Name: in vivo methods to assess retinal ganglion cell and optic nerve function and structure in large animals
Uploaded: 2022-02-26T13:00:00
Description: 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

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...

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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 ...

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