

Effects of 5-hydroxydopamine and 6-hydroxydopamine on the ultrastructure of type I cells in paraganglia of the rat recurrent laryngeal nerve.

The ultrastructure of the Type I cells in paraganglia of rat recurrent laryngeal nerve (RLN) was studied after the administration of 5-hydroxydopamine (5-OHDA) and 6-hydroxydopamine (6-OHDA). Normal Type I cells of RLN-paraganglia contained abundant organelles and their cytoplasm was characterized by the presence of numerous membrane-bounded dense-cored vesicles (DCVs). The DCVs were round in profile (diameter 107.67 +/- 0.06 nm, all values expressed as mean +/- s.e.m. in the present study) and possessed dense cores of moderate to low electron density. After 5-OHDA treatment (single injection, 100 mg/kg b.w., i.v.), the majority of DCVs were filled with a material of high electron density. No significant difference was observed between the profile diameter of the DCVs in 5-OHDA-treated rats (104.96 +/- 0.06 nm) and that in normal rats. After 6-OHDA treatment (three injections, 100 mg/kg b.w. each at 12 h intervals i.p.), no significant alteration in the electron density of the core was noted. However, most of the DCVs were enlarged and round, elliptical or irregular in profile (190.57 +/- 2.77 nm x 130.34 +/- 2.09 nm). The dense core of DCVs was centrally or eccentrically located in DCVs. The results of the present study indicate that: 1) there is only one type of granulated glomus cell (i.e., Type I cells) in the rat RLN-paraganglia under normal physiological condition; and 2) since the ultrastructural morphology of DCVs in Type I cells of rat RLN-paraganglia is altered after 5-OHDA or 6-OHDA treatment, these cells may possess mechanisms for the uptake of false adrenergic neurotransmitter and/or neurotoxin.

Central projection of the sensory fibres of the recurrent laryngeal nerve of the cat.

There have been few studies of the central projection of the sensory fibres of the recurrent laryngeal nerve into the medulla oblongata. In the present study terminal degeneration was observed in both the nucleus of the tractus solitarius and the nucleus of the spinal tract of the trigeminal nerve after transection of the nerve and after injection of a lectin, Ricinus communis agglutinin (RCA). Degeneration was observed to be more extensive after RCA injection than after nerve transection. In both instances, however, degeneration was observed bilaterally.

Neurite regeneration in the cat recurrent laryngeal nerve: an immunohistochemical study.

The recurrent laryngeal nerve (RLN) consists of various motor, sensory and autonomic nerve fibers, although it has not been established whether different neuronal types exhibit a similar ability to regenerate. To address this question, freezing was used to injure the cat RLN fibers and the presence or absence of immunoreactivity for neuropeptides or transmitter-synthesizing enzymes was then examined as a marker to classify the fibers. In the control RLN, calcitonin gene-related peptide-immunoreactive (CGRP-IR) fibers were the highest in number and were distributed throughout the nerve fascicles. The number of substance P-immunoreactive (SP-IR) fibers was about 40% that of CGRP-IR fibers, while a portion of CGRP-IR fibers was found to contain SP immunoreactivity. Relatively low numbers of tyrosine hydroxylase-immunoreactive (TH-IR) and neuropeptide Y (NPY-IR) nerve fibers were seen which tended to form clusters. The distribution pattern of NPY-IR fibers was very similar to that of TH-IR fibers. In the regenerating RLN 1 week after the freezing injury, the fastest growing axons were CGRP-IR, while the regenerating rates of SP-IR, TH-IR and NPY-IR fibers were slower than that of CGRP-IR fibers. These results suggest that the ability for neurite regeneration varies among neuron types and that CGRP-IR fibers possess the most rapid ability to regenerate.

Connective tissues in the optic nerve and in peripheral nerves: ultrastructural observations.

The connective tissues of the bovine optic nerve and left recurrent nerve have been investigated at the ultrastructure level by TEM and freeze-fracture. The outermost of three concentric sheaths enveloping the nerve appear very similar; the intermediate sheaths appear different enough to suggest different functionalities, while the innermost sheaths are completely dissimilar. The ultrastructural aspects of the extracellular matrix suggest that all the connective tissues may mainly play a passive role as a packing material for the nerve fibers, while none of the sheaths seems apt to withstand mechanical stresses.

Regeneration of the recurrent laryngeal nerve in the guinea pig: reorganization of motoneurons after freezing injury.

The purpose of this study was to clarify the morphologic changes resulting from reinnervation after a freezing injury. We chose the freezing injury as the most promising nerve regeneration model in order to examine the mechanism behind the production of misdirected reinnervation. The left recurrent laryngeal nerve of the adult guinea pig was injured by freezing (-80 degrees C) at the level of the 10th tracheal ring. At intervals ranging from 2 weeks to 6 months after the injury, horseradish peroxidase was injected into the left posterior cricoarytenoid muscle to ascertain the presence of retrograde-labeled perikarya in the medulla oblongata. Projections to the individual laryngeal muscles and to the entire recurrent laryngeal nerve served as normal controls. In addition, we observed by electron microscopy the degeneration and regeneration processes of the recurrent laryngeal nerve following injury. From 2 to 6 months after the freezing injury, the number of labeled neurons in the nucleus ambiguus increased gradually from 20 to 90. In addition, the area occupied by neurons which project to the posterior cricoarytenoid muscle was expanded, but was confined within the region of perikarya projecting to the normal recurrent laryngeal nerve. Most axons degenerated within 3 days and showed regenerative sprouting with growth cones by 7 days postinjury. Despite the fact that freezing injury preserved the basal lamina tunnel with minimal disturbance of the recurrent laryngeal nerve fiber structure, target-specific reinnervation was incomplete.

[Auxiliary motor innervation of the laryngeal muscles via the internal branch of the superior laryngeal nerve]. / Untersuchungen zur zusätzlichen motorischen Innervation der Kehlkopfmuskulatur über den Ramus internus des Nervus laryngeus superior.

According to our present knowledge of the neuromuscular innervation of the intrinsic laryngeal muscles, the cricothyroid muscle is innervated by the external branch of the superior laryngeal nerve (NLS), whereas all other remaining muscles get their supply from the inferior laryngeal (recurrent) nerve. Mainly in the phoniatric literature, however, opinions differ concerning an additional motoric laryngeal innervation. In human larynges, excised for large unilateral carcinoma, horseradish peroxidase (HRP) was injected into the internal branch of the NLS. Anterograde labelling of axons was demonstrated histochemically. In adjacent sections of the different muscles, end plates and axons were stained histochemically with silver impregnation and acetylcholinesterase. Evidence is presented of motor innervation of the internal branch of the NLS in some laryngeal muscles. With retrograde HRP-tracing in sheep, motoneurons were detected in the nucleus ambiguus, although the recurrent nerve and the external branch had been divided and excised. Thus, histologically an additional neuromuscular supply via the internal branch of the NLS is demonstrated.

Ultrastructural observations of organelle accumulation in the equine recurrent laryngeal nerve.

The left recurrent laryngeal nerves from five horses with sub-clinical neuropathy were examined by light and electron microscopy in a study designed to examine accumulation of axonal organelles at paranodal and internodal locations. Transverse sections of the nerve showed scattered fibres with split myelin sheaths and axonal accumulation of organelles. On longitudinal sections these collections were seen to result from an axonal outpouching in which dense lamellar bodies and mitochondria had accumulated. These paranodal collections, which could be found on both sides of the node, were often associated with infoldings of the terminal loops of myelin and with occasional paranodal demyelination. The fact that many of the organelles in the outpouches were lysosomal in nature was confirmed by their positive staining for cathepsin D activity. Longitudinal sections demonstrated a number of axons which were swollen over a long distance and which contained focal accumulations of similar organelles. In places, however, there was a clear separation between these organelles and the cytoskeletal proteins. In each case these swollen axons were surrounded by Schwann cell nuclei and their processes, forming well-ordered onion bulbs. The possibility that these two types of changes, i.e. the paranodal accumulations and the axonal swellings could result from a disturbance in axonal transport in this distal axonopathy is discussed.

The recurrent laryngeal nerve in spastic dysphonia. A light and electron microscopic study.

Sections of right or left recurrent laryngeal nerve (RLN), removed from patients with spastic dysphonia (SD) at the time of surgery, were studied using light and electron microscopy and were compared with control RLNs. In both groups, small, medium and large-sized myelinated nerve fibres were found in the RLN. Furthermore, numerous unmyelinated axons intermingled with the myelinated fibres were detected in the control group as well as in SD specimens. Slight morphometric differences were also found between the two groups, but these cannot explain the causation of spastic dysphonia.

Morphometric analysis of glomus cells within the recurrent laryngeal nerve of the rat.

An endoneurial paraganglion located in the recurrent laryngeal nerve of the rat was found in 17 of 20 nerves studied. The median size of the paraganglia was estimated to be 0.8 X 10(6) micron 3. The number of type I like cells within a paraganglion ranged between 8 and 24. Altogether, 120 endoneurial dense-cored vesicle cells from 8 paraganglia were subjected to a morphometric analysis at the ultrastructure level. The mean vesicle profile diameter was estimated to be 101.3 nm and only one type of granulated cell could be distinguished. The volume density of the dense-cored vesicles was estimated to be about 6%, a value amounting to two to three times that in the carotid glomus. Moreover, the mean cell profile area of vesiculated cells of recurrent laryngeal nerve exceeds that of the cells of the carotid glomus.

Carotid-body-like tissue within the recurrent laryngeal nerve: an endoneural chemosensitive micro-organ?

Paraganglia associated with the ninth and tenth cranial nerves have been observed in several locations in the head and neck region. The most studied of these organs, both morphologically and functionally, is the carotid body, a chemoreceptor organ located in the carotid bifurcation. In the present study a microglomus (microparaganglion) composed of islands of carotid-body-like cells was found in the endoneural space of the recurrent laryngeal nerve of rats, 5 to 10 mm from the nerve entrance into the larynx. This microglomus was surrounded by bundles of myelinated and unmyelinated nerve fibers. Fenestrated blood vessels were observed between cells. The cells were characterized by their cytoplasmic membrane-bound granules and dense-core vesicles. Ultrastructurally, these cells seemed to be identical to carotid body chemoreceptor cells and thus to indicate that the cells of the microglomus store catecholamines. Nerve terminals impinging on the cells, classified as both afferent and efferent, were also identified. The recurrent laryngeal endoneural microglomus may have a chemosensitive function influencing the laryngeal physiologic reflex mechanisms.

Recurrent laryngeal nerve pathology in spasmodic dysphonia.

Since it was first described in 1871, spasmodic (spastic) dysphonia has been considered a disease of psychogenic origin. Unsupported theories of possible organic etiology have appeared sporadically in the literature. In 1976 sectioning of the recurrent laryngeal nerve for patients with this disease was reported with resultant improvement in voice production. This was attempted because the spasmodic dysphonic has, in effect, already compensated vocal cords bilaterally. It was reasoned, therefore, that if one of these was paralyzed the patient would immediately be converted to a state approximating that of well-compensated unilateral vocal cord paralysis which situation, as is well known, usually carries with it a fairly good voice. A controlled study to evaluate the efficacy of this surgical approach has been undertaken at the Cleveland Clinic during the past year. In an attempt to elucidate the possible organic etiology of spasmodic dysphonia, a section of nerve was removed in every case and examined by both light and electron microscopy. Special stains for myelin were also used on the light microscopy specimens. Demyelinization has been found in most of the cases examined by electron microscopy. Possible correlation between this disease entity and other cranial nerve syndromes of unknown etiology is noted. Such conditions as trigeminal neuralgia, glossopharyngeal neuralgia, belpharospasm, hemifacial spasm, and even possibly Bell's palsy may exhibit a similar etiology.

Fiber components of the recurrent laryngeal nerve in the cat.

Experimental neuroanatomical methods were employed in 21 adult cats to determine 1) the number and size of myelinated motor and sensory fibers in the recurrent laryngeal nerve (RLN), and 2) the fiber components originating in the nucleus ambiguus (NA) and retrofacial nucleus (RFN) of the brain stem. Intracranial transection of the X and XI cranial nerves and selective destruction of the NA or RFN were the experimental lesions inflicted in order to obtain the following results. About 55% (312) of the right RLN (565 fibers) is composed of myelinated motor nerve fibers which measure 4 mu - 9 mu in diameter. Nine percent come from the RFN and are smaller (4-6 mu) than the 46% which emanate from the NA and measure 6-9 mu in diameter. The remaining 45% of the RLN is made up of sensory neurons which can be divided into three groups. 1) The largest numerical group (32%) is very small in caliber (1-3 mu) and supplies extralaryngeal regions (trachea, esophagus). 2) The intermediate size fiber group (4-9 mu) comprises 11% of the RLN and probably supplies the subglottic mucosa. 3) The smallest group (2%) of sensory fibers is the largest in diameter (10-15 mu) and may represent either the innervation of muscle spindles or afferents from the superior laryngeal nerve coursing down into the chest.