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Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator. Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage. This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.


Introduction
RLN is a common pathology of the upper airway in horses characterized by varying degrees of arytenoid paralysis. The left side of the larynx is most commonly affected. The prevalence of the disease can reach up to 35% in certain horse populations. Although several hypotheses tried to explain the etiology and pathogenesis of this disease, the exact cause of RLN remains uncertain. The pathology is described as a distal axonopathy of the recurrent laryngeal nerve with lesions of demyelination but also some degree of remyelination. This axonopathy leads to the denervation of intrinsic laryngeal muscles and concomitant atrophy 1,2 . This pathology is often slowly progressive and may lead to a total loss of the arytenoid abduction of the affected side 3 .
Affected horses emit abnormal respiratory sounds during exercise and, sometimes, show exercise intolerance in more severe cases. The definitive diagnosis is made by the endoscopic examination on a non-sedated standing horse where a partial or total loss of laryngeal abduction is observed 1,2,4 . Currently, the most common treatment is laryngoplasty (also known as "tie-back"), which is sometimes associated with a ventriculo-cordectomy. Although the overall success rate of these surgeries is considered as good to excellent 5 , post-operative complications are very common. The most common complication is a gradual loss of abduction. Barnett et al. 6 reported a loss of at least one grade of abduction within the first six weeks following the surgery in at least 76% of horses. Other complications, such as prosthesis failure, coughing, and airway contamination, are also reported 5 .
Mesenchymal stem cells (MSCs) have been a part of equine medicine in research and practice for more than a decade, although proven studies on their efficiency are still scarce. The two most commonly exploited sources of adult mesenchymal stem cells in horses are bone marrow and adipose tissue . However, under specific environmental conditions, they can differentiate into nonmesenchymal lineages such as neurons, astrocytes, and myelinating cells of the peripheral nervous system and the spinal cord 9,10 . MSCs have already been used in several neuropathy models 10,11 . Their ability to migrate into areas of degenerated nervous tissue and to regenerate neural cells has been demonstrated after a systemic and local administration 11 . Moreover, Schwann-like cells derived from MSCs can recruit macrophages to remove cellular debris and secrete neurotrophic factors promoting axonal growth and remyelination 9 .
The aim of this study is to describe the technique of a nerve stimulator-guided injection of muscle-derived autologous stem cells near the left recurrent laryngeal nerve in healthy horses. Typically, a nerve stimulator connected to an injection needle is used for the electro-localization of peripheral nerves in order to apply local anesthetics to that area 12 . A weak direct current impulse is supplied in proximity to the nerve to induce a motor response. The ability to produce this motor response depends on several parameters, such as the conductive area of the needle, any impedance of the tissue, the current applied, the pulse duration, and the distance from the needle to the nerve. Nerve stimulator needles are designed to have a very restrictive conductive area at the tip of the needle, while the rest of the needle is insulated. This design helps to precisely localize the nerve. Modern nerve stimulators adapt to varying tissue impedances and deliver the constant amperage set on the machine. Furthermore, most machines use a pulse duration of 0.1 s, so that the determinative parameters are the current applied and the distance to the needle. The relationship between the needle-to-nerve distance and the current necessary to produce a motor response is described by Coulomb's law: E = kQ/r; where E is the required stimulation charge, k is Coulomb's constant, Q is the minimal required stimulation charge, and r is the distance between the two electrodes. In the electro-localization of nerves, the distance between the two electrodes is considered to be the needle-to-nerve distance 12 . Electrical charge dissipates following the rule of the inverse square of the needle-to-nerve distance 13 . Clinical practice has shown that the motor nerve stimulation at 0.5 mA is highly correlated to the successful nerve block and that a loss of motor signal at lower currents will prevent the user from administering accidental intraneuronal injections. The aim of this study is to test the feasibility and safety of this technique in a limited number of horses. If the feasibility and safety of this technique are confirmed, it can be easily transferred to affected horses. Further, equine RLN can serve as a model for peripheral degenerative neuropathy.

Protocol
The commission for the ethical use of animals of the University of Liege approved the study protocol.  NOTE: The specimen becomes visible at the tip of the biopsy needle. 9. With the help of a second person, open the sample tube that contains the sample medium. Use a 19 G hypodermic needle to remove the tiny muscle piece from the biopsy needle. Place the muscle sample in the sampling medium. Close the tube with the screw cap and gently tilt it 2x to ensure that the sample is floating in the medium. 10. Arm the biopsy needle. Take the cannula from the sterile surface and reassemble the armed biopsy needle and cannula. Introduce the armed needle and the cannula through the skin incision as previously described. Repeat the sample procedure 2-3x until approximately 20 mg of muscle tissue is sampled. Close the sampling tube. Gently turn the tube 2x to mix the muscle pieces with the sampling medium. 11. Ship the samples to the laboratory at a constant temperature between 4 to 8 °C.

Muscle Microbiopsy
NOTE: The laboratory will then process the sample. The protocol can be paused here. 12. The procedure is well tolerated and soreness at the sampling side is not observed. In the unlikely event of muscle soreness observed at the sampling site, administer appropriate analgesic treatment.

Treatment of the Cells
NOTE: The stem cells used in this study have been prepared according to the method described by Ceusters et al. 7 . Their article also describes the characterization of the cells.
1. Start by preparing the specific culture medium, DF20, by adding 20% of heat-inactivated fetal bovine serum, 5 mL of penicillin (1,000 IU/mL)streptomycin (10,000 µg/mL), and 2.5 mL of amphotericin B (250 µg/mL) to a bottle of 500 mL of a commercially available culture medium with glutamine and phenol red.

Injection of the Cells
NOTE: Two people are required to perform the injection of the stem cells.
1. Warm the cell suspension progressively to room temperature. Aspirate the suspension into a 2 mL syringe. 2. Sedate the horse by injecting detomidine 10 µg/kg into the jugular vein with a 19 G hypodermic needle. Wait for 5 min to see the full effect of the sedation, which becomes visible by the characteristic head-down position of the horse.

Representative Results
The commission for the ethical use of animals of the University of Liege approved the study protocol. Six horses from the herd of research horses at the Mont-le-Soie Equine Research Centre were included in this study. In the first horse, the recurrent laryngeal nerve was localized by electrostimulation. Figure 2 shows the setting used, with the stimulation needle inserted into the left dorsolateral aspect of the larynx of the horse in the nerve stimulator. When the loss of movement of the arytenoid cartilage is observed at 0.5 mA, 1 mL of 2% lidocaine is injected. This resulted in an absence of movement at higher currents and a transitory paralysis of the left arytenoid. The control endoscopy, 24 h later, revealed a complete recovery of the arytenoid function. The full protocol was successfully repeated in five horses. None of the horses showed a negative reaction to the muscle microbiopsy. In all horses, a sufficient number of cells were grown in the set time. The upper airway endoscopy was performed in all five horses and the laryngeal function scores of I or II.1 according to Robinson et al. 14 were determined. All horses tolerated the nerve stimulation of the recurrent laryngeal nerve very well. No adverse reaction was observed during or after the stimulation or the injection of the stem cells.
All endoscopies were recorded and scored by two blinded clinicians. There was no difference between the pre-injection scores of the laryngeal functions and the scores obtained on day 1, 7, and 28 after the stem cell injection, as tested by Wilcoxon rank analysis 15 . Table 1 summarizes the laryngeal scores of all horses at different time points, as well as the time from the insertion of the needle to the injection of the stem cells and the current that provoked the arytenoid movement when the cells where injected.
The cells that have been used in the present study were prepared according to a protocol published previously 7 . The cells from all the fractions were able to trilineage differentiation, expressed CD90 and CD44, did not express CD45 and MHC II, and had clonogenic capacities. The cells of the 15-25% fraction have been chosen for further processing because they showed the greatest expression of CD90 and the biggest proliferative capacities.
The aim of the present study was not to test the efficiency of the stem cell application to regenerate a damaged peripheral nerve but only to test the feasibility of the procedure and to assess the safety of the stem cell injection to the recurrent laryngeal nerve in a small number of healthy horses. An absence of any functional changes on the endoscopic imaging in the five horses up to 28 days after the injection and an absence of any clinical signs in four of the five horses up to 1 year after the injection confirms the feasibility and the safety of the procedure. One horse had to be euthanized in the follow-up period for reasons unrelated to the study. Although the techniques prove to be safe in a small number of horses, the number of horses included in the present study is too low to demonstrate an absence of rare events.  Table 1: Signalment and laryngeal function scores of the horses. This table shows the time of injection, the lowest current provoking any arytenoid movement before the injection, and the laryngeal scores of five healthy horses before and at day 1, 7, and 28 after the injection of autologous muscle-derived mesenchymal stem cells in proximity to the left laryngeal recurrent nerve. Horse #5 was not available for the control at day 28 after the injection for reasons unrelated to this study. The scores refer to the scores described by Robinson et al. 14 .

Score before injection
Here, score I means that the movements of both arytenoid cartilages were synchronous and symmetrical, and a complete abduction of both arytenoid cartilages could be obtained and maintained. Score II.1 means that the movements of the arytenoid cartilages were asynchronous or may be asymmetric at certain times; however, a complete abduction of both arytenoid cartilages could be obtained and maintained.

Discussion
This protocol describes the successful application of muscle-derived autologous stem cells to the recurrent laryngeal nerves in horses using a nerve stimulator-guided approach. The harvest of the muscle microbiopsy specimen, as well as the isolation, culture, and characterization of the stem cells, have been described in detail before, while the injection of these cells in proximity to a peripheral nerve is original. The electrolocalization of the nerve with an electrical nerve stimulator has served for the present study and was used to inject mesenchymal stem cells in direct proximity to the recurrent laryngeal nerve. The motor response was monitored by video endoscopy through the nasopharynx. If, during the positioning process of the needle, other nerves were stimulated, the corresponding movement was visible on the endoscopy screen. A successful stimulation of the recurrent laryngeal nerve was characterized by the typical abduction of the arytenoid cartilage. In one horse, local anesthetic was injected to induce a transient paralysis of the arytenoid muscle. Although the successful implantation of the cells in proximity to the nerve has not been tested specifically in the present study, the electro-localization of the nerve and the injection at a low current proved that the stem cells were applied near the nerve. The proximity of the injectate to the nerve was further demonstrated by a successful motor block induced by an injection of a local anesthetic.
In the remaining five horses, the time from the first stimulation until the successful stem cell injection varied from 3 to 12 min. Horses were slightly sedated during the procedure, which increased their compliance with the electrical stimulation. None of the horses showed adverse reactions at any time, indicating that the duration of the stimulation can be prolonged if necessary to obtain good needle positioning. A steep learning curve is expected to be observed if the operator is using this technique regularly and in a larger number of horses with varying anatomy.
Horses commonly suffer from RLN, which is characterized by varying degrees of paralysis of the left arytenoid cartilage leading to abnormal respiratory sounds and exercise intolerance. A histology of affected nerves reveals typical lesions of peripheral neuropathy 16 . Peripheral neuropathy is a term used to describe various types of peripheral nerve lesions leading to impaired sensations, movements, or organ functions. The causes are highly variable and may include diabetes, nutrient deficiencies, a treatment with specific drugs, a traumatic injury, ischemia, or an infection, or they can be idiopathic. Independent of the cause, peripheral neuropathies share common histopathological signs, such as Wallerian degeneration, distal axonopathy, or segmental demyelination. It has been shown that the administration of mesenchymal stem cells to damaged peripheral nerves may be beneficial for their regeneration; however, the underlying mechanisms are not well understood. Traditionally, we believe that mesenchymal stem cells will only differentiate into progenitors of adipose, muscle, cartilage, and bone tissue, but under specific conditions, they have been shown to differentiate into myocytes 17 , astrocytes 18 , and myelinating cells of the peripheral 19 and central nervous system 19 . During an in vitro culture, an exposure to neuropeptides will favor the differentiation of mesenchymal stem cells into cells expressing neuronal markers 21,22 . Several in vivo studies have demonstrated the beneficial effect of mesenchymal stem cells on nerve regeneration and a functional recovery in rat models of sciatic nerve injury 10,23 . This is probably caused by favorable environmental conditions that contribute to the differentiation of stem cells into tissue-specific cells 24 . Future studies should also investigate this technique for the administration of predifferentiated cells in RLN-affected horses.
To the best of our knowledge, this is the first report that describes the use of a nerve stimulator to localize a peripheral nerve in order to inject a specific treatment into it. Other peripheral nerve pathologies in various species, such as neuropathic pain, can be treated by the administration of stem cells in proximity to the nerve 25,26 . Future studies should test the effect of this technique in clinical patients and investigate the risk of migration and the potential of differentiation of the injected cells.

Disclosures
The authors have nothing to disclose.