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)...

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

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.

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Research

JoVE Journal

Neuroscience

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

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

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

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

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

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

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

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

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 nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

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 faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

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 axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric 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 ...