Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.
This method can help answer key questions about the stem cell-based treatment of peripheral neuropathies, particularly for Equine Recurrent Laryngeal Neuropathy. The main advantages of this technique are that the stem cell harvest is minimally invasive, and that the injection method can also be transferred to other peripheral neuropathies. The implications of this technique extend to other studies of other cell types, as the stem cells can be differentiated into other cells of interest before the injection.
Visual demonstration of this method is critical, as the video endoscopic control of the laryngeal movements and the manipulation of the nerve stimulator techniques can be difficult to learn. Identify the sample site in the long head of the triceps muscle by visual inspection and palpation, about midline between the point of the elbow and the point of the shoulder. Use electric clippers to shave an approximately two by two centimeter area, and apply a surgical scrub consisting of poly-iodide liquid soap and alcohol compresses for one minute to the exposed skin.
Using a 25 gauge needle, subcutaneously inject one milliliter of 2%lidocaine solution into the center of the disinfected area. After disinfecting with clean antibacterial hand soap, and donning sterile gloves, place a semi-automatic 14 gauge biopsy needle on a disposable sterile drape, and pull on the spring mechanism to arm the needle. Assemble a cannula and obturator in the locked position to form a trochar, and introduce the trochar perpendicularly through the desensitized skin, approximately 1.5 centimeters into the underlaying muscle tissue.
Then remove the trochar, and place the obturator on a sterile drape. Introduce the needle through the cannula. Next, introduce the needle and the cannula through the skin incision into the muscle, until the tip of the needle is in the middle of the long head of the triceps muscle.
Then release the biopsy needle, pull the needle and cannula out of the muscle together, and remove the needle from the cannula. Pull the spring with one hand while holding the needle with the other hand, until the specimen becomes visible at the tip of the biopsy needle. With the help of an assistant, open a sample tube containing the sample medium, and use a 19-gauge hypodermic needle to transfer the tiny muscle piece from the biopsy needle into the sampling medium.
Close the tube with the screw cap, and gently tilt the tube two times to ensure that the sample is floating in the medium. Then re-arm the biopsy needle and reintroduce the cannula through the skin incision to collect two to three more samples until approximately 20 milligrams of muscle tissue has been acquired. To inject the cells, after warming the cell suspension to room temperature, aspirate one milliliter of cells into a two milliliter syringe and confirm that the horse is in the head-down position that is indicative of full sedation.
Next, clip a twenty-by-ten centimeter area over the left laryngeal region, and apply a surgical scrub of poly-iodide soap and alcohol to the exposed region. Place a flexible standard video endoscope through the left nare of the horse toward the nasopharynx, adjusting the position of the endoscope until a full view of both the arytenoid cartilages and the epiglottis is obtained. Connect the stimulation injection needle to the negative electrode of the nerve stimulator.
Connect the syringe containing the cell suspension to the injection line, and pre-fill the tube to flush air out of the system. Connect the positive electrode of the nerve stimulator to an electrode patch that will be stuck to the area of exposed skin. Load a syringe with a volume of sterile saline solution equal to the prefilled volume of the tube, and introduce the stimulation injection needle toward the dorsolateral aspect of the larynx, starting the nerve stimulator in the one Hertz stimulation mode, with a current of two milliamps upon approaching the dorsal lateral aspect of the larynx.
Gently move the needle toward the recurrent laryngeal nerve. As soon as the abduction movement of the left arytenoid cartilage is visible, reduce the current, until the movement disappears, while keeping the needle in place. When a loss of motor response at 0.5 milliamps is observed, maintain the needle position, and inject one milliliter of the autologous stem cells, followed by the entire volume of pre-prepared saline.
Maintain the needle in a stable position during this process, as the distance to the nerves is controlled by the currents that produces a motor response and tiny movements of the needle may produce a sudden loss of response. Then remove the needle and endoscope, and allow the horse to recover with monitoring. In previous studies, no adverse reactions have been observed during or after the sampling, nor has any inflammation been observed during or after the stimulation or the injection of the stem cells.
Further, no differences between the pre-injection scores of the laryngeal functions, and the scores obtained on days one, seven, and 28 after the stem cell injection have been measured. While attempting this procedure, it's important to remember that the stem cells collection procedure is noninvasive and allows the culture of a huge number of autologous mesenchymal stem cells in a short amount of time. Following this procedure, other methods, like the injection of pre-differentiated cells, can be performed to answer additional questions about whether Schwann cells are more likely to contribute to the determination of nerves.
After its development, this technique paved the way for researchers in regenerative medicine to explore stem cell use in Equine Recurrent Laryngeal Neuropathy.
