Publications by authors named "Justin Perkins"

35 Publications

Splenic Nerve Neuromodulation Reduces Inflammation and Promotes Resolution in Chronically Implanted Pigs.

Front Immunol 2021 29;12:649786. Epub 2021 Mar 29.

Clinical Science & Services, The Royal Veterinary College, Hatfield, United Kingdom.

Neuromodulation of the immune system has been proposed as a novel therapeutic strategy for the treatment of inflammatory conditions. We recently demonstrated that stimulation of near-organ autonomic nerves to the spleen can be harnessed to modulate the inflammatory response in an anesthetized pig model. The development of neuromodulation therapy for the clinic requires chronic efficacy and safety testing in a large animal model. This manuscript describes the effects of longitudinal conscious splenic nerve neuromodulation in chronically-implanted pigs. Firstly, clinically-relevant stimulation parameters were refined to efficiently activate the splenic nerve while reducing changes in cardiovascular parameters. Subsequently, pigs were implanted with a circumferential cuff electrode around the splenic neurovascular bundle connected to an implantable pulse generator, using a minimally-invasive laparoscopic procedure. Tolerability of stimulation was demonstrated in freely-behaving pigs using the refined stimulation parameters. Longitudinal stimulation significantly reduced circulating tumor necrosis factor alpha levels induced by systemic endotoxemia. This effect was accompanied by reduced peripheral monocytopenia as well as a lower systemic accumulation of CD16CD14 pro-inflammatory monocytes. Further, lipid mediator profiling analysis demonstrated an increased concentration of specialized pro-resolving mediators in peripheral plasma of stimulated animals, with a concomitant reduction of pro-inflammatory eicosanoids including prostaglandins. Terminal electrophysiological and physiological measurements and histopathological assessment demonstrated integrity of the splenic nerves up to 70 days post implantation. These chronic translational experiments demonstrate that daily splenic nerve neuromodulation, implanted electronics and clinically-relevant stimulation parameters, is well tolerated and is able to prime the immune system toward a less inflammatory, pro-resolving phenotype.
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http://dx.doi.org/10.3389/fimmu.2021.649786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043071PMC
March 2021

Imaging of focal seizures with Electrical Impedance Tomography and depth electrodes in real time.

Neuroimage 2021 Mar 20;234:117972. Epub 2021 Mar 20.

Medical Physics and Biomedical Engineering, University College London, UK.

Intracranial EEG is the current gold standard technique for localizing seizures for surgery, but it can be insensitive to tangential dipole or distant sources. Electrical Impedance Tomography (EIT) offers a novel method to improve coverage and seizure onset localization. The feasibility of EIT has been previously assessed in a computer simulation, which revealed an improved accuracy of seizure detection with EIT compared to intracranial EEG. In this study, slow impedance changes, evoked by cell swelling occurring over seconds, were reconstructed in real time by frequency division multiplexing EIT using depth and subdural electrodes in a swine model of epilepsy. EIT allowed to generate repetitive images of ictal events at similar time course to fMRI but without its significant limitations. EIT was recorded with a system consisting of 32 parallel current sources and 64 voltage recorders. Seizures triggered with intracranial injection of benzylpenicillin (BPN) in five pigs caused a repetitive peak impedance increase of 3.4 ± 1.5 mV and 9.5 ± 3% (N =205 seizures); the impedance signal change was seen already after a single, first seizure. EIT enabled reconstruction of the seizure onset 9 ± 1.5 mm from the BPN cannula and 7.5 ± 1.1 mm from the closest SEEG contact (p<0.05, n =37 focal seizures in three pigs) and it could address problems with sampling error in intracranial EEG. The amplitude of the impedance change correlated with the spread of the seizure on the SEEG (p <0.001, n =37). The results presented here suggest that combining a parallel EIT system with intracranial EEG monitoring has a potential to improve the diagnostic yield in epileptic patients and become a vital tool in improving our understanding of epilepsy.
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http://dx.doi.org/10.1016/j.neuroimage.2021.117972DOI Listing
March 2021

Model-based geometrical optimisation and in vivo validation of a spatially selective multielectrode cuff array for vagus nerve neuromodulation.

J Neurosci Methods 2021 Mar 28;352:109079. Epub 2021 Jan 28.

Medical Physics and Biomedical Engineering, University College London, UK.

Background: Neuromodulation by electrical stimulation of the human cervical vagus nerve may be limited by adverse side effects due to stimulation of off-target organs. It may be possible to overcome this by spatially selective stimulation of peripheral nerves. Preliminary studies have shown this is possible using a cylindrical multielectrode human-sized nerve cuff in vagus nerve selective neuromodulation.

New Method: The model-based optimisation method for multi-electrode geometric design is presented. The method was applied for vagus nerve cuff array and suggested two rings of 14 electrodes, 3 mm apart, with 0.4 mm electrode width and separation and length 0.5-3 mm, with stimulation through a pair in the same radial position on the two rings. The electrodes were fabricated using PDMS-embedded stainless steel foil and PEDOT: pTS coating.

Results: In the cervical vagus nerve in anaesthetised sheep, it was possible to selectively reduce the respiratory breath rate (RBR) by 85 ± 5% without affecting heart rate, or selectively reduce heart rate (HR) by 20 ± 7% without affecting respiratory rate. The cardiac- and pulmonary-specific sites on the nerve cross-sectional perimeter were localised with a radial separation of 105 ± 5 degrees (P < 0.01, N = 24 in 12 sheep).

Conclusions: Results suggest organotopic or function-specific organisation of neural fibres in the cervical vagus nerve. The optimised electrode array demonstrated selective electrical neuromodulation without adverse side effects. It may be possible to translate this to improved treatment by electrical autonomic neuromodulation for currently intractable conditions.
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http://dx.doi.org/10.1016/j.jneumeth.2021.109079DOI Listing
March 2021

Optimisation and validation of immunohistochemical axonal markers for morphological and functional characterisation of equine peripheral nerves.

Equine Vet J 2020 Dec 18. Epub 2020 Dec 18.

Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.

Background: Horses are affected by various peripheral nerve disorders but defining their aetiology and pathophysiology is hampered by limited understanding of associated morphological and pathological changes and involvement of specific axonal types.

Objectives: To investigate the hypothesis that selected antibody markers, used in conjunction with various tissue processing methods, would enable identification of axons with different functional modalities within a range of equine peripheral nerves.

Study Design: Optimisation and validation study.

Methods: A range of antibodies were evaluated immunohistochemically via fluorescence confocal microscopy in cadaver equine nerve samples of primary motor, mixed or primary sensory functions (recurrent laryngeal, phrenic and plantar digital) within formalin-fixed paraffin-embedded (FFPE) and formalin-fixed frozen (FFF) tissues subjected to different antigen retrieval protocols.

Results: Immunohistochemistry of FFPE-derived nerve samples with selected antibodies and specific antigen retrieval methods enabled identification of myelinated and unmyelinated axons, cholinergic, sympathetic and peptidergic axons. The recurrent laryngeal and phrenic nerves are composed of myelinated cholinergic (motor), myelinated sensory fibres, unmyelinated adrenergic (sympathetic) axons and unmyelinated peptidergic (sensory) axons. In contrast, as expected, the plantar digital nerve had no myelinated motor fibres being mainly composed of myelinated sensory fibres, unmyelinated sympathetic and unmyelinated peptidergic sensory axons.

Main Limitation: Attempts specifically to label parasympathetic fibres were unsuccessful in any nerve examined in both FFPE and FFF tissues.

Conclusions: A panel of antibody markers can be used to reveal morphological and functional properties of equine nerves. Future work should enable better characterisation of morphological changes in equine neuropathies at various stages of disease development.
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http://dx.doi.org/10.1111/evj.13403DOI Listing
December 2020

Imaging fascicular organization of rat sciatic nerves with fast neural electrical impedance tomography.

Nat Commun 2020 12 7;11(1):6241. Epub 2020 Dec 7.

Department of Medical Physics and Biomedical Engineering, University College London, London, UK.

Imaging compound action potentials (CAPs) in peripheral nerves could help avoid side effects in neuromodulation by selective stimulation of identified fascicles. Existing methods have low resolution, limited imaging depth, or are invasive. Fast neural electrical impedance tomography (EIT) allows fascicular CAP imaging with a resolution of <200 µm, <1 ms using a non-penetrating flexible nerve cuff electrode array. Here, we validate EIT imaging in rat sciatic nerve by comparison to micro-computed tomography (microCT) and histology with fluorescent dextran tracers. With EIT, there are reproducible localized changes in tissue impedance in response to stimulation of individual fascicles (tibial, peroneal and sural). The reconstructed EIT images correspond to microCT scans and histology, with significant separation between the fascicles (p < 0.01). The mean fascicle position is identified with an accuracy of 6% of nerve diameter. This suggests fast neural EIT can reliably image the functional fascicular anatomy of the nerves and so aid selective neuromodulation.
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http://dx.doi.org/10.1038/s41467-020-20127-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721735PMC
December 2020

Thyrohyoideus muscle innervation in the horse.

Vet Surg 2021 Jan 6;50(1):53-61. Epub 2020 Nov 6.

Clinique vétérinaire de Grosbois, Boissy-St-Léger, France.

Objective: To describe the innervation of the thyrohyoideus (TH) muscle and to confirm our findings with stimulation of first cervical (C1) nerve branches.

Study Design: Ex vivo phase 1 and clinical phase 2.

Animals: Fourteen head and neck specimens and 17 client-owned horses.

Methods: In phase 1, the cranial nerve (CN) XII and the C1 nerve were dissected with their branches in 20 dissections were performed on 14 specimens (6 left and right side and 8 only left or right) Anatomy was noted. Samples of nerve bifurcations were collected for histological confirmation of anatomical findings. First cervical nerve branches were stimulated in horses undergoing cervical nerve graft to treat laryngeal hemiplegia.

Results: The nerve innervating the TH muscle arose directly from the C1 nerve in 17 of 20 dissections, from an anastomotic branch between CN XII and the C1 nerve in two of 20 dissections, and from the C1 nerve and the anastomotic branch in one of 20 dissections. No direct connection between the TH muscle and CN XII was found. Histological examination revealed that the anastomosis was composed of C1 nerve fibers passing over to CN XII. First cervical stimulation resulted in TH muscle contraction in 16 of 17 horses.

Conclusions: The innervation of the TH muscle originated from the C1 nerve according to dissection, histological, and conduction studies, with variation in the branching pattern.

Clinical Significance: Care should be taken to preserve the C1 nerve during prosthetic laryngoplasty. The surgical technique for C1 nerve grafts should be reconsidered in light of these findings, along with new options to treat dorsal displacement of the soft palate..
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http://dx.doi.org/10.1111/vsu.13536DOI Listing
January 2021

Quantification of clinically applicable stimulation parameters for precision near-organ neuromodulation of human splenic nerves.

Commun Biol 2020 Oct 16;3(1):577. Epub 2020 Oct 16.

Galvani Bioelectronics, Gunnels Wood Road, Stevenage, SG1 2NY, UK.

Neuromodulation is a new therapeutic pathway to treat inflammatory conditions by modulating the electrical signalling pattern of the autonomic connections to the spleen. However, targeting this sub-division of the nervous system presents specific challenges in translating nerve stimulation parameters. Firstly, autonomic nerves are typically embedded non-uniformly among visceral and connective tissues with complex interfacing requirements. Secondly, these nerves contain axons with populations of varying phenotypes leading to complexities for axon engagement and activation. Thirdly, clinical translational of methodologies attained using preclinical animal models are limited due to heterogeneity of the intra- and inter-species comparative anatomy and physiology. Here we demonstrate how this can be accomplished by the use of in silico modelling of target anatomy, and validation of these estimations through ex vivo human tissue electrophysiology studies. Neuroelectrical models are developed to address the challenges in translation of parameters, which provides strong input criteria for device design and dose selection prior to a first-in-human trial.
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http://dx.doi.org/10.1038/s42003-020-01299-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568572PMC
October 2020

Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling.

Pflugers Arch 2020 10 1;472(10):1435-1446. Epub 2020 Sep 1.

Faculty of Medicine, National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt) increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude - 106.9%, S-endpoint gradient + 54.9%, S point - 39.4%, RS ratio + 38.6% and (-dV/dt) - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.
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http://dx.doi.org/10.1007/s00424-020-02446-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476990PMC
October 2020

Ex vivo modeling of the airflow dynamics and two-and three-dimensional biomechanical effects of suture placements for prosthetic laryngoplasty in horses.

Am J Vet Res 2020 Aug;81(8):665-672

Objective: To identify the degree of left arytenoid cartilage (LAC) abduction that allows laryngeal airflow similar to that in galloping horses, assess 2-D and 3-D biomechanical effects of prosthetic laryngoplasty on LAC movement and airflow, and determine the influence of suture position through the muscular process of the arytenoid cartilage (MPA) on these variables.

Sample: 7 equine cadaver larynges.

Procedures: With the right arytenoid cartilage maximally abducted and inspiratory airflow simulated by vacuum, laryngeal airflow and translaryngeal pressure and impedance were measured at 12 incremental LAC abduction forces (0% to 100% [maximum abduction]) applied through laryngoplasty sutures passed caudocranially or mediolaterally through the left MPA. Cross-sectional area of the rima glottis and left-to-right angle quotient were determined from photographs at each abduction force; CT images were obtained at alternate forces. Arytenoid and cricoid cartilage markers allowed calculation of LAC roll, pitch, and yaw through use of Euler angles on 3-D reconstructed CT images.

Results: Translaryngeal pressure and impedance decreased, and airflow increased rapidly at low abduction forces, then slowed until a plateau was reached at approximately 50% of maximum abduction force. The greatest LAC motion was rocking (pitch). Suture position through the left MPA did not significantly affect airflow data. Approximately 50% of maximum abduction force, corresponding to a left arytenoid angle of approximately 30° and left-to-right angle quotient of 0.79 to 0.84, allowed airflow of approximately 61 ± 6.5 L/s.

Conclusions And Clinical Relevance: Ex vivo modeling results suggested little benefit to LAC abduction forces > 50%, which allowed airflow similar to that reported elsewhere for galloping horses.
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http://dx.doi.org/10.2460/ajvr.81.8.665DOI Listing
August 2020

MicroCT optimisation for imaging fascicular anatomy in peripheral nerves.

J Neurosci Methods 2020 05 13;338:108652. Epub 2020 Mar 13.

EIT and Neurophysiology Lab, Medical Physics and Biomedical Engineering, University College London, Gower Street, London, WC1E 6BT, United Kingdom.

Background: Due to the lack of understanding of the fascicular organisation, vagus nerve stimulation (VNS) leads to unwanted off-target effects. Micro-computed tomography (microCT) can be used to trace fascicles from periphery and image fascicular anatomy.

New Method: In this study, we present a simple and reproducible method for imaging fascicles in peripheral nerves with iodine staining and microCT for the determination of fascicular anatomy and organisation.

Results: At the determined optimal pre-processing steps and scanning parameters, the microCT protocol allowed for segmentation and tracking of fascicles within the nerves. This was achieved after 24 hours and 120 hours of staining with Lugol's solution (1% total iodine) for rat sciatic and pig vagus nerves, respectively, and the following scanning parameters: 4 μm voxel size, 35 kVp energy, 114 μA current, 4 W power, 0.25 fps in 4 s exposure time, 3176 projections and a molybdenum target.

Comparison With Existing Method(s): This optimised method for imaging fascicles provides high-resolution, three-dimensional images and full imaging penetration depth not obtainable with methods typically used such as histology, magnetic resonance imaging and optical coherence tomography whilst obviating time-consuming pre-processing methods, the amount of memory required, destruction of the samples and the cost associated with current microCT methods.

Conclusion: The optimised microCT protocol facilitates segmentation and tracking of the fascicles within the nerve. The resulting segmentation map of the functional anatomical organisation of the vagus nerve will enable selective VNS ultimately allowing for the avoidance of the off-target effects and improving its therapeutic efficacy.
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http://dx.doi.org/10.1016/j.jneumeth.2020.108652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181190PMC
May 2020

Feasibility of kilohertz frequency alternating current neuromodulation of carotid sinus nerve activity in the pig.

Sci Rep 2019 12 2;9(1):18136. Epub 2019 Dec 2.

The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK.

Recent research supports that over-activation of the carotid body plays a key role in metabolic diseases like type 2 diabetes. Supressing carotid body signalling through carotid sinus nerve (CSN) modulation may offer a therapeutic approach for treating such diseases. Here we anatomically and histologically characterised the CSN in the farm pig as a recommended path to translational medicine. We developed an acute in vivo porcine model to assess the application of kilohertz frequency alternating current (KHFAC) to the CSN of evoked chemo-afferent CSN responses. Our results demonstrate the feasibility of this approach in an acute setting, as KHFAC modulation was able to successfully, yet variably, block evoked chemo-afferent responses. The observed variability in blocking response is believed to reflect the complex and diverse anatomy of the porcine CSN, which closely resembles human anatomy, as well as the need for optimisation of electrodes and parameters for a human-sized nerve. Overall, these results demonstrate the feasibility of neuromodulation of the CSN in an anesthetised large animal model, and represent the first steps in driving KHFAC modulation towards clinical translation. Chronic recovery disease models will be required to assess safety and efficacy of this potential therapeutic modality for application in diabetes treatment.
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http://dx.doi.org/10.1038/s41598-019-53566-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889394PMC
December 2019

Investigation into pathophysiology of naturally occurring palatal instability and intermittent dorsal displacement of the soft palate (DDSP) in racehorses: Thyro-hyoid muscles fatigue during exercise.

PLoS One 2019 25;14(10):e0224524. Epub 2019 Oct 25.

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

Exercise induced intermittent dorsal displacement of the soft palate (DDSP) is a common cause of airway obstruction and poor performance in racehorses. The definite etiology is still unclear, but through an experimental model, a role in the development of this condition was identified in the dysfunction of the thyro-hyoid muscles. The present study aimed to elucidate the nature of this dysfunction by investigating the spontaneous response to exercise of the thyro-hyoid muscles in racehorses with naturally occurring DDSP. Intramuscular electrodes were implanted in the thyro-hyoid muscles of nine racehorses, and connected to a telemetric unit for electromyographic monitoring implanted subcutaneously. The horses were recruited based on upper airway function evaluated through wireless endoscopy during exercise. Five horses, with normal function, were used as control; four horses were diagnosed as DDSP-affected horses based on repeated episodes of intermittent dorsal displacement of the soft palate. The electromyographic activity of the thyro-hyoid muscles recorded during incremental exercise tests on a high-speed treadmill was analyzed to measure the mean electrical activity and the median frequency of the power spectrum, thereafter subjected to wavelet decomposition. The affected horses had palatal instability with displacement on repeated exams prior to surgical implantation. Although palatal instability persisted after surgery, only two of these horses displaced the palate after instrumentation. The electromyographic traces from this group of four horses showed, at highest exercise intensity, a decrease in mean electrical activity and median power frequency, with progressive decrease in the contribution of the high frequency wavelets, consistent with development of thyro-hyoid muscle fatigue. The results of this study identified fatigue as the main factor leading to exercise induced palatal instability and DDSP in a group of racehorses. Further studies are required to evaluate the fiber type composition and metabolic characteristics of the thyro-hyoid muscles that could predispose to fatigue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224524PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814218PMC
March 2020

Assessing pathological changes within the nucleus ambiguus of horses with recurrent laryngeal neuropathy: An extreme, length-dependent axonopathy.

Muscle Nerve 2019 12 9;60(6):762-768. Epub 2019 Oct 9.

Comparative Neuromuscular Disease Laboratory, Department of Clinical Sciences and Services, Royal Veterinary College, University of London, London, UK.

Introduction: Equine recurrent laryngeal neuropathy (RLN) is a naturally occurring model of length-dependent axonopathy characterized by asymmetrical degeneration of recurrent laryngeal nerve axons (RLn). Distal RLn degeneration is marked, but it is unclear whether degeneration extends to include cell bodies (consistent with a neuronopathy).

Methods: With examiners blinded to RLN severity, brainstem location, and side, we examined correlations between RLN severity (assessed using left distal RLn myelinated axon count) and histopathological features (including chromatolysis and glial responses) in the nucleus ambiguus cell bodies, and myelinated axon count of the right distal RLn of 16 horses.

Results: RLN severity was not associated with RLn cell body number (P > .05), or degeneration. A positive correlation between the left and right distal RLn myelinated axon counts was identified (R = 0.57, P < .05).

Discussion: We confirm that RLN, a length-dependent distal axonopathy, occurs in the absence of detectable neuronopathy.
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http://dx.doi.org/10.1002/mus.26699DOI Listing
December 2019

Functional electrical stimulation following nerve injury in a large animal model.

Muscle Nerve 2019 06 25;59(6):717-725. Epub 2019 Mar 25.

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

Introduction: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis.

Methods: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined.

Results: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation.

Discussion: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.
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http://dx.doi.org/10.1002/mus.26460DOI Listing
June 2019

Electrode fabrication and interface optimization for imaging of evoked peripheral nervous system activity with electrical impedance tomography (EIT).

J Neural Eng 2019 02 16;16(1):016001. Epub 2018 Nov 16.

Department of Medical Physics and Biomedical Engineering, University College London, Gower Street, London WC1E 6BT, United Kingdom.

Objective: Non-invasive imaging techniques are undoubtedly the ideal methods for continuous monitoring of neural activity. One such method, fast neural electrical impedance tomography (EIT) has been developed over the past decade in order to image neural action potentials with non-penetrating electrode arrays.

Approach: The goal of this study is two-fold. First, we present a detailed fabrication method for silicone-based multiple electrode arrays which can be used for epicortical or neural cuff applications. Secondly, we optimize electrode material coatings in order to achieve the best accuracy in EIT reconstructions.

Main Results: The testing of nanostructured electrode interface materials consisting of platinum, iridium oxide, and PEDOT:pTS in saline tank experiments demonstrated that the PEDOT:pTS coating used in this study leads to more accurate reconstruction dimensions along with reduced phase separation between recording channels. The PEDOT:pTS electrodes were then used in vivo to successfully image and localize the evoked activity of the recurrent laryngeal fascicle from within the cervical vagus nerve.

Significance: These results alongside the simple fabrication method presented here position EIT as an effective method to image neural activity.
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http://dx.doi.org/10.1088/1741-2552/aae868DOI Listing
February 2019

Functional Electrical Stimulation Leads to Increased Volume of the Aged Thyroarytenoid Muscle.

Laryngoscope 2018 12 3;128(12):2852-2857. Epub 2018 Oct 3.

Department of Phoniatrics , Medical University Graz, Graz, Austria.

Objectives/hypothesis: To reverse sarcopenia and increase the volumes of atrophied laryngeal muscles by functional electrical stimulation (FES) using a minimal invasive surgical procedure in an aged ovine model.

Study Design: Prospective animal study.

Methods: A stimulation electrode was placed unilaterally near the terminal adduction branch of the recurrent laryngeal nerve (RLN) adjacent to the right cricothyroid joint. The electrode was connected to an implant located subcutaneously at the neck region. Predesigned training patterns were automatically delivered by a bidirectional radio frequency link using a programming device and were repeated automatically by the implant every other day over 11 weeks in the awake animal. Outcome parameters comprised volumetric measurements based on three-dimensional reconstructions of the entire thyroarytenoid muscle (TAM), as well as gene expression analyses.

Results: We found significant increases of the volumes of the stimulated TAM of 11% and the TAM diameter at the midmembranous parts of the vocal folds of nearly 40%. Based on gene expression, we did not detect a shift of muscle fiber composition.

Conclusions: FES of the terminal branches of the RLN is a secure and effective way to reverse the effects of age-related TAM atrophy and to increase volumes of atrophied muscles.

Level Of Evidence: NA Laryngoscope, 128:2852-2857, 2018.
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http://dx.doi.org/10.1002/lary.27342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586045PMC
December 2018

Anatomy of the vestibulum esophagi and surgical implications during prosthetic laryngoplasty in horses.

Vet Surg 2018 Oct 19;47(7):942-950. Epub 2018 Sep 19.

Equine Clinic of Grosbois, Boissy St Leger, France.

Objective: To describe the anatomy of the entry to the equine esophagus (vestibulum esophagi) and to assess the risk of penetrating its adventitia and/or lumen during laryngoplasty.

Study Design: Ex vivo cadaveric study.

Sample Population: Five isolated equine larynges and 39 equine head and neck specimens.

Methods: The anatomy of the vestibulum esophagi was studied by dissection of 5 cadaver specimens. Then, a bilateral laryngoplasty was performed, including 5 suture placements through the muscular processes, caudal, rostral, and sagittal, with straight and curved needles. Two of the 3 surgeons performing the implantations were unaware of the goals of the study. Suture positions and iatrogenic trauma to the lumen and/or adventitia of the vestibulum esophagi were identified during dissection of the specimens. Risk factors for penetrating the adventitia were evaluated with a multivariate regression model.

Results: The vestibulum esophagi spans between both wings of the thyroid cartilage over the entire width of the larynx, covering the rostral spine (arcuate crest) of the arytenoid cartilages. It is covered by the thyropharyngeus and cricopharyngeus muscles. Masked surgeons were associated with a significantly higher number of adventitia penetrations (72%) compared to the nonmasked surgeon (9%). The lumen of the vestibulum esophagi was penetrated in 4.6% of suture placements and only by the 2 masked surgeons.

Conclusion: Penetration of the adventitia was more common when surgeons were unaware of the anatomical extent of the vestibulum esophagi.

Clinical Significance: Anatomical knowledge of the extent of the vestibulum esophagi reduces the risk of penetrating its lumen or adventitia during suture placement on the muscular process of the arytenoid cartilage.
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http://dx.doi.org/10.1111/vsu.12944DOI Listing
October 2018

Ex vivo biomechanical stability of 5 cricoid-suture constructs for equine laryngoplasty.

Vet Surg 2017 Jul 12;46(5):705-713. Epub 2017 May 12.

August Winkhausstr. 62, Telgte, Germany.

Objective: To determine the biomechanical properties of 5 suture constructs in the equine cricoid under cyclic loading and load to failure testing.

Study Design: Ex vivo study.

Samples: Seventy-five equine cadaver larynges.

Methods: Each larynx was implanted with 1 of 5 cricoid-suture constructs. The standard laryngoplasty, where a suture is passed once through the cricoid, including its caudal edge, was used in 2 constructs: 1 with 5 USP Ethibond (ES) and 1 with 2 mm Fibertape (FS). In the third construct, the 2 mm Fibertape was passed twice through the cricoid including its caudal edge (Double Loop-DL). Constructs 4 and 5 used 2 mm Fibertape in a U-shaped loop passed through the cricoid but excluding its caudal edge. One construct was supported with a metallic button (MB) on the caudo-ventral aspect of the cricoid while the other included only the U-shaped loop (U). Constructs were subjected to cyclic loading and to single cycle to failure. Reduction of the left-to-right arytenoid angle quotient (LRQ), suture migration, and load at failure were compared.

Results: LRQ reduction after cyclic loading was lower in MB and U than ES constructs. During cyclic loading, suture migration was reduced in MB, U, and DL compared to ES constructs. Mean load at failure was lower in FS and U than in ES constructs.

Conclusion: Loss of abduction after equine laryngoplasty may be reduced and pullout forces increased by applying a MB construct in the cricoid cartilage. In vivo testing is required to verify these results.
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http://dx.doi.org/10.1111/vsu.12671DOI Listing
July 2017

Reversing Age Related Changes of the Laryngeal Muscles by Chronic Electrostimulation of the Recurrent Laryngeal Nerve.

PLoS One 2016 28;11(11):e0167367. Epub 2016 Nov 28.

Department of Phoniatrics, ENT University Hospital, Medical University of Graz, Graz, Austria.

Age related atrophy of the laryngeal muscles -mainly the thyroarytenoid muscle (TAM)- leads to a glottal gap and consequently to a hoarse and dysphonic voice that significantly affects quality of life. The aim of our study was to reverse this atrophy by inducing muscular hypertrophy by unilateral functional electrical stimulation (FES) of the recurrent laryngeal nerve (RLN) in a large animal model using aged sheep (n = 5). Suitable stimulation parameters were determined by fatiguing experiments of the thyroarytenoid muscle in an acute trial. For the chronic trial an electrode was placed around the right RLN and stimulation was delivered once daily for 29 days. We chose a very conservative stimulation pattern, total stimulation time was two minutes per day, or 0.14% of total time. Overall, the mean muscle fiber diameter of the stimulated right TAM was significantly larger than the non-stimulated left TAM (30μm±1.1μm vs. 28μm±1.1 μm, p<0.001). There was no significant shift in fiber type distribution as judged by immunohistochemistry. The changes of fiber diameter could not be observed in the posterior cricoarytenoid muscle (PCAM). FES is a possible new treatment option for reversing the effects of age related laryngeal muscle atrophy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167367PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125708PMC
August 2017

Feasibility, repeatability, and safety of ultrasound-guided stimulation of the first cervical nerve at the alar foramen in horses.

Am J Vet Res 2016 Nov;77(11):1245-1251

OBJECTIVE To develop and assess the feasibility, repeatability, and safety of an ultrasound-guided technique to stimulate the first cervical nerve (FCN) at the level of the alar foramen of the atlas of horses. ANIMALS 4 equine cadavers and 6 clinically normal Standardbreds. PROCEDURES In each cadaver, the FCN pathway was determined by dissection, and any anastomosis between the first and second cervical nerves was identified. Subsequently, each of 6 live horses underwent a bilateral ultrasound-guided stimulation of the FCN at the alar foramen 3 times at 3-week intervals. After each procedure, horses were examined daily for 5 days. RESULTS In each cadaver, the FCN passed through the alar foramen; a communicating branch between the FCN and the accessory nerve and anastomoses between the ventral branches of the FCN and second cervical nerve were identified. The anastomoses were located in the upper third of the FCN pathway between the wing of the atlas and the nerve's entry in the omohyoideus muscle. Successful ultrasound-guided electrical stimulation was confirmed by twitching of the ipsilateral omohyoideus muscle in all 6 live horses; this finding was observed bilaterally during each of the 3 experimental sessions. No complications developed at the site of stimulation. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that ultrasound-guided stimulation of the FCN at the alar foramen appears to be a safe and straightforward procedure in horses. The procedure may have potential for use in horses with naturally occurring recurrent laryngeal neuropathy to assess reinnervation after FCN transplantation or nerve-muscle pedicle implantation in the cricoarytenoideus dorsalis muscle.
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http://dx.doi.org/10.2460/ajvr.77.11.1245DOI Listing
November 2016

COMPUTED TOMOGRAPHIC APPEARANCE OF MELANOMAS IN THE EQUINE HEAD: 13 CASES.

Vet Radiol Ultrasound 2016 May 22;57(3):246-52. Epub 2016 Jan 22.

Department of Clinical Sciences and Services, The Royal Veterinary College, Hawkshead Lane, Hatfield, AL9 7TA, UK.

Melanomas are one of the most common neoplasms in the horse and are frequently found in the head region. There is a genetic predisposition in horses with a gray hair coat. Computed tomography (CT) is frequently used in referral practice to evaluate the equine head but there are few reports describing the CT appearance of melanomas in this location. The aim of this retrospective, case series study was to describe characteristics in a group of horses with confirmed disease. Case records from two referral hospitals were reviewed, and 13 horses were identified that had undergone CT of the head, with a diagnosis of melanoma based on cytology, histopathology, or visual assessment of black (melanotic) tissue. A median of 11 melanomas was identified per horse (range 3-60), with a total of 216 masses. Melanomas were found most frequently in the parotid salivary gland, guttural pouches, surrounding the larynx and pharynx and adjacent to the hyoid apparatus. In noncontrast CT images, all melanomas were hyperattenuating (median; 113.5 Hounsfield units (HU), IQR; 26 HU) compared to masseter musculature (median; 69 HU, IQR; 5.5 HU). Fifty-six (25.9%) masses were partially mineralized and 41 (19.4%) included hypoattenuating areas. Histopathological assessment of these melanomas suggested that the hyperattenuation identified was most likely a result of abundant intracytoplasmic melanin pigment. Melanomas of the equine head appeared to have consistent CT features that aided detection of mass lesions and their distribution, although histopathological analysis or visual confirmation should still be obtained for definitive diagnosis.
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http://dx.doi.org/10.1111/vru.12345DOI Listing
May 2016

Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.

Artif Organs 2015 Oct;39(10):876-85

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

Bilateral vocal fold paralysis (BVCP) is a life-threatening condition that follows injury to the Recurrent Laryngeal nerve (RLn) and denervation of the intrinsic laryngeal musculature. Functional electrical stimulation (FES) enables restoration and control of a wide variety of motor functions impaired by lower motor neuron lesions. Here we evaluate the effects of FES on the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle in a large animal model of RLn injury. Ten horses were instrumented with two quadripolar intramuscular electrodes in the left PCA muscle. Following a 12-week denervation period, the PCA was stimulated using a once-daily training session for 8 weeks in seven animals. Three animals were used as unstimulated controls. Denervation produced a significant increase in rheobase (P < 0.001). Electrical stimulation produced a 30% increase in fiber diameter in comparison with the unstimulated control group (33.9 ± 2.6 µm FES+, 23.6 ± 4.2 µm FES-, P = 0.04). A trend toward a decrease in the proportion of type 1 (slow) fibers and an increase in type 2a (fast) fibers was also observed. Despite these changes, improvement in PCA function at rest was not observed. These data suggest that electrical stimulation using a relatively conservative set of stimulation parameters can reverse the muscle fiber atrophy produced by complete denervation while avoiding a shift to a slow (type 1) fiber type.
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http://dx.doi.org/10.1111/aor.12624DOI Listing
October 2015

Ultrasonography detects early laryngeal muscle atrophy in an equine neurectomy model.

Muscle Nerve 2016 Apr;53(4):583-92

Department of Clinical Sciences and Services, Royal Veterinary College, University of London, London, UK.

Introduction: A unilateral neurectomy model was used to study the relationship between histologic and ultrasonographic tissue characteristics during muscle atrophy over time.

Methods: This investigation was an in vivo experimental study in an equine model (n = 28). Mean pixel intensity of ultrasonographic images was measured, a muscle appearance grade was assigned weekly, and muscles were harvested from 4 to 32 weeks. Minimum fiber diameter, fiber density per unit area, percent collagen, percent fat, and fiber type profile were measured from muscle cryosections and correlated with the ultrasonographic parameters.

Results: A significant relationship was identified between collagen content, minimum fiber diameter, and ultrasonographic muscle appearance by as early as 8 weeks. There was no apparent association between fat content of muscle and the ultrasonographic appearance of atrophy before 28 weeks.

Conclusions: Early muscle atrophy before fatty infiltration is detectable with ultrasound. The effect of muscle collagen content on echointensity may be mediated by reduced fiber diameter.
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http://dx.doi.org/10.1002/mus.24785DOI Listing
April 2016

In vitro effect of ventriculocordectomy before laryngoplasty on abduction of the equine arytenoid cartilage.

Vet Surg 2011 Apr 11;40(3):305-10. Epub 2011 Feb 11.

Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, UK.

Objective: To determine whether ventriculocordectomy (VCE) performed before prosthetic laryngoplasty (PL) results in increased rima glottidis size compared with PL alone.

Study Design: Experimental study.

Animals: Equine cadaver larynges (n=13).

Methods: Right arytenoid cartilages were maximally abducted using a standard PL technique. Standard PLs were then performed on the left side and the force required to maximally abduct the left arytenoid cartilage recorded (F(max) ). Photographs were taken of the rima glottidis at zero force and at five equal levels of force up to F(max) . The force applied was released, left VCE performed, and photographs repeated. Arytenoid left:right angle quotients (LRQ) and glottic cross-sectional area ratios (CSAR) were calculated at each force level in each condition (PL and VCE-PL).

Results: Mean LRQ and CSAR for both PL and VCE-PL increased with increasing force, initially rapidly before plateauing at ~50% of F(max) . LRQ and CSAR were significantly greater for VCE-PL than for PL (P<.001). When VCE was performed before PL, 12% less force was required to achieve an LRQ of 0.8, and 45% less for a CSAR of 0.8.

Conclusions: In vitro, VCE performed before PL enables the arytenoid cartilage to be abducted to a greater degree for a given PL suture force.
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http://dx.doi.org/10.1111/j.1532-950X.2011.00796.xDOI Listing
April 2011

Three-dimensional biomechanics of simulated laryngeal abduction in horses.

Am J Vet Res 2010 Sep;71(9):1003-10

Department of Veterinary Clinical Sciences, Royal Veterinary College, North Mymms, Hatfield, Hertfordshire AL9 7TA, England.

Objective: To investigate the influence of simulated contraction of the cricoarytenoideus dorsalis (CAD) muscle on the 3-D motion of the arytenoid cartilage.

Sample Population: 5 larynges from equine cadavers.

Procedures: Serial computed tomographic scans of each larynx were conducted at 7 incremental forces simulating contraction of medial, lateral, and combined bellies of the left CAD muscle. Three-dimensional reconstruction of radiopaque markers placed at anatomic landmarks on the left arytenoid and cricoid cartilages enabled quantification of marker displacement according to a Cartesian coordinate system. Rotation (roll, pitch, and yaw) of dorsal and ventral arytenoid planes was calculated relative to a plane formed by the coordinates of 3 markers on the cricoid cartilage by use of Euler angles.

Results: Displacement and rotational data showed that rocking motion occurs throughout arytenoid abduction and most of the rotational component is attributable to pitch; greater pitch was associated with action of the lateral belly. Roll of the ventral arytenoid plane was principally associated with action of the medial belly, which counteracted the tendency of the arytenoid cartilage to rotate medially into the rima glottidis lumen. The distance between markers on the arytenoid cartilage was not constant during contraction because of slight deformation of the corniculate process of the arytenoid cartilage, therefore indicating that the arytenoid cartilage is not a rigid body during abduction.

Conclusions And Clinical Relevance: Arytenoid cartilage abduction was dependent on the rocking motion elicited by the lateral belly of the CAD muscle; therefore, laryngoplasty suture placement should mimic the action of the lateral, rather than the medial, muscle belly.
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http://dx.doi.org/10.2460/ajvr.71.9.1003DOI Listing
September 2010

Full-thickness skin grafting to cover equine wounds caused by laceration or tumor resection.

Vet Surg 2010 Aug 6;39(6):708-14. Epub 2010 May 6.

Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA.

Objective: To describe and evaluate full-thickness skin grafting of equine wounds.

Study Design: Case series.

Animals: Adult horses (n=6).

Methods: A full-thickness graft was harvested from the pectoral region with the horse anesthetized or standing and sedated after local anesthetic infiltration. Grafts were attached to the cutaneous margin of the wound with staples and/or sutures if the horse was anesthetized or if the recipient site was desensitized. Cyanoacrylate glue was used to attach the grafts to the cutaneous margin of the wound of 3 horses. Medical records were reviewed for history, physical examination findings, grafting technique, postoperative complications, and outcome.

Results: Three horses had full-thickness skin grafting to cover a fresh defect created by excision of a cutaneous neoplasm, and 3 horses had full-thickness skin grafting to cover a fresh or granulating laceration. Grafts were completely accepted in 5 horses. The superficial layers of all grafts sloughed, but the final cosmetic appearance of accepted grafts was good.

Conclusions: Full-thickness skin grafting can be performed in standing sedated horses with good cosmesis, especially when the meshed graft is expanded minimally.

Clinical Relevance: Good acceptance of a full-thickness graft can be expected, regardless of whether the graft is applied to a fresh or granulating wound.
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http://dx.doi.org/10.1111/j.1532-950X.2010.00691.xDOI Listing
August 2010

RRD1, a component of the TORC1 signalling pathway, affects anaesthetic response in Saccharomyces cerevisiae.

Yeast 2009 Dec;26(12):655-61

Division of Mathematics and Natural Sciences, Pennsylvania State University Altoona, College, PA 16601, USA.

The molecular mechanisms of action of volatile anaesthetics remain unknown despite clinical use for over 150 years. While many effects of these agents have been characterized, clear insight into how these effects relate to the physiological state of anaesthesia has not been established. Volatile anaesthetics arrest cell division in Saccharomyces cerevisiae in a manner that parallels the anaesthetic actions of these drugs in mammals. To gain additional insight into the cellular activities of these drugs, we isolated genes that, when present on multi-copy plasmids, render S. cerevisiae resistant to the volatile anaesthetic isoflurane. One of these genes, RRD1, encodes a subunit of the Tap42p-Sit4p-Rrd1p phosphatase complex that functions in the target of rapamycin complex 1 (TORC1) signalling pathway. In addition, we show that mutations in two other genes encoding components of the TORC1 pathway, GLN3 and URE2, also affect yeast anaesthetic response. These findings suggest that TORC1-mediated signalling is involved in cellular response to volatile anaesthetics in S. cerevisiae.
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http://dx.doi.org/10.1002/yea.1712DOI Listing
December 2009

Sinoscopic treatment of rostral maxillary and ventral conchal sinusitis in 60 horses.

Vet Surg 2009 Jul;38(5):613-9

Department of Veterinary Clinical Sciences, Royal Veterinary College, North Mymms, UK.

Objective: To evaluate the use of sinoscopy for detection and treatment of ventral conchal sinus (VCS) and/or rostral maxillary sinus (RMS) disease in horses.

Study Design: Case series

Animals: Horses (n=60) with suspected paranasal sinus disease.

Methods: Horses were evaluated by sinoscopy through a conchofrontal sinus (CFS) portal with ventral conchal bulla (VCB) fenestration. Other endoscopic sinus approaches and adjunctive diagnostic tests; oral examination, computed tomography, radiography, scintigraphy and endoscopic examination of the upper portion of the respiratory tract were used in some horses.

Results: The CFS approach permitted adequate observation of the RMS and VCS in 53 horses (88%). Hemorrhage caused by VCB fenestration prevented examination of the RMS and/or VCS in 12 horses (21%). Observation of lesions was possible in all horses diagnosed with neoplasia, sinus cysts, and progressive ethmoidal hematomas. Endoscopy of the paranasal sinuses was useful diagnostically in 82% of horses with primary sinusitis. Other diagnostic modalities were usually required to confirm a diagnosis of dental sinusitis.

Conclusions: Trephination into the CFS with VCB fenestration is a minimally invasive technique that provides consistent access to the RMS and VCS. It facilitates diagnosis of many sinus disorders and endoscopically guided treatment of many horses with sinus cysts and primary sinusitis, in combination with sinus lavage.

Clinical Relevance: Many diseases affecting the RMS and VCS can be diagnosed and resolved endoscopically using a CFS approach with VCB fenestration, thus avoiding the need for osteoplastic sinus surgery and its associated risks and complications.
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http://dx.doi.org/10.1111/j.1532-950X.2009.00556.xDOI Listing
July 2009

Comparison of sinoscopic techniques for examining the rostral maxillary and ventral conchal sinuses of horses.

Vet Surg 2009 Jul;38(5):607-12

Department of Veterinary Clinical Sciences, Royal Veterinary College, Hertfordshire, UK.

Objective: To develop a reliable technique for sinoscopic examination of the ventral conchal (VCS) and rostral maxillary sinuses (RMS) of horses

Study Design: Descriptive study

Animals: Cadaveric equine heads (n=40)

Methods: The VCS and RMS were examined endoscopically using: (1) rostral trephination of the RMS (rostral RMS approach); (2) caudal trephination of the RMS (caudal RMS approach); (3) conchofrontal sinus (CFS) trephination followed by RMS trephination at a site identified by endoscopic transillumination of the maxillary bone at the most rostral aspect of the caudal maxillary sinus (CMS) (light-indicated RMS approach); (4) CFS trephination with fenestration of the ventral conchal bulla (VCB; frontal VCB approach); (5) CMS trephination with VCB fenestration (caudal VCB approach); and (6) CFS and CMS trephination with VCB fenestration (combined VCB approach).

Results: Success in observing the rostral and caudal aspects of the VCS and RMS with each approach were: (1) rostral RMS approach (0 horses; 16 horses [40%]); (2) caudal RMS approach (0 horses; 11 horses [28%]); (3) light-indicated RMS approach (3 horses [8%]; 24 horses [60%]); (4) frontal VCB approach (24 horses [60%]; 29 horses [73%] respectively); (5) caudal VCB approach (16 horses [40%] both structures); and (6) combined VCB approach (27 horses [68%]; 35 horses [88%]).

Conclusions: Trephination into the CFS coupled with fenestration of the VCB provided consistent access to both the rostral and caudal aspects of the RMS and VCS. A trephine hole into the RMS provided poor access to the VCS, and placed the reserve crowns of the maxillary cheek teeth at risk of damage.

Clinical Relevance: The RMS and VCS can be best examined endoscopically using approaches with fenestration of the VCB.
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http://dx.doi.org/10.1111/j.1532-950X.2009.00555.xDOI Listing
July 2009