Equine laryngeal hemiplegia. Part II. An electron microscopic study of peripheral nerves

The recurrent laryngeal nerves were examined by electron microscopy in five control, four subclinical and four clinical laryngeal hemiplegic horses. In addition, the peroneal nerve was examined in two horses in the latter group. The distally distributed loss of large myelinated fibres in the left recurrent laryngeal nerve seen by light microscopy was confirmed. In addition, active axonal pathology was found to be more evident than indicated by light microscopic investigations. The onion bulb formations observed indicated the repetitive nature of the damaging influence to nerve fibres. Although the pathological changes were most obvious in the distal left recurrent laryngeal nerve, alterations similar in type and distribution were present in other areas of the left and right nerves, and in the distal hindlimb nerves. The observation of fibres with inappropriately thick myelin sheaths relative to their axonal calibre, was confirmed statistically by determining the regressions of axis cylinder perimeter against the number of myelin lamellae. In conclusion, the peripheral nerve pathology of equine laryngeal hemiplegia was demonstrated to be a distally distributed loss of myelinated fibres, with considerable active axonal damage, in conjunction with axonal atrophy. These features suggest that this disease may be classified as a distal axonopathy.     

Equine laryngeal hemiplegia. Part I. A light microscopic study of peripheral nerves

This light microscopic investigation of 15 Thoroughbred horses provided substantial evidence for the classification of equine laryngeal hemiplegia as a distal axonopathy. Morphologic and morphometric examinations were performed on resin embedded recurrent laryngeal nerves from control, subclinical and clinical laryngeal hemiplegic animals. In the latter group of animals some distal hindlimb nerves were also examined. A distally graded loss of myelinated fibres selectively affecting those of large diameter was demonstrated in both left and right recurrent laryngeal nerves. Morphologic evidence of similar pathological changes in long hindlimb nerves was also present. An explanation for the early involvement of the cricoarytenoideus lateralis muscle in the course of laryngeal hemiplegia, was offered by the demonstration of more large diameter fibres in the branch of the recurrent laryngeal nerve innervating it, thus making it more susceptible to the disease process.     

Equine laryngeal hemiplegia. Part IV. Muscle pathology

This study confirmed that neurogenic muscle pathology exists in intrinsic laryngeal muscles supplied by the recurrent laryngeal nerves in horses subclinically and clinically affected with laryngeal hemiplegia. An important additional observation was the occurrence in three out of four laryngeal hemiplegic horses of neurogenic muscle changes in a hindlimb muscle, the extensor digitorum longus, a muscle supplied by another long peripheral nerve. This finding suggests that a polynenropathy exists in laryngeal hemiplegic horses, and supports the classification of this disease as a distal axonopathy. Comparison of the degree of pathology in the intrinsic laryngeal muscles and that of the recurrent laryngeal nerves innervating them, demonstrated a strong correlation between the extent of damage in the distal left recurrent laryngeal nerve and the overall degree of muscle pathology. The muscle damage in clinically affected horses is a reflection of the nerve damage present in the most distal portion of the recurrent laryngeal nerve. The more variable pathological changes found in proximal levels of the left and right recurrent laryngeal nerves probably reflects the ongoing nature of the pathological process affecting nerve fibres. The existence of a subclinically affected group of horses, the earliest involvement of an adductor, the left cricoarytenoideus lateralis muscle, and the presence of changes in the right intrinsic laryngeal muscles all confirmed the findings of previous workers.     

Preferential denervation of the adductor muscles of the equine larynx. II: Nerve pathology

The terminal branches of the recurrent laryngeal nerve (RLN) of three normal ponies and six horses with sub-clinical laryngeal disease were examined qualitatively and quantitatively in an attempt to explain the preferential denervation of the laryngeal adductor muscles in the neuropathy of idiopathic laryngeal hemiplegia (ILH). The myelinated fibre spectra of all the motor nerve fibres in the left and right abductor and adductor branches of the RLN in three normal ponies were measured. The density of myelinated fibres was also calculated. There was no significant difference between the larger group of myelinated fibres in the adductor or abductor branches. In the six horses with laryngeal hemiparesis, however, there was a marked preferential loss of the medium/large size myelinated fibres in the left adductor branch, although nerve fibre densities were not significantly different. While no simple morphometrical feature was found to explain the selective muscle denervation, the greater loss of large diameter myelinated fibres in the adductor branches confirms the earlier observation of adductor muscle susceptibility in the neuropathy of ILH.     

Bilateral laryngeal paralysis in the horse

Four two-year-old Thoroughbreds suffered an acute gastrointestinal illness shortly after dosing with mineral oil which was thought to have been contaminated with an organophosphate compound. Three weeks later all four were noted to be dyspnoeic and endoscopic examination showed that they had developed bilateral laryngeal paralysis. Two of the horses died during severe bouts of dyspnoea six and eight months later and the third was killed shortly thereafter. Examination of the left and right recurrent laryngeal nerves from these horses showed a severe loss of myelinated fibres distally, especially in the left nerve. A similar but less severe lesion was seen in other long peripheral nerves, including the phrenic and digital nerves of the third horse. The spinal cord in two horses showed evidence of mild axonal degeneration which was not related to a particular tract or location. The fourth horse had bilateral laryngeal paralysis two years later. The acute clinical signs and delayed neurological sequelae seen in these horses were strongly suggestive of accidental organophosphate toxicity.     

Equine laryngeal hemiplegia. Part V. Central nervous system pathology

Evidence of long central nerve fibre degeneration (axonal spheroids) in the lateral cuneate nuclei was found in all eight Thoroughbreds affected clinically and subclinically with equine laryngeal hemiplegia, but in only one of six control animals. It was considered that these spheroids may signify a central nervous component of the disease process of laryngeal hemiplegia although until further investigations are performed no firm conclusions regarding the relationship of these findings with laryngeal hemiplegia could be made. Examination of the left and right nucleus ambiguus of clinical and subclinical laryngeal hemiplegic horses revealed no pathological alterations.     

Pathological aspects of Australian Stringhalt.

Nine horses with clinical signs of Australian Stringhalt were killed and tissues collected for a detailed pathological study. Lesions were limited to peripheral nerves and muscles. The most severely affected nerves were the superficial and deep peroneal, distal tibial, plantar digital, volar and recurrent laryngeal nerve with changes characterised by a selective loss of large diameter myelinated fibres with various degrees of demyelination, fibrosis, Schwann cell proliferation and onion-bulb formation. A routine evaluation of the brain and spinal cord by light microscopy failed to reveal any consistent abnormalities. Morphometric analysis of deep peroneal and recurrent laryngeal nerves confirmed the reduced number of large diameter myelinated axons. Teased fibre preparations of these nerves did not show any abnormalities in internodal distance. The most severe muscle lesions were in the long and lateral digital extensors, cranial tibial, dorsal cricoarytenoid, gracilis and lateral deep digital flexor with extensive atrophy of fibres and diffuse fibrosis. Histochemical evaluation of the long digital extensor from 3 affected horses showed an abnormally wide distribution in fibre size and a reduction in type II fibres compared with controls. These lesions are consistent with a distal axonopathy leading to neurogenic muscle atrophy. The distribution of neuromuscular lesions in Australian Stringhalt may be explained by the susceptibility of longer, larger myelinated nerve fibres to injury, but the cause for this distal axonopathy remains unknown.     

Morphometric study of the recurrent laryngeal nerve in young ‘normal’ horses

Quantitative measurements were made on cross-sectional preparations of the distal part of the recurrent laryngeal nerve (RLN) from nine young mixed-breed horses to establish reference values for the total number of myelinated fibres, mean fibre diameter and percentage of thickest fibres (over 9·5 gm) and to delineate diameter distribution curves. The total number of myelinated fibres, mean fibre diameter and percentage of thickest fibres for the left RLN were significantly lower than those of the right RLN (P<0–005). The distribution of fibres was unimodal. The fibre diameter ranged from 1 μm to 17 μm. Approximately, 95 per cent of fibres had a diameter larger than 5 μm.     

Peripheral nerve pathology in two rottweilers with neuronal vacuolation and spinocerebellar degeneration

Neuronal vacuolation and spinocerebellar degeneration in young Rottweiler dogs is a neurodegenerative condition characterized by neuronal vacuolation of several nuclei in the central nervous system and degeneration of the spinal cord white matter. Here, we describe the morphologic and ultrastructural findings in laryngeal muscles and peripheral nerves of a 16-week-old female and a 32-week-old female Rottweiler dog affected by progressive ataxia and tetraparesis associated with laryngeal paralysis. Lesions were characterized by neurogenic muscle atrophy of the intrinsic laryngeal muscles, and a loss of large myelinated fibers in the recurrent laryngeal nerve, accompanied by demyelinating/remyelinating features affecting the small myelinated fibers. No significant changes were detected in the cranial laryngeal, vagus, phrenic, ulnar, or peroneal nerves. These findings were indicative of a selective distal neuropathy of the recurrent laryngeal nerve with early severe axonal degeneration, mainly of the large myelinated fibers.     

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.     

Histologic evaluation of nerve muscle pedicle graft used as a treatment for left laryngeal hemiplegia in standardbreds.

A nerve muscle pedicle (NMP) graft was placed in the cricoarytenoideus dorsalis (CAD) muscle of 6 horses with induced left laryngeal hemiplegia. The NMP graft was created by use of the first cervical nerve and omohyoideus muscle. In 1 horse (control), the first cervical nerve was transected after placement of the NMP graft. One year after the surgical procedure, horses were examined endoscopically and then anesthetized. While the larynx was observed endoscopically, the first cervical nerve was stimulated. Horses were subsequently euthanatized, and the larynx was harvested. Prior to anesthesia, the endoscopic appearance of the larynx of all horses was typical of laryngeal hemiplegia. During anesthesia, stimulation of the first cervical nerve produced vigorous abduction of the left arytenoid in principal horses but not in the control horse. The right cricoarytenoideus lateralis and CAD muscles were grossly and histologically normal. Also, the left cricoarytenoideus lateralis was atrophic in all horses as was the left CAD muscle of the control horse. In contrast, the left CAD muscle harvested from principal horses had evidence of reinnervation with type 1 or type 2 fiber grouping. One year after the NMP graft procedure, horses with left laryngeal hemiplegia had reinnervation of the left CAD muscle. In another study, reinnervation was sufficient to allow normal laryngeal function during exercise. Combined, these data suggest that the NMP graft procedure is a viable technique for the treatment of left laryngeal hemiplegia in horses.     

Stereological characteristics of the equine accessory nerve

Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.     

Attempts to restore abduction of the paralyzed equine arytenoid cartilage. III. Nerve anastomosis.

The purpose of this project was to attempt restoration of abduction of a recently denervated left dorsal cricoarytenoid muscle in the horse by anastomosing the first cervical nerve to the abductor branch of the left recurrent laryngeal nerve. Ten horses were used in the study. In six horses the left recurrent laryngeal nerve was transected and ligated while the ventral branch of the left first cervical nerve was anastomosed to the abductor branch of the left recurrent laryngeal nerve. The remaining four horses also had the left recurrent laryngeal nerve transected and ligated but had no nerve anastomosis performed. Each horse was evaluated preoperatively, and at one week, three and six months after surgery, by endoscopy and determination of upper airway resistance. The endoscopy was performed with the horses breathing room air and while breathing 10% carbon dioxide. All ten horses showed endoscopic signs of complete laryngeal hemiplegia immediately postoperatively. Starting at three months postoperatively clonic movements of the left arytenoid cartilage were observed in four of the six reinnervated horses but not in the sham operated horses. At the sixth postoperative month five reinnervated horses had clonic movements of the left arytenoid cartilage. The comparison of upper airway resistance measurements before surgery and at one week, three and six months after surgery showed no significant differences in either control or experimental horses. Following euthanasia at six months postoperatively, the left and right dorsal crioarytenoid muscles were compared for evidence of reinnervation. No significant difference in weight was noted in the reinnervated horses but the left dorsal cricoarytenoid muscle weighed less than the control horses.     

Further evidence for a central nervous system component in equine laryngeal hemiplegia

Evidence for a central nervous system component of the distal axonopathy associated with equine laryngeal hemiplegia was found in this survey of 33 horses (control, 15; subclinical, 8; clinical, 10). Significantly higher numbers of axonal spheroids were present in sections of the lateral cuneate nuclei in layngeal hemiplegic horses than in those unaffected by the disease.     

Further evidence for a central nervous system component in equine laryngeal hemiplegia

Evidence for a central nervous system component of the distal axonopathy associated with equine laryngeal hemiplegia was found in this survey of 33 horses (control, 15; subclinical, 8; clinical, 10). Significantly higher numbers of axonal spheroids were present in sections of the lateral cuneate nuclei in layngeal hemiplegic horses than in those unaffected by the disease.     

Attempts to restore abduction of the paralyzed equine arytenoid cartilage. I. Nerve-muscle pedicle transplants.

The purpose of this project was to adapt a surgical technique from humans and dogs to horses in which a portion of an accessory muscle of respiration and its nerve supply is transplanted to a denervated dorsal cricoarytenoid muscle. Anatomical dissections in seven horses revealed two possible donor nerve-pedicle grafts: the omohyoid and the sternothyrohyoid, both innervated by a branch of the first and second cervical nerves. Histochemical evaluations in two ponies of the dorsal cricoarytenoid, omohyoid and sternothyrohyoid muscles revealed similar proportions of fiber types 1 and 2 in all three muscles. Electromyographic studies in these two ponies revealed that the omohyoid and sternothyrohyoid muscles contract synchronously with respirations during forced inspiration under general anesthesia. Based on surgical ease of access, a 1 cm2 portion of the omohyoid muscle at the point of penetration of the second cervical nerve was used as a nerve-muscle pedicle graft in an attempt to reinnervate the left dorsal cricoarytenoid muscle in four ponies. These four ponies (as well as three others which served as controls) had previously undergone left recurrent laryngeal nerve transection. All seven ponies endoscopically showed signs of complete left laryngeal hemiplegia immediately postoperatively. Animals were monitored endoscopically for 30 weeks after surgery. The three control ponies showed no abduction of the arytenoid cartilage. In addition, in these three ponies, histological and histochemical expected changes of muscle fiber atrophy and fibrosis were present in the dorsal cricoarytenoid muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin heavy chain composition in normal and atrophic equine laryngeal muscle

The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. Left and right thyroarytenoideus (TA) and cricoarytenoideus dorsalis (CAD) were obtained from 7 horses affected with left-sided intrinsic laryngeal muscle atrophy and from 2 normal horses. Frozen sections were evaluated histologically for degree of atrophy and fiber type composition. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of muscle protein. Histologic atrophy was seen in all atrophic muscles and some right-sided muscles of 3 affected horses, as well as the left TA of 1 normal horse. Fiber type grouping or loss of type I muscle fibers was observed in the left-sided laryngeal muscles in all but 1 affected horse, as well as in the right muscles of 2 affected horses, and the left TA of 1 normal horse. SDS-PAGE showed 2 bands corresponding to the type I and type IIB myosin isoforms in the CAD and TA of the 2 normal horses. Affected horses demonstrated a trend toward increased expression of the type IIB isoform and decreased expression of the type I isoform in atrophic muscles. This study confirmed the presence of histologic abnormalities in grossly normal equine laryngeal muscle, and it demonstrated an increased expression of type IIB MHC with a concurrent decreased expression of type I MHC in affected muscles. Evaluation of muscle fiber changes at the cellular level under denervated and reinnervated conditions may aid in assessing future strategies for reinnervation or regeneration of atrophic laryngeal muscle.     

The pathogenesis of equine laryngeal hemiplegia--a review

Recent research on the muscular and nervous changes which occur in idiopathic equine laryngeal hemiplegia has indicated that many of the traditional concepts of the aetiology of this disease are erroneous. In light of the new knowledge gained, the various predispositions and possible causes of laryngeal hemiplegia are discussed, and it is suggested that the underlying mechanism of axonal damage in this neuropathy of horses may be related to abnormal energy metabolism in the axon.     

Partial Arytenoidectomy in the Horse Using an Extralaryngeal Approach

An extralaryngeal approach to partial arytenoidectomy in the horse was developed by in vitro experiments on isolated larynges and then on intact equine cadavers. The goals of the approach were to preserve the laryngeal mucosa, eliminate the need for a laryngotomy or tracheotomy, and minimize postoperative complications. The new approach was evaluated in seven horses with normal upper respiratory tracts. Left laryngeal hemiplegia was surgically created, and, after a 30-day convalescence, left partial arytenoidectomy was performed using an extralaryngeal approach. The left-to-right hemilaryngeal ratio was calculated before and after left recurrent laryngeal neurectomy and 60 days after partial arytenoidectomy. Left partial arytenoidectomy was successfully completed in all horses without performing a laryngotomy or tracheotomy. Preservation of the laryngeal mucosa (6/7 horses) and apparent stabilization of the adjacent soft tissue (6/7 horses) was achieved. In one horse, a 1 -cm laryngeal mucosal tear healed without complication, and in another some collapse of adjacent soft tissue occurred when a retention suture failed. The mean left-to-right hemilaryngeal ratio was significantly increased compared to horses with left laryngeal hemiplegia, but it remained significantly less than the pre-recurrent laryngeal neurectomy ratio (p < .05). Coughing, aspiration, and airway narrowing were not observed. Partial arytenoidectomy could be reliably performed through an extralaryngeal approach in horses with a normal underlying arytenoid cartilage with preservation of the laryngeal mucosa.     

A study of the weights of some intrinsic laryngeal and palatine muscles in the thoroughbred horse

The weights of some intrinsic laryngeal and palatine muscles from 46 thoroughbred horses of varying ages are presented. Thesehorses had no clinical history of wind affliction. The muscles studied were the dorsal cricoarytenoid, the lateral cricoarytenoid, the cricothyroid, the palatine levator and the palatine tensor.

The muscles supplied by the left recurrent laryngeal nerve tended to be lighter in older horses than the muscles supplied by the right recurrent laryngeal nerve. However there was no significant difference in weight between the left and right side for all muscles studied except the lateral cricoarytenoid. In this muscle the left side was significantly lighter than the right (P<0.001).