This study was funded by the following grants: National Key R&#38;D Program of China (2021YFA1101200); Medical Research Project of Wenzhou (Y20170188), National Key R&#38;D Program of China (2016YFC1101200); National Natural Science Foundation of China (81770926;81800842); Key R&#38;D Program of Zhejiang Province (2018C03G2090634); and Key R&#38;D Program of Wenzhou Eye Hospital (YNZD1201902). The sponsor or funding organization had no role in the design or conduct of this research.

Experiments were conducted strictly in accordance with the ARRIVE guidelines and the National Institutes of Health guide for the care and use of Laboratory animals, and adhere to the protocols approved by the Institutional Animal Care and Use Committee in Wenzhou Medical University (WMU) and Joinn Laboratory (Suzhou). The male Saanen goats, aged from 4 to 6 months with weight of 19-23 kg, were housed in the WMU animal facility. The male Rhesus macaques, aged from 5 to 6 years with weight of 5-7 kg, were housed in the Joi...

<ol>
	<li>Benowitz, L., Yin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewiring+the+injured+CNS:+lessons+from+the+optic+nerve.">Rewiring the injured CNS: lessons from the optic nerve.</a> Experimental Neurology. 209 (2), 389-398 (2008).</li><li>Park, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+axon+regeneration+in+the+adult+CNS+by+modulation+of+the+PTEN/mTOR+pathway.">Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.</a> Science. 322 (5903), 963-966 (2008).</li><li>Duan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-specific+regeneration+of+retinal+ganglion+cells+following+axotomy:+effects+of+osteopontin+and+mTOR+signaling.">Subtype-specific regeneration of retinal ganglion cells following axotomy: effects of osteopontin and mTOR signaling.</a> Neuron. 85 (6), 1244-1256 (2015).</li><li>Bei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+visual+function+by+enhancing+conduction+in+regenerated+axons.">Restoration of visual function by enhancing conduction in regenerated axons.</a> Cell. 164 (1-2), 219-232 (2016).</li><li>He, Z., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+control+of+axon+regeneration.">Intrinsic control of axon regeneration.</a> Neuron. 90 (3), 437-451 (2016).</li><li>Yang, S. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+promote+long-distance+optic+nerve+regeneration.">Strategies to promote long-distance optic nerve regeneration.</a> Frontiers in Cellular Neuroscience. 14, 119 (2020).</li><li>Foroozan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+treatments+for+nonarteritic+anterior+ischemic+optic+neuropathy.">New treatments for nonarteritic anterior ischemic optic neuropathy.</a> Neurologic Clinics. 35 (1), 1-15 (2017).</li><li>Singman, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+traumatic+optic+neuropathy.">Indirect traumatic optic neuropathy.</a> Military Medical Research. 3, 2 (2016).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+evaluation+of+retinal+ganglion+cells+and+optic+nerve's+integrity+in+large+animals+by+multi-modality+analysis.">In vivo evaluation of retinal ganglion cells and optic nerve's integrity in large animals by multi-modality analysis.</a> Experimental Eye Research. 197, 108117 (2020).</li><li>Tolbert, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Rhesus+macaque+to+human:+structural+evolutionary+pathways+for+immunoglobulin+G+subclasses.">From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses.</a> mAbs. 11 (4), 709-724 (2019).</li><li>Preuss, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Specializations of the human visual system: the monkey model meets human reality. , 231-259 (2004).</li><li>Friedli, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pronounced+species+divergence+in+corticospinal+tract+reorganization+and+functional+recovery+after+lateralized+spinal+cord+injury+favors+primates.">Pronounced species divergence in corticospinal tract reorganization and functional recovery after lateralized spinal cord injury favors primates.</a> Science Translational Medicine. 7 (302), (2015).</li><li>Porciatti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+assessment+of+retinal+ganglion+cell+function.">Electrophysiological assessment of retinal ganglion cell function.</a> Experimental Eye Research. 141, 164-170 (2015).</li><li>Smith, C. A., Vianna, J. R., Chauhan, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+retinal+ganglion+cell+damage.">Assessing retinal ganglion cell damage.</a> Eye. 31 (2), 209-217 (2017).</li><li>Schuman, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+and+histologic+measurements+of+nerve+fiber+layer+thickness+in+normal+and+glaucomatous+monkey+eyes.">Optical coherence tomography and histologic measurements of nerve fiber layer thickness in normal and glaucomatous monkey eyes.</a> Investigative Ophthalmology &amp; Visual Science. 48 (8), 3645-3654 (2007).</li><li>You, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+reproducibility+of+VEP+recording+in+rats:+electrodes,+stimulus+source+and+peak+analysis.">Improving reproducibility of VEP recording in rats: electrodes, stimulus source and peak analysis.</a> Documenta Ophthalmologica. 123 (2), 109-119 (2011).</li><li>Odom, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISCEV+standard+for+clinical+visual+evoked+potentials:+(2016+update).">ISCEV standard for clinical visual evoked potentials: (2016 update).</a> Documenta Ophthalmologica. 133 (1), 1-9 (2016).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+oil-induced+ocular+hypertension+and+glaucomatous+neurodegeneration+in+mouse.">Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse.</a> eLife. 8, 45881 (2019).</li><li>Seidman, S. H., Telford, L., Paige, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical,+horizontal,+and+torsional+eye+movement+responses+to+head+roll+in+the+squirrel+monkey.">Vertical, horizontal, and torsional eye movement responses to head roll in the squirrel monkey.</a> Experimental Brain Research. 104 (2), 218-226 (1995).</li><li>Porciatti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+pattern+electroretinogram.">The mouse pattern electroretinogram.</a> Documenta Ophthalmologica. 115 (3), 145-153 (2007).</li></ol>

The authors do not have any conflicts of interest to disclose.

In this study, we present a protocol of VEP, PERG, and OCT in goat and rhesus macaque. These in vivo methods can be applied in large animal models of various optic neuropathies, such as glaucoma, ischemic, or traumatic optic neuropathy and optic neuritis9.
PVEP is more stable and sensitive than FVEP17; however, it can't be elicited in goat9. As such, FVEP is performed in goat and PVEP is performed in rhesus macaqu...

The optic nerve (ON), which consists of axons from the retinal ganglion cells (RGC), transmits visual signal from the retina to the brain. ON diseases, such as glaucoma, traumatic or ischemic optic neuropathy, often caused irreversible ON/RGC degeneration and devastating visual loss. Although there are currently many breakthroughs in ON regeneration and RGC protection in rodent models1,2,3,4,5,6, clinical treatments for most of the ON diseases remained essentially the same over the last half century with unsatisfactory outcome7,8. To fill the gap between basic research and clinical practice, translational studies using large animal model of ON diseases are often necessary and beneficial because of their closer anatomical similarity to humans than rodent models.
Goat and rhesus macaques are two large animal species used in our lab to model human&#39;s ON disease. The size of a goat&#39;s eyeball, ON, and the adjacent structure (orbital and nasal cavity, skull base, etc.) is similar to that of a human based on skull CT scan9. As such, goat model provides an opportunity to evaluate and refine therapeutic devices or surgical procedures prior to use in humans. The rhesus macaque, as non-human primate (NHP), has human-like unique visual system that does not exist in other species10,11. In addition, pathophysiological responses to injuries and treatments in NHP is much similar to that in humans12.
In vivo tests to assess the ON and RGC&#39;s structure and function longitudinally are important in large animal studies. Pattern electroretinogram (PERG) has been used to evaluate the RGC function. Flash visual evoked potential (FVEP) reflects the integrity of retino-geniculo-cortical pathway in the visual system. Thus, PERG combined with FVEP can reflect the ON function9,13,14 . The retinal optic coherence tomography (OCT) imaging can show the retinal structure with high temporal and spatial resolution, which enables measurement of the thickness of the retinal ganglion complex (GCC)9,15. For electrophysiological examinations in this study, monitoring vital signs (heat rate, breach rate, blood pressure) and level of oxygen saturation (SpO2) before testing are crucial since these parameters have potent impacts on ocular blood flow and thus the function of the visual system. However, we didn&#39;t monitor the vital signs when carrying out OCT retinal imaging for the sake of simplicity. According to our previous study9, the GCC thickness measured by OCT retinal imaging is quite stable, with inter-session coefficient of variation close to 3%. These in vivo tests in goat and rhesus macaque have been described in detail in our previous study9. Here we present these methods to help increase experimental transparency and reproducibility.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>47.6 x 26.8 cm monitors</td>
			<td>DELL Inc.</td>
			<td>E2216HV</td>
			<td>The visual stimuli of contrast-reversal black-white checkerboards were displayed on screens</td>
		</tr>
		<tr>
			<td>6.0 mm tracheal tube</td>
			<td>Henan Tuoren Medical Device Co., Ltd</td>
			<td>PVC 6.0</td>
			<td>ensure the airway</td>
		</tr>
		<tr>
			<td>alligator clip</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>atropine</td>
			<td>Guangdong Jieyang Longyang Animal pharmaceutical Co.,Ltd.</td>
			<td></td>
			<td>reduce bronchial secretion and protect heart from vagal nerve activation</td>
		</tr>
		<tr>
			<td>Carbomer Eye Gel</td>
			<td>Fabrik GmbH Subsidiary of Bausch &#38; Lomb</td>
			<td></td>
			<td>moisten the cornea and stabilize the recording electrodes</td>
		</tr>
		<tr>
			<td>ERG-Jet recording electrodes</td>
			<td>Roland Consult Stasche&#38;Finger GmbH</td>
			<td>2300 La Chaux-De-Fonds</td>
			<td>ERG recording</td>
		</tr>
		<tr>
			<td>eye speculum</td>
			<td>Shanghai Jinzhong Medical Device Co., Ltd</td>
			<td>ZYD020</td>
			<td>open palpebral fissure</td>
		</tr>
		<tr>
			<td>Heidelberg Spectralis OCT system</td>
			<td>Heidelberg Engineering</td>
			<td></td>
			<td>OCT system</td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td>(https://www.heidelbergengineering.com/media/e-learning/Totara-US/files/pdf-tutorials/2238-003_Spectralis-Training-Guide.pdf)</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>RWD Life Science Co., Ltd</td>
			<td>R510-22</td>
			<td>isoflurane anesthesia</td>
		</tr>
		<tr>
			<td>male Saanen goats</td>
			<td>Caimu Livestock Company, country (Hangzhou, China)</td>
			<td></td>
			<td>The male Saanen goats, aged from 4 to 6 months with weight of 19&#8211;23 kg</td>
		</tr>
		<tr>
			<td>needle electrode</td>
			<td>Roland Consult Stasche&#38;Finger GmbH</td>
			<td>U51-426-G-D</td>
			<td>use for FVEP ground electrode and PERG reference electrodes</td>
		</tr>
		<tr>
			<td>periphery venous catheter intravenously</td>
			<td>BD shanghai Medical Device Co., Ltd</td>
			<td>383019</td>
			<td>intravenous access for atropine and propofol</td>
		</tr>
		<tr>
			<td>propofol</td>
			<td>Xian Lipont Enterprise Union Management Co.,Ltd.</td>
			<td></td>
			<td>induce Isoflurane anesthesia in goat</td>
		</tr>
		<tr>
			<td>Tropicamide Phenylephrine Eye Drops</td>
			<td>SANTEN OY, Japan</td>
			<td></td>
			<td>5% tropicamide and 5% phenylephrine hydrochloride</td>
		</tr>
		<tr>
			<td>visual electrophysiology device</td>
			<td>Gotec Co., Ltd</td>
			<td>GT-2008V-III</td>
			<td>use for FVEP &#38; PERG</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>Huamu Animal Health Products Co., Ltd.</td>
			<td></td>
			<td>xylazine anesthesia: intramuscular injection of xylazine 3mg/kg</td>
		</tr>
		<tr>
			<td>zoletil50</td>
			<td>Virbac</td>
			<td></td>
			<td>induce Isoflurane anesthesia in monkey</td>
		</tr>
	</tbody>
</table>

in vivo methods to assess retinal ganglion cell and optic nerve function and structure in large animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

Figure 1A shows representative results of FVEP in goat. Although the waveforms in same flash intensity have relative similarity, we still recommend to examine the waveforms twice. Electromagnetic waves generated by electronic devices will interfere with the collected electrical signals, resulting in high baseline noise and poor repeatability of the waveform. Therefore, it is recommended to ensure that there are no redundant electronic devices plugged into the surrounding environment during e...

Watch this Scientific Journal Video about In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals at JoVE.com

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

Hi, everyone. This is Yikui Zhang from Wenzhou Eye Hospital. The upper nerve lacks Exxons from the orexin ganglion cells, and then transmit the visual signal to the brain.Larger model of upper nerve injury are essential to translate the novel treatment from robot model to lymph application. Here, we describe some in vivo methods to evaluate the structure and function of the orexin ganglion cells in the upper nerve in large animals. By presenting this method step-by-step, we hope to increase experimental reproductive disabilities and facilitate the usage of larger models of optic neurosis.Shave the with electronic razor. And, prep it with 70%alcohol three times, to clean the skin and expose the subcutaneous vein. Insert a peripheral venous castor intravenously.Then drip atropine and propofol. Intubate the goat with a six millimeter tracheal tube and connect to an artificial respirator. Anesthesia was maintained with 3.5%isoflurane in oxygen at a constant two liter managed flow rate.Place the temperature sensor under the goat's tongue. Clip the oxygen saturation cleave to the proximal end of the goat ear. Tie the blood pressure cuff to the base of the thigh.Clamp ECG cleaves on the limbs in order. Then, we can monitor the goat's vital indicators. Shave the hair and disinfect the skin with betadine and 70%alcohol three times at the center of the frontal bone.Open the sterilizer bag containing scissors and the nails. Make skin incisions to expose the skull with a scissor. Steel screws for implanted at the center of the frontal bone.Shave the hair and disinfect the skin with betadine and 70%alcohol three times at a middle point of two ears in occipital bone. Make skin incisions. Steel screws for implanted at the middle point of two ears in occipital bone.The ground needle electrode is inserted subcutaneously beneath the frontal skull screw. The active electrode and the reference electrode are connected to a frontal and occipital screws respectively with alligator clips to reduce the electrode impotence. Patch one eye.Apply topical anesthesia eye drops to the ocular surface. Pupils on both sides are dilated by mydriasis eye drops. Place the goat's head in a gas-filled stimulator aimed ambulant light.Place the black blanket over the stimulator and the goat head for five minutes adaptation. Eyelid speculum is used to fully expose its pupil. Check the electrode tissue contact impotence.Ensure that impotence is below 10 kilometers for each to avoid electromagnetic interference from other electrical devices in the same room. Check the baseline noise without light stimulation. Start FVEP recording.Note that FVEP recording at each light intensity is performed twice. Note that moist the cornea with artificial tear if it appears dry. Repeat about steps for the contralateral eye.Start FVEP recording at different light intensities consecutively. For each recording, 64 traces are averaged to yield one wave form. The first positive and active peak in the FVEP wave form was designated as P1 and N1.The sterilized ground electrode is positioned in the earlobe.The sterilized active and reference electrodes are inserted subcutaneously along the midline and the frontal and occipital bone respectively. Eyelid speculum is used to fully expose its pupil. Patch one eye.Record pattern VEP responses of the other eye at different spatial frequencies consecutively. Connect to the ground electrode with an alligator clip. Two sterilized needle reference electrodes are placed under the skin one centimeter behind the lateral canthus on both sides.Eyelid speculum is used to fully expose its pupil. Two disinfected ERG recording electrodes are centered both cornea after topical application of artificial tear. Two stimulators are placed in front of each eye with a viewing distance of 50 centimeters.Ensure that impotence is below 10 kilometer. Track the baseline noise without light stimulation. Aim the ambulant light.Start pattern ERG recording from both eyes simultaneously at different spatial frequencies consecutively. Finally, return of the screen to record pattern ERG without visual stimulators and an active control. The bandpass filter is set to range from one to 75 Hertz for three CPD pattern ERG.The bandpass filter is from one to 50 Hertz to smooth the shape without affecting its amplitude. The first positive and active peaks in the wave form are designated as P1 and N1.The pattern ERG amplitude measured from N1 to P1.To reduce electrode impotence, a sterilized needle reference is placed under the frontal skin. And a sterilized needle reference electrode is placed under the skin one centimeter behind the lateral canthus on one side.A disinfected ERG recording electrode is placed on the cornea on a topical application of artificial tear. Patch one eye. A display is placed at viewing distance of 50 centimeters.Eyelid speculum is used to fully expose its pupil. Then, the goat is placed on the chin rest. Align the goat with the camera to send his optic nerve head in the confocal scan laser ophthalmoscopy image by modifying its head position.Click the start button to enter the detection end phase. Wait for the machine to finish loading. Press the yellow start button to start the formal imaging.Adjust the joystick to evenly illuminate the infrared image to improve image quality. Slide the camera forward until the upright retinal OCT image is shown on upper right screen. Modify the joystick to have it evenly dance in a horizontal retinal OCT image.Press the button on the joystick to catch your image automatically. Hold this joystick to maintain image quality on the live image screen until image execution is completed. Then press acquire.Select a high quality initial photo. Right click and select set reference. Press the follow up button to allow program to automatically match the current scan to the baseline scan.Once matched, pressed the button on the joystick to activate automatic real time tracking. Click the measurement button to enter the measurement platform. Choose the eraser a tool and wipe the RFL line, which is automatically labeled by machine.Choose the line drawing tool to manually outline the boundary between IPL and INL. Between the red and blue lines is the GCC complex. Retina altered imaging in rhesus macaque is performed by using the same equipment and procedure as those used in goat.Figure A shows representative FVEP in goat. Figure B shows representative pattern ERG wave form in goat. Figure C shows representative pattern FVEP in rhesus macaque.And, figure D shows representative pattern ERG wave form in rhesus macaque. Figure two shows representative retinal OCT images around optic nerve head and the thickness of GCC complex in different peripheral regions in goat and the rhesus macaque. Large animal model plays an important role in translational research.In many cases, promising therapeutic effects in small animal models failed to be translated to human patients. In this study, we presented video protocol of FVEP, PERG, and OCT in goat and the rhesus macaque. This in vivo method can be applied in larger model of various optic neurosis, such as glaucoma ischemic or traumatic optic neuropathy and optic neuritis.

in-vivo-methods-to-assess-retinal-ganglion-cell-optic-nerve-function

Research

JoVE Journal

Neuroscience

9.7K 조회수.  Wenzhou Medical University. 안녕 여러분. 원저우안과병원 이쿠이 장입니다. 상부 신경은 오렉스신 신경절 세포에서 엑손이 부족하고, 그 후 뇌에 시각적 신호를 전송합니다.상부 신경 상해의 더 큰 모형은 로봇 모형에서 림프 응용으로 새로운 처리를 번역하기 위하여 필수적입니다. 여기서, 우리는 큰 동물에서 상부 신경에서 orexin 신경절 세포의 구조 와 기능을 평가하는 생체 내 방법을 설명합니?...