Name: ethanol-induced cervical sympathetic ganglion block applications for promoting canine inferior alveolar nerve regeneration using an artificial nerve
Uploaded: 2018-11-30T13:00:00
Description: Watch this Scientific Journal Video about Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve at JoVE.c

This work was supported by the Department of Bioartificial Organs in Kyoto University Institute for Frontier Medical Science. We would like to thank the veterinary staff of the Institute for Frontier Medical Science.

This study was conducted in accordance with the Guiding Principles for the Care and Use of Animals and approved by the Committee for Animal Research of Kyoto University (Kyoto, Japan; authorization number: R-16-16). All efforts were made to minimize animal suffering, and all sections of this report adhere to the ARRIVE (Animal Research: Reporting of in Vivo Experiments) guidelines.
1. Fabrication of the PGA-C tube
<ol>
	<li>To fabricate the artificial nerve conduit by means of an ab...

<ol>
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We present an efficient method for IAN regeneration by using a bioabsorbable nerve tube in combination with ethanol-induced CSGB. For this study we used dogs, since other animal models, like mice, rats, and rabbits, have a short life expectancy and small body size, and hence cannot be used to perform the precise surgical procedures. As the IAN is located within the mandibular canal surrounded by bone, a surgical technique is necessary to avoid nerve and blood vessel damage when performing nerve reconstruction. An importa...

In many cases, traumatic injury of the inferior alveolar nerve (IAN) is iatrogenic, being frequently caused by the extraction of the third molar or the placement of dental implants1,2,3. Injury of the IAN can lead to deficits in thermal and touch sensations as well as paresthesia, dysesthesia, hypoesthesia, and allodynia. Nerve injury is treated not only by conservative therapy but also by other methods, including suturing and autograft placement. However, these methods have drawbacks, which often include the lack of symptom improvement and neurological defects at the donor site4,5,6.
The artificial nerve &#8212; polyglycolic acid-collagen (PGA-C) tube was originally developed in Japan. It is a bio-absorbable tube with its inner lumen filled with a spongiform collagen7. In animal experiments, this tube was used to enhance nerve regeneration in beagle dogs with peroneal nerve defect, and was shown to promote higher level of recovery than autologous nerve transplantation8. The clinical application of the PGA-C tube began in 2002 in patients with peripheral nerve injuries. Moreover, favorable clinical outcomes have been achieved in the treatment of trigeminal neuropathy (IAN and lingual nerve)9,10,11. A critical factor for successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue8. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region and increases blood flow to the respective innervated area12; thus, it has been used in the treatment of complex regional pain syndrome and circulatory insufficiency13,14,15. However, there have been only a few experimental investigations on the efficacy of CSGB in increasing blood flow16,17. To ensure adequate CSGB efficiency, the blockade must be applied together with local anesthetics once or twice daily for several weeks, thus posing a challenge when generating animal models to investigate this technique. To address this limitation, in a previous study, we developed a canine model of long-term increased blood flow in the orofacial region18. The model was generated by performing a CSGB by injecting 99.5% ethanol. We evaluated the oral mucosal blood flow and nasal skin temperature by laser Doppler flowmetry and infrared thermography once per week for 12 weeks. We found that the blood flow of the orofacial region was increased for 7 &#8211; 10 weeks in this model.
In the present study, we evaluated the effects of ethanol-induced CSGB on nerve regeneration. 
The PGA-C tube was implanted into beagle dogs across a 10-mm gap in the left IAN. A week later, CSGB was performed by injecting ethanol. Three months after surgery, we performed a variety of electrophysiological, histological, and morphological studies to evaluate the effects of CSGB on nerve regeneration. We provide a detailed protocol for IAN reconstruction using a PGA-C tube and ethanol-induced CSGB.

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			<td style="width:228px;">Nippon Meatpackers</td>
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			<td height="21" style="height:21px;width:396px;">Disposable scalpel (No.15)</td>
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			<td style="width:224px;">219ABBZX00073000</td>
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			<td style="width:228px;">Nakanishi</td>
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			<td height="21" style="height:21px;width:396px;">8-0 nylon sutures</td>
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			<td style="width:224px;">620005641</td>
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			<td height="21" style="height:21px;width:396px;">Disposable scalpel (No.10)</td>
			<td style="width:228px;">Kai medical&#160;</td>
			<td style="width:224px;">219ABBZX00073000</td>
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			<td height="21" style="height:21px;width:396px;">30-gauge needle&#160;</td>
			<td style="width:228px;">Nipro</td>
			<td>1134</td>
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			<td height="21" style="height:21px;width:396px;">1-0 absorbable stitches</td>
			<td style="width:228px;">Ethicon</td>
			<td style="width:224px;">J347H</td>
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			<td height="21" style="height:21px;width:396px;">3-0 Nylon stitches</td>
			<td style="width:228px;">Ethicon</td>
			<td style="width:224px;">8872H</td>
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			<td height="21" style="height:21px;width:396px;">Neo Thermo</td>
			<td style="width:228px;">NEC Avio</td>
			<td style="width:224px;">TVS-700</td>
			<td>Infrared thermography&#160;</td>
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			<td style="width:228px;">NIHON KOHDEN</td>
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			<td style="width:224px;">T3260-5G</td>
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			<td height="21" style="height:21px;width:396px;">Light microscope</td>
			<td style="width:228px;">Keyence</td>
			<td style="width:224px;">BZ-9000</td>
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			<td style="width:228px;">Hitachi High Technologies</td>
			<td style="width:224px;">Hitachi H-7000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Dynamic cell count</td>
			<td style="width:228px;">Keyence</td>
			<td style="width:224px;">BZ-H1C</td>
			<td>Software for morphological evaluation</td>
		</tr>
	</tbody>
</table>

ethanol-induced cervical sympathetic ganglion block applications for promoting canine inferior alveolar nerve regeneration using an artificial nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We observed an increase in the facial skin temperature of the blocked side 1 week after the left CSGB (Figure 8).
At 3 months post-reconstruction, the PGA-C tube at the reconstruction area was absorbed and regeneration of the inferior alveolar nerve was observed in the reconstruction-only and reconstruction + CSGB groups (Figure 9A, B)...

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Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

This method can help answer key questions about nerve regeneration in the artificial organ field using an artificial nerve conduit. The main advantage of this technique is that the piezoelectric surgery can be used for inferior alveolar nerve reconstruction to avoid nerve and blood vessel damage. The implications of this technique extend towards treatment of orofacial neuropathy, as it has the potential to promote nerve regeneration.After confirming anesthesia by a lack of response to pedal reflex, apply gel over the anterior surface of the eyes to avoid corneal abrasion. Place the dog in the right lateral position. Use a 27-gauge needle to inject three milliliters of 1%lidocaine into the left mandibular gingiva.Next, use a number 15 scalpel blade to make a five-centimeter transverse incision in the left mandibular gingiva to expose the mandibles. Use piezoelectric ultrasonic vibrations at a 28-to 32-kilohertz frequency to grind the proximal aspect of the mandible into a three-centimeter by eight-millimeter rectangle through the posterior mental foramen. As the inferior alveolar nerve is located within the mandibular canal and surrounded by bone, we use piezoelectric surgery which consists of ultrasonic waves for the bone processing to minimize the risk of nerve and vessel injury.Remove the frontal part of the mandibular bone plate to expose the left inferior alveolar nerve. Use a scalpel to transect the inferior alveolar nerve to allow removal of a 10-millimeter segment. Insert the proximal and distal stumps of the severed nerve into a nerve tube to a depth of two millimeters.Use 8-0 nylon sutures and a surgical microscope at an 8X magnification to suture the tube to the proximal and distal nerve ends. Then, return the bone plate to its original site in the mandible, and close the wound with 4-0 nylon sutures. One day after surgery, perform computed tomography imaging of the facial bone under anesthesia to confirm that the mandibular bone plate is in its proper position.One week after the reconstruction procedure, shave the surgical field. Then, draw an incision line on the left side of the surgical area, and use a 21-gauge needle to inject five milliliters of 1%lidocaine into the shaved chest as a local anesthetic and analgesic. Using a number 10 scalpel blade, make an incision over the marked line, and use an electric scalpel to incise the fat layer to expose the muscle fascia.Expose the serratus ventralis and scalenus muscles. Raise the muscles from ventral to dorsal to expose the second and third ribs. Perform a left lateral thoracotomy at the second and third intercostal space to expose the left cervical sympathetic ganglion.Use a 30-gauge needle to inject 2 milliliters of 99.5%ethanol into the cervical sympathetic ganglion. The cervical sympathetic ganglion is degeneratively changed by ethanol. Then, close the intercostal space with interrupted 1-0 absorbable stitches.Close the skin with interrupted 3-0 nylon stitches, and measure the facial skin temperature with infrared thermography one week after the cervical sympathetic ganglion block, or CSGB. At three months post-reconstruction, the polyglycolic acid-collagen tube at the reconstruction area is absorbed, and regeneration of the inferior alveolar nerve is observed in both the reconstruction-only and reconstruction-plus-CSGB groups. The sensory nerve action potential is also measurable in both reconstruction sides of the reconstruction-plus-CSGB and nerve reconstruction groups, with a significantly higher recovery index and sensory nerve conduction velocity observed in the reconstruction-plus-CSGB animals than in the reconstruction-only group.Myelinated nerve fibers are observed at the central and distal segments of the regenerated inferior alveolar nerve in the reconstruction-only and reconstruction-plus-CSGB groups, although both groups demonstrated smaller regenerated myelinated nerve diameters compared to the normal control group. The presence of regenerated axons and Schwann cells is further confirmed at the central and distal segments of reconstruction-plus-CSGB groups by staining with anti-neurofilament and anti-S100 antibodies, respectively. Morphological analysis reveals a significantly higher myelinated nerve fiber density and nerve tissue percentage in both the center and distal segments of the regenerated left inferior alveolar nerve in the CSGB group compared to the reconstruction-alone animals, with a significantly smaller G-ratio observed with the CSGB procedure.Cervical sympathetic ganglion block results in an improved nerve regeneration, suggesting that an increased blood flow effectively promote nerve regeneration. This technique contributes to the development of research in the field of in situ tissue engineering for exploring surgical treatment for the orofacial neuropathy using an artificial nerve.

Research

JoVE Journal

Neuroscience

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

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Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

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Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

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Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

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Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...