Ex vivo investigation of the effect of the transverse arytenoid ligament on abduction of the arytenoid cartilage when performing equine laryngoplasty.

Aims: To investigate the effect of the transverse arytenoid ligament (TAL) on abduction of the arytenoid cartilage when performing laryngoplasty. Methods: Modified prosthetic laryngoplasty was performed on right and left sides of 13 cadaver larynges. Increasing force was sequentially applied to the left arytenoid cartilage at 3 N intervals from 0-24 N, when the force on the right arytenoid cartilage was either 0 or 24 N, before and after TAL transection. Digital photographs of the rostral aspect of the larynx were used to determine the left arytenoid abduction angles for these given force combinations and results compared before and after TAL transection. Longitudinal and transverse sections of the TAL from seven other equine larynges were also examined histologically. Results: Increasing force on the left arytenoid cartilage from 0-24 N produced a progressive increase in the angle of the left arytenoid cartilage (p < 0.001) and increasing force on the right arytenoid cartilage from 0-24 N reduced the angle of the left arytenoid cartilage (p < 0.001). Following transection of the TAL the mean angle of the left arytenoid increased from 36.7 (95% CI = 30.5-42.8)° to 38.4 (95% CI = 32.3-44.5)°. Histological examination showed that the TAL was not a discrete ligament between the arytenoid cartilages but was formed by the convergence of the ligament and the left and right arytenoideus transversus muscles. Conclusions: Transection of the TAL in ex vivo equine larynges enabled greater abduction of the left arytenoid cartilage for a given force. These results indicate that TAL transection in conjunction with prosthetic laryngoplasty may have value, but the efficacy and safety of TAL transection under load in vivo, and in horses clinically affected with recurrent laryngeal neuropathy must be evaluated. Abbreviations: Fmax: Force needed to maximally abduct the left or right arytenoid; TAL: Transverse arytenoid ligament.

Nerve-specific extracellular matrix hydrogel promotes functional regeneration following nerve gap injury.

Nerve transection requires surgical intervention to restore function. The standard of care involves coaptation when a tension-free repair is achievable, or interposition of a graft or conduit when a gap remains. Despite advances, nerve gap injury is associated with unsatisfactory recovery. This study investigates the use of a decellularized, porcine nerve-derived hydrogel filler (peripheral nerve matrix, PNM) for conduits in an 8 mm rat sciatic nerve gap model. The decellularized tissue maintained multiple nerve-specific matrix components and nerve growth factors. This decellularized tissue was used to formulate hydrogels, which were deployed into conduits for nerve gap repair. Nerve recovery was assessed up to 24 weeks post injury by gait analysis, electrophysiology, and axon counting. Deployment of PNM within conduits was shown to improve electrophysiologic response and axon counts compared with those of empty conduit controls. These results indicate that PNM has potential benefits when used as a filler for conduits in nerve gap injuries.

Successful treatment of persistent dorsal displacement of the soft palate and evaluation of laryngohyoid position in 15 racehorses.

REASONS FOR PERFORMING STUDY: Caudal descent of the larynx has been proposed to be associated with intermittent dorsal displacement of the soft palate (DDSP) as it leads to a loss of the seal between the epiglottis and soft palate but further investigation of this theory is required. OBJECTIVES: To evaluate laryngohyoid position of horses with persistent DDSP in comparison to horses with intermittent DDSP and evaluate the outcome of treatment. HYPOTHESES: Horses with persistent DDSP have a different laryngohyoid position compared to those with intermittent DDSP. Horses with persistent DDSP can be returned successfully to racing with a laryngeal tie-forward procedure. METHODS: Medical records of 15 racehorses presented for persistent DDSP between 2002 and 2007 were reviewed. Age, sex and breed matched horses diagnosed with intermittent DDSP were used as a comparison group. Treatment of all horses was performed by laryngeal tie-forward, 8/15 horses with persistent DDSP had a subsequent laser staphylectomy. Preoperative laryngohyoid position was compared between the 2 groups using a radiographic reference system. Surgical effect on position was assessed by comparing pre- and post operative radiographic measurements. Outcome was assessed by return to racing and comparison of pre- and post operative race earnings ($). RESULTS: Thirteen of 15 horses with persistent DDSP returned to racing. Seven of 15 horses were treated with laryngeal tie-forward alone while 8/15 horses were also treated with a laser staphylectomy. Horses with persistent DDSP had a more caudal larynx (ossification of the thyroid cartilage) (13 mm, P = 0.014), a more caudal (10 mm, P = 0.044) and dorsal (7 mm, P = 0.01) basihyoid bone, and a more dorsal thyrohyoid-thyroid articulation (10 mm, P = 0.002) than horses with intermittent DDSP. CONCLUSIONS AND POTENTIAL RELEVANCE: Racehorses with persistent DDSP can be treated successfully by laryngeal tie-forward or by laryngeal tie-forward followed by laser staphylectomy. Horses with persistent DDSP have a more caudal larynx and more caudal and dorsal basihyoid bone than horses with intermittent DDSP.

Relationships between race earnings and horse age, sex, gait, track surface and number of race starts for Thoroughbred and Standardbred racehorses in North America.

REASONS FOR PERFORMING STUDY: There is no consensus on objective outcome measures that can be used to determine if a medical or surgical treatment affects race performance. OBJECTIVE: To determine the association between 2 commonly used outcome measures (total starts and total earnings) and age, sex, gait and race surface. METHODS: A cross-sectional study was performed using the race performance data for all Thoroughbred horses age 2, 3, 4 and 5 years racing in the United States, and Standardbred horses of the same ages racing in the United States and Canada during the year 2006. Median earnings and starts were determined for each combination of age, sex and track surface (for Thoroughbred) or gait (for Standardbred). The effect these variables had on starts on race earnings ($) was determined using linear regression. RESULTS: Race records for 68,649 Thoroughbreds and 25,830 Standardbreds were obtained. All independent variables (age, breed, sex, gait, track surface and total number of starts) had a significant impact on total earnings (P<0.0001). CONCLUSIONS: The data show considerable variation across age groups and track surfaces for Thoroughbreds and across age groups for Standardbreds. They also show that the decision to use earnings or starts as outcome measures could have a marked effect on reported success for a particular treatment. POTENTIAL RELEVANCE: Both earning and start data should be reported in studies evaluating outcome following surgery or other intervention. Considerations of age, breed, sex, track surface and gait should be included in the design of these studies.

Incidence of swallowing during exercise in horses with dorsal displacement of the soft palate.

REASONS FOR PERFORMING STUDY: The relationship between dorsal displacement of the soft palate (DDSP) and swallowing is unclear. OBJECTIVE: To quantify the relationship between DDSP and swallowing in horses at exercise. HYPOTHESES: The frequency of swallowing increases immediately prior to DDSP in horses at exercise. METHODS: Videoendoscopic and upper airway pressure data were collated from horses with a definitive diagnosis of DDSP at exercise. Horses with no upper airway abnormalities were matched by age, breed and sex and used as controls. Sixty-nine horses were identified with a definitive diagnosis of DDSP during the study interval. Airway pressure data were available for 42 horses. RESULTS: The majority of horses displaced at high exercising speeds while accelerating; a smaller number displaced during deceleration after peak speed had been reached. Horses swallowed significantly more frequently in the 1 min immediately preceding DDSP than in the control horses at equivalent speeds. DDSP at exercise results in a significant increase in tracheal expiratory pressure, a significant decrease in pharyngeal expiratory pressure and a significantly less negative pharyngeal inspiratory pressure compared to matched controls and compared to the pressures during the 1 min interval prior to DDSP. There was no significant difference between any measure of airway pressure before or after a swallow when examined at each time interval in the DDSP population. CONCLUSIONS: The frequency of swallowing decreases with increasing speed in normal horses. In contrast, the frequency of swallowing increases immediately prior to onset of DDSP. This is not a result of pharyngeal and tracheal pressure changes. POTENTIAL RELEVANCE: The increased frequency of swallowing observed prior to DDSP may be related to the aetiology of the disease.

Considerations for pacing of the cricoarytenoid dorsalis muscle by neuroprosthesis in horses.

REASONS FOR PERFORMING STUDY: The success rate of prosthetic laryngoplasty is limited and may be associated with significant sequelae. Nerve muscle pedicle transplantation has been attempted but requires a year before function is restored. OBJECTIVE: To determine the optimal parameters for functional electrical stimulation of the recurrent laryngeal nerve in horses. METHODS: An experimental in vivo study was performed on 7 mature horses (2-21 years). A nerve cuff was placed on the distal end of the common trunk of the recurrent laryngeal nerve (RLN). In 6 horses the ipsilateral adductor branch of RLN was also transected. The electrodes were connected to programmable internal stimulator. Stimulation was performed using cathodic phase and then biphasic pulses at 24 Hz with a 0.427 ms pulse duration. Stimulation-response experiments were performed at monthly intervals, from one week following implantation. The study continued until unit failure or the end of project (12 months). Two of the horses were stimulated continuously for 60 min to assess onset of fatigue. RESULTS: Excellent arytenoid cartilage abduction (mean arytenoid angle of 52.7 degrees, range 48.5-56.2 degrees) was obtained in 6 horses (laryngeal grades I or II (n = 3) and III (n = 2). Poor abduction was obtained in grade IV horses (n = 2). Arytenoid abduction was maintained for up to a year in one horse. Technical implant failure resulted in loss of abduction in 6 horses at one week to 11 months post operatively. Mean tissue impedance was 1.06 kOhm (range 0.64-1.67 kOhm) at one week, twice this value at 2 months (mean 2.32, range 1.11-3.75 kOhm) and was stable thereafter. Maximal abduction was achieved at a stimulation range of 0.65-7.2 mA. No electrical leakage was observed. Constant stimulation of the recurrent laryngeal nerve for 60 min led to full abduction without evidence of muscle fatigue. CONCLUSIONS: Functional electrical stimulation of the recurrent laryngeal nerve leading to full arytenoid abduction can be achieved. The minimal stimulation amplitude for maximal abduction angle is slightly higher than those for man and dogs. CLINICAL RELEVANCE: This treatment modality could eventually be applicable to horses with recurrent laryngeal neuropathy.

Characterization of cognition in mild cognitive impairment with and without Parkinson's disease.

•Screening tests can diagnose PD-MCI but do not give detailed cognitive profiles.•Criteria based on a complete neuropsychological battery identify more PD patients with MCI.•The overall cognitive profile is similar in PD-MCI and MCI.•Neuropsychological batteries and definition of impairment cut-offs should be refined.

Dictyostelium RasG is required for normal motility and cytokinesis, but not growth.

RasG is the most abundant Ras protein in growing Dictyostelium cells and the closest relative of mammalian Ras proteins. We have generated null mutants in which expression of RasG is completely abolished. Unexpectedly, RasG- cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG- cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.

Meta-analysis evaluating resting laryngeal endoscopy as a diagnostic tool for recurrent laryngeal neuropathy in the equine athlete.

BACKGROUND: Resting endoscopy has commonly been used as a method to predict laryngeal function at exercise. OBJECTIVES: To perform a meta-analysis to determine the sensitivity and specificity of resting laryngeal endoscopy to predict clinical recurrent laryngeal neuropathy at exercise. STUDY DESIGN: Meta-analysis. METHODS: Manuscripts were included if data were available for both resting and exercising airway function on all or a subset of horses. Normal resting endoscopy was defined as laryngeal grades 1 or 2 on a 4-/7-point scale or 1, 2 or 3 on a 5-point scale and normal dynamic endoscopy as a dynamic laryngeal grade A. RESULTS: Twelve studies including 1827 horses were evaluated. A small proportion of horses with grade 1 or grade 2 laryngeal function at rest, 3.5 and 11.9%, respectively, were identified as having abnormal laryngeal function at exercise. Within the horses with grade 3 laryngeal function at rest, 16% were classified as grade A, 26.4% as grade B and 57.6% as grade C at exercise. Worsening subgrades within resting grade 3 demonstrated an increasing proportion of complete or partial paralysis at exercise. The sensitivity and specificity of resting endoscopy was 74.4 and 95.1%, respectively, and the positive and negative predictive values were 85.6 and 90.5% respectively. MAIN LIMITATIONS: Use of two separate grading systems for evaluating resting laryngeal function. Other forms of dynamic airway collapse were not evaluated. CONCLUSIONS: Resting endoscopy is sensitive and highly specific for predicting laryngeal function at exercise. Dynamic endoscopy is important to assess multiple causes of airway collapse.

Development of equine upper airway fluid mechanics model for Thoroughbred racehorses.

REASON FOR PERFORMING STUDY: Computational fluid dynamics (CFD) models provide the means to evaluate airflow in the upper airways without requiring in vivo experiments. HYPOTHESIS: The physiological conditions of a Thoroughbred racehorse's upper airway during exercise could be simulated. METHODS: Computed tomography scanned images of a 3-year-old intact male Thoroughbred racehorse cadaver were used to simulate in vivo geometry. Airway pressure traces from a live Thoroughbred horse, during exercise was used to set the boundary condition. Fluid-flow equations were solved for turbulent flow in the airway during inspiratory and expiratory phases. The wall pressure turbulent kinetic energy and velocity distributions were studied at different cross-sections along the airway. This provided insight into the general flow pattern and helped identify regions susceptible to dynamic collapse. RESULTS: The airflow velocity and static tracheal pressure were comparable to data of horses exercising on a high-speed treadmill reported in recent literature. The cross-sectional area of the fully dilated rima glottidis was 7% greater than the trachea. During inspiration, the area of highest turbulence (i.e. kinetic energy) was in the larynx, the rostral aspect of the nasopharynx was subjected to the most negative wall pressure and the highest airflow velocity is more caudal on the ventral aspect of the nasopharynx (i.e. the soft palate). During exhalation, the area of highest turbulence was in the rostral and mid-nasopharynx, the maximum positive pressure was observed at the caudal aspect of the soft palate and the highest airflow velocity at the front of the nasopharynx. CONCLUSIONS AND CLINICAL RELEVANCE: In the equine upper airway collapsible area, the floor of the rostral aspect of the nasopharynx is subjected to the most significant collapsing pressure with high average turbulent kinetic during inhalation, which may lead to palatal instability and explain the high prevalence of dorsal displacement of the soft palate (DDSP) in racehorses. Maximal abduction of the arytenoid cartilage may not be needed for optimal performance, since the trachea cross-sectional area is 7% smaller than the rima glottidis.

Implications of different degrees of arytenoid cartilage abduction on equine upper airway characteristics.

REASON FOR PERFORMING STUDY: The necessary degree of arytenoid cartilage abduction (ACA) to restore airway patency at maximal exercise has not been determined. OBJECTIVES: Use computational fluid dynamics modelling to measure the effects of different degrees of ACA on upper airway characteristics of horses during exercise. HYPOTHESIS: Maximal ACA by laryngoplasty is necessary to restore normal peak airflow and pressure in Thoroughbred racehorses with laryngeal hemiplegia. METHODS: The upper airway was modeled with the left arytenoid in 3 different positions: maximal abduction; 88% cross-sectional area of the rima glottis; and 75% cross-sectional area of the rima glottis. The right arytenoid cartilage was maximally abducted. Two models were assumed: Model 1: no compensation of airway pressures; and Model 2: airway pressure compensation occurs to maintain peak airflow. The cross-sectional pressure and velocity distributions for turbulent flow were studied at peak flow and at different positions along the airway. RESULTS: Model 1: In the absence of a change in driving pressure, 12 and 25% reductions in cross-sectional area of the larynx resulted in 4.11 and 5.65% reductions in peak airflow and 3.68 and 5.64% in tidal volume, respectively, with mild changes in wall pressure. Model 2: To maintain peak flow, a 6.27% increase in driving tracheal pressure was required to compensate for a cross-sectional reduction of 12% and a 13.63% increase in driving tracheal pressure was needed for a cross-sectional area reduction of 25%. This increase in negative driving pressure resulted in regions with low intraluminal and wall pressures, depending on the degree of airway diameter reduction. CONCLUSION: Assuming no increase in driving pressure, the decrease in left ACA reduced airflow and tidal volume. With increasing driving pressure, a decrease in left ACA changed the wall pressure profile, subjecting the submaximally abducted arytenoid cartilage and adjacent areas to airway collapse. CLINICAL RELEVANCE: The surgical target of ACA resulting in 88 % of maximal cross-sectional area seems to be appropriate.

Intra-articular stabilisation of the equine cricoarytenoid joint.

REASONS FOR PERFORMING STUDY: The success of laryngoplasty is limited by abduction loss in the early post operative period. OBJECTIVE: To determine the efficacy of polymethylmethacrylate (PMMA) in stabilising the cricoarytenoid joint (CAJ) and reducing the force on the laryngoplasty suture. HYPOTHESIS: Injection into the cricoarytenoid joint resists the forces produced by physiological laryngeal air flows and pressures thereby reducing the force experienced by the laryngoplasty suture. METHODS: Ten cadaver larynges were collected at necropsy and PMMA was injected into one CAJ at selected random. Each larynx was subjected to physiological conditions with with constant (static) or cycling (dynamic) flow. The specimens were tested sequentially in each of 4 conditions: 1) bilateral full abduction (Control 1); 2) transection of the suture on the side without PMMA; 3) bilateral abduction achieved by replacing the suture (Control 2); and 4) cutting the suture on the PMMA side. Tracheal pressure and flow and pressure in the flow chamber were recorded using pressure and flow transducers. The strain experienced by each suture during bilateral abduction (Controls 1 and 2) was measured. Statistical comparison of the 4 conditions was performed using a mixed effect model with Tukey's post hoc test for multiple comparisons. The strain gauge data were analysed by paired comparison of the regression slopes. RESULTS: In the static and dynamic states, tracheal pressure increased and tracheal flow decreased when the suture on the non-cement side was cut (P < 0.05). There was no significant difference in any outcome measure between PMMA injected into the CAJ and bilaterally abducted specimens (Controls 1 and 2) for either condition. The rate of increase in strain with increasing translaryngeal pressure was significantly less on the suture with PMMA placed in the CAJ (P = 0.03). CONCLUSIONS: These data provide strong evidence that injecting PMMA into the CAJ resists the collapsing effect of physiological airflows and pressures in vitro and reduces the force experienced by the laryngoplasty suture during maximal abduction. POTENTIAL RELEVANCE: Augmentation of prosthetic laryngoplasty with this technique may reduce arytenoid abduction loss in the early post operative period.

Neuroanatomy of the equine dorsal cricoarytenoid muscle: surgical implications.

REASON FOR PERFORMING STUDY: Studies are required to define more accurately and completely the neuroanatomy of the equine dorsal cricoarytenoid muscle as a prerequisite for developing a neuroprosthesis for recurrent laryngeal neuropathy. OBJECTIVES: To describe the anatomy, innervation, fibre types and function of the equine dorsal cricoarytenoid muscle. METHODS: Thirty-one larynges were collected at necropsy from horses with no history of upper airway disease and 25 subjected to gross dissection. Thereafter, the following preparations were made on a subset of larynges: histochemical staining (n = 5), Sihler's and acetylcholinesterase staining for motor endplates (n = 2). An additional 6 larynges were collected and used for a muscle stimulation study. RESULTS: Two neuromuscular compartments (NMC), each innervated by a primary nerve branch of the recurrent laryngeal nerve, were identified in all larynges. Stimulation of the lateral NMC produced more lateral displacement of the arytenoid cartilage than the medial NMC (P<0.05). The medial NMC tended to rotate the arytenoid cartilage dorsally. Motor endplates were identified at the junction of the middle and caudal thirds of each NMC. If fibre type grouping was present it was always present in both NMCs. CONCLUSIONS: The equine dorsal cricoarytenoid muscle has 2 distinct muscle NMCs with discrete innervation and lines of action. The lateral NMC appears to have a larger role in increasing cross-sectional area of the rima glottidis. POTENTIAL RELEVANCE: This information should assist in planning surgical reinnervation procedures and development of a neuroprosthesis for recurrent laryngeal neuropathy.

Racing performance following the laryngeal tie-forward procedure: a case-controlled study.

REASONS FOR PERFORMING STUDY: The laryngeal tie-forward procedure (LTFP) is becoming widely used for correction of dorsal displacement of the soft palate (DDSP) despite the absence of an evidence-based assessment of its efficacy. HYPOTHESES: The LTFP returns racing performance to preoperative baseline levels and to that of matched controls; and post operative laryngohyoid position is associated with post operative performance. DESIGN AND POPULATION: Case-controlled study of racehorses undergoing a LTFP for dorsal displacement of the soft palate at Cornell University between October 2002 and June 2007. METHODS: The presence of at least one post operative start and race earnings ($) were used as outcome variables. Controls were matched by age, breed and sex from the third race prior to surgery. A novel radiographic reference system was used to determine laryngohyoid position pre- and post operatively. Data for definitively and presumptively diagnosed cases were analysed separately. RESULTS: During the study interval, 263 racehorses presented, of which 106 were included in the study; 36 had a definitive diagnosis of DDSP and 70 a presumptive diagnosis. Treated horses were equally likely to race post operatively as controls in the equivalent race. Treated horses had significantly lower earnings in the race before surgery than matched controls. The procedure moved the basihyoid bone dorsally and caudally and the larynx dorsally and rostrally. A more dorsal post operative basihyoid position and more dorsal and less rostral laryngeal position were associated with an increased probability of racing post operatively. CONCLUSIONS: Horses undergoing a LTFP are as likely to race post operatively as matched controls. The procedure restores race earnings to preoperative baseline levels and to those of matched controls. POTENTIAL RELEVANCE: This study provides strong evidence supporting the use of the LTFP in racehorses. Further work is needed to determine the relationship between laryngohyoid conformation and nasopharyngeal stability in horses.

Transoesophageal ultrasound and computer tomographic assessment of the equine cricoarytenoid dorsalis muscle: Relationship between muscle geometry and exercising laryngeal function.

REASONS FOR PERFORMING STUDY: Early detection of recurrent laryngeal neuropathy (RLN) is of considerable interest to the equine industry. OBJECTIVES: To describe two imaging modalities, transoesophageal ultrasound (TEU) and computed tomography (CT) with multiplanar reconstruction to assess laryngeal muscle geometry, and determine the relationship between cricoarytenoid dorsalis (CAD) geometry and function. STUDY DESIGN: Two-phase study evaluating CAD geometry in experimental horses and horses with naturally occurring RLN. METHODS: Equine CAD muscle volume was determined from CT scan sets using volumetric reconstruction with LiveWire. The midbody and caudal dorsal-ventral thickness of the CAD muscle was determined using a TEU in the same horses; and in horses with a range of severity of RLN (n = 112). RESULTS: Transoesophageal ultrasound was able to readily image the CAD muscles and lower left:right CAD thickness ratios were observed with increasing disease severity. Computed tomography based muscle volume correlated very closely with ex vivo muscle volume (R2 = 0.77). CONCLUSIONS: Computed tomography reconstruction can accurately determine intrinsic laryngeal muscle geometry. A relationship between TEU measurements of CAD geometry and laryngeal function was established. These imaging techniques could be used to track the response of the CAD muscle to restorative surgical treatments such as nerve muscle pedicle graft, nerve anastomosis and functional electrical stimulation.

Laryngeal advancement surgery improves swallowing function in a reversible equine dysphagia model.

REASONS FOR PERFORMING STUDY: Pharyngeal dysphagia is a debilitating, sometimes fatal condition in horses, with multiple aetiologies. The pathophysiology is complex and not fully understood. Treatment is largely supportive. Laryngeal advancement surgery may diminish symptoms of dysphagia and improve swallowing in affected horses. OBJECTIVES: 1) to induce reversible moderate and marked pharyngeal dysphagia by regional anaesthesia of branches of the glossopharyngeal (IX), vagus (X) and hypoglossal (XII) nerves; 2) to characterise the dysphagia produced by each model; and 3) to determine whether laryngeal advancement surgery improves swallowing in these models. STUDY DESIGN: Experimental design using 6 adult horses. METHODS: Two dysphagia models were produced by blocking IX, the pharyngeal branch of X and XII unilaterally (moderate model) and only the pharyngeal branch of X bilaterally (marked model) within the guttural pouches. Both models were performed on each horse before and after surgery in order to assess the effectiveness of the surgical procedure as a potential treatment for pharyngeal dysphagia. Dysphagia was scored by partly blinded observers on a scale of 0-12 based on observations of eating (nonblinded), endoscopic examinations and fluoroscopic swallowing (blinded), where 0 = normal swallow and 12 = severe dysphagia with tracheal aspiration. Data were analysed by 3-factor ANOVA, with significance taken as P<0.05. RESULTS: Dysphagia models were reversible, and horses swallowed normally within 3 h of model induction. The marked dysphagia model impaired movement of feed from the base of the tongue to the oesophagus and caused severe airway contamination. The average dysphagia score (mean ± s.d.) for the marked dysphagia model was 10.6 ± 1.1 before surgery and 6.1 ± 4.3 after surgery (P = 0.007). Laryngeal advancement surgery did not significantly improve the dysphagia scores in the moderate model (P = 0.5). CONCLUSIONS: Laryngeal advancement surgery may improve swallowing and reduce aspiration in horses affected with diseases that cause pharyngeal dysphagia.

Acid-induced fusion of liposomes: studies with 2,3-seco-5 alpha-cholestan-2,3-dioic acid.

The effect of 2,3-seco-5 alpha-cholestan-2,3-dioic acid on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine is markedly dependent on pH. Above pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid raises the temperature of this transition, i.e., it stabilizes the bilayer phase. At pH 6.56 there is little effect of this sterol derivative on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. However, below pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid markedly lowers the temperature of this transition. The promotion of hexagonal phase formation increases both with increasing mol fraction of this sterol derivative and with lower pH, particularly in the range between pH 6.56 and pH 5.0. Below about pH 6, 2,3-seco-5 alpha-cholestan-2,3-dioic acid also induces vesicle fusion as measured both by lipid mixing as well as by mixing of aqueous contents. For these assays vesicles made of phosphatidylethanolamine (made from egg phosphatidylcholine) and extruded through 0.2 micron pore membranes were used. At higher concentrations or at lower pH the 2,3-seco-5 alpha-cholestan-2,3-dioic acid induces some leakage of the contents of these vesicles. Nevertheless, with vesicles containing only 2 weight% sterol derivative, it was possible to demonstrate substantial mixing of aqueous contents of the vesicles over the pH range 3.5 to 5.5. Several of the properties of 2,3-seco-5 alpha-cholestan-2,3-dioic acid indicate that this compound may be useful in sensitizing vesicles to acid-induced fusion for the purpose of endocytic drug delivery.

Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction.

Cholesterol sulphate is a potent stabilizer of membrane bilayer structure in both dielaidoylphosphatidylethanolamine and egg phosphatidylethanolamine model membranes, however, the addition of calcium abolishes this bilayer stabilization. Calcium also induces fusion and leakage of egg phosphatidylethanolamine large unilamellar vesicles containing cholesterol sulphate, but has no effect on fusion or leakage of egg phosphatidylcholine large unilamellar vesicles containing cholesterol sulphate. With egg phosphatidylethanoiamine liposomes, the initial rate, and extent of fusion, at constant calcium concentration, vary inversely with the mol percentage of cholesterol sulphate present in the vesicle membrane. The interaction of calcium and cholesterol sulphate, which causes membrane destabilization and fusion in phosphatidylethanolamine containing model systems, may play a role in the acrosome reaction in human sperm.

Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A resolution.

The structure of the tri-N-acetylchitotriose inhibitor complex of hen egg-white lysozyme has been refined at 1.75 A resolution, using data collected from a complex crystal with ligand bound at less than full occupancy. To determine the exact value of the inhibitor occupancy, a model comprising unliganded and sugar-bound protein molecules was generated and refined against the 1.75 A data, using a modified version of the Hendrickson & Konnert least-squares procedure. The crystallographic R-factor for the model was found to fall to a minimum at 55% bound sugar. Conventional refinement assuming unit occupancy was found to yield incorrect thermal and positional parameters. Application of the same refinement procedures to an earlier 2.0 A data set, collected independently on different complex crystals by Blake et al. gave less consistent results than the 1.75 A refinement. From an analysis of the high resolution structure a detailed picture of the protein-carbohydrate interactions in the non-productive complex has emerged, together with the conformation and mobility changes that accompany ligand binding. The specificity of interaction between the protein and inhibitor, bound in subsites A to C of the active site, is seen to be generated primarily by an extensive network of hydrogen bonds, both to the protein itself and to bound solvent molecules. The latter also play an important role in maintaining the structural integrity of the active site cleft in the apo-protein.

1H nuclear magnetic resonance studies of hen lysozyme-N-acetylglucosamine oligosaccharide complexes in solution. Application of chemical shifts for the comparison of conformational changes in solution and in the crystal.

Two-dimensional 1H nuclear magnetic resonance spectroscopy has been used to examine the complexes formed in solution between hen egg-white lysozyme and N-acetylglucosamine (GlcNAc) oligosaccharides. Changes in chemical shift have been measured for resonances of the majority of residues of lysozyme on binding the monomer, dimer and trimer of GlcNAc. The three inhibitors induce very similar changes in chemical shift, and these increase slightly with the length of the oligosaccharide. The largest changes are confined principally to the vicinity of site C in the active site cleft of the enzyme. These changes in chemical shift have been compared with differences in the ring current chemical shifts calculated from the crystal structures of unbound and GlcNAc3 bound lysozyme. This comparison suggests that the major conformational changes of residues in the vicinity of site C of the enzyme, that are caused by the binding of GlcNAc3, observed in the diffraction studies are at least consistent with the changes that occur in solution. Small changes in chemical shift are observed in the enzyme in regions remote from the active site, which indicate that the effects of inhibitor binding are felt throughout the enzyme. These changes in chemical shift correlate to a lesser extent than those near site C with the changes in chemical shift predicted from changes in conformation observed in the crystal structures. The results illustrate that chemical shifts are useful in assessing the significance of small conformational changes in proteins, although the usefulness of this approach will be limited by the resolution of the crystallographic structures, as well as the uncertainties in the origins of the chemical shift. Although conformational changes in site C account for many of the changes in the NMR spectrum of lysozyme, evidence is, however, presented for multiple binding sites for the GlcNAc oligosaccharides in solution, perhaps involving partial occupancy of site D.