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.

<ol>
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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. 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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><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 &amp;amp; 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&amp;amp;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&amp;ndash;23 kg</td></tr><tr><td>needle electrode</td><td>Roland Consult Stasche&amp;amp;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 &amp;amp; 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 ...

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

Research

JoVE Journal

Neuroscience

9.8K Views.  Wenzhou Medical University. Hallo allemaal. Dit is Yikui Zhang van het Wenzhou Eye Hospital. De bovenste zenuw mist Exxons van de orexine ganglioncellen en stuurt vervolgens het visuele signaal naar de hersenen.Een groter model van bovenste zenuwletsel is essentieel om de nieuwe behandeling van robotmodel naar lymfetoepassing te vertalen. Hier beschrijven we enkele in vivo methoden om de structuur en functie van de orexine ganglioncellen in de bovenste zenuw bij grote dieren te evalueren. Door deze methode stap v...

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Medicine

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

A New Optokinetic Testing Method to Measure Rat Vision

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits&#160;movement so that the&#160;rat&#39;s head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat&#39;s visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental rats.

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

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

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