Name: in vivo vascular injury readouts in mouse retina to promote reproducibility
Uploaded: 2022-04-21T13:50:00
Description: Watch this Scientific Journal Video about In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility at JoVE.com

This work was supported by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) grant DGE - 1644869(to CKCO), the National Eye Institute (NEI) 5T32EY013933 (to AMP), the National Institute of Neurological Disorders and Stroke (RO1 NS081333, R03 NS099920 to CMT), and the Department of Defense Army/Air Force (DURIP to CMT).

This protocol follows the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Rodent experiments were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
NOTE: Imaging was done on 2 month old C57BL/6J male mice that weighed approximately 23 g.
1. Preparation of reagents for retinal imaging
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	<li>Preparation of injec...

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An update of the literature.</a> Retina. 33 (5), 901-910 (2013).</li><li>Ho, M., Liu, D. T. L., Lam, D. S. C., Jonas, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vein+occlusions,+from+basics+to+the+latest+treatment.">Retinal vein occlusions, from basics to the latest treatment.</a> Retina. 36 (3), 432-448 (2016).</li><li>Zhang, H., 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=Development+of+a+new+mouse+model+of+branch+retinal+vein+occlusion+and+retinal+neovascularization.">Development of a new mouse model of branch retinal vein occlusion and retinal neovascularization.</a> Japanese Journal of Ophthalmology. 51 (4), 251-257 (2007).</li><li>Ebneter, A., Agca, C., Dysli, C., Zinkernagel, M. 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(174), e62980 (2021).</li><li>Schmidt-Erfurth, U., 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=Guidelines+for+the+management+of+retinal+vein+occlusion+by+the+European+society+of+retina+specialists+(EURETINA).">Guidelines for the management of retinal vein occlusion by the European society of retina specialists (EURETINA).</a> Ophthalmologica. 242 (3), 123-162 (2019).</li><li>Yoshimura, 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=Comprehensive+analysis+of+inflammatory+immune+mediators+in+vitreoretinal+diseases.">Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases.</a> PLoS One. 4 (12), 8158 (2009).</li><li>Mezu-Ndubuisi, O. 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Retina. 2 (11), 1125-1132 (2018).</li><li>Nicholson, 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=Diagnostic+accuracy+of+disorganization+of+the+retinal+inner+layers+in+detecting+macular+capillary+non-perfusion+in+diabetic+retinopathy.">Diagnostic accuracy of disorganization of the retinal inner layers in detecting macular capillary non-perfusion in diabetic retinopathy.</a> Clinical &amp; Experimental Ophthalmology. 43 (8), 735-741 (2015).</li><li>Obrosova, I., Chung, S., Kador, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+cataracts:+mechanisms+and+management.">Diabetic cataracts: mechanisms and management.</a> Diabetes/Metabolism Research and Reviews. 26 (3), 172-180 (2010).</li><li>Hegde, K., Henein, M., Varma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+mouse+as+a+model+animal+for+the+study+of+diabetic+cataracts.">Establishment of the mouse as a model animal for the study of diabetic cataracts.</a> Ophthalmic Research. 35 (1), 12-18 (2003).</li><li>Takahashi, H., 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=Time+course+of+collateral+vessel+formation+after+retinal+vein+occlusion+visualized+by+OCTA+and+elucidation+of+factors+in+their+formation.">Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation.</a> Heliyon. 7 (1), 05902 (2021).</li><li>Haj Najeeb, B., 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=Fluorescein+angiography+in+diabetic+macular+edema:+A+new+approach+to+its+etiology.">Fluorescein angiography in diabetic macular edema: A new approach to its etiology.</a> Investigation Ophthalmology &amp; Visual Science. 58 (10), 3986-3990 (2017).</li><li>Alam, M., 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=Quantitative+optical+coherence+tomography+angiography+features+for+objective+classification+and+staging+of+diabetic+retinopathy.">Quantitative optical coherence tomography angiography features for objective classification and staging of diabetic retinopathy.</a> Retina. 40 (2), 322-332 (2020).</li><li>Uddin, M., Jayagopal, A., McCollum, G., Yang, R., Penn, J. <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+imaging+of+retinal+hypoxia+using+HYPOX-4-dependent+fluorescence+in+a+mouse+model+of+laser-induced+retinal+vein+occlusion+(RVO).">In vivo imaging of retinal hypoxia using HYPOX-4-dependent fluorescence in a mouse model of laser-induced retinal vein occlusion (RVO).</a> Investigation Ophthalmology &amp; Visual Science. 58 (9), 3818-3824 (2017).</li><li>Qiang, W., Wei, R., Chen, Y., Chen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+pathological+features+and+current+animal+models+of+type+3+macular+neovascularization.">Clinical pathological features and current animal models of type 3 macular neovascularization.</a> Frontiers in Neuroscience. 15, 734860 (2021).</li><li>Park, 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=Imaging+laser-induced+choroidal+neovascularization+in+the+rodent+retina+using+optical+coherence+tomography+angiography.">Imaging laser-induced choroidal neovascularization in the rodent retina using optical coherence tomography angiography.</a> Investigation Ophthalmology &amp; Visual Science. 57 (9), 331 (2016).</li><li>Chen, J., Qian, H., Horai, R., Chan, C., Caspi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+optical+coherence+tomography+and+electroretinography+to+evaluate+retinal+pathology+in+a+mouse+model+of+autoimmune+uveitis.">Use of optical coherence tomography and electroretinography to evaluate retinal pathology in a mouse model of autoimmune uveitis.</a> PLoS One. 8 (5), 63904 (2013).</li></ol>

Noninvasive rodent retinal imaging presents an avenue to study pathology and develop interventions. Previous studies have developed and optimized a mouse model of RVO, limiting variability and allowing for reliable translation of common clinical pathologies in the murine retina5,7,13. Developments in ophthalmic imaging technology further allow for the use of clinical in vivo imaging techniques such as FA and OCT in expe...

Neurovascular disease is a major healthcare problem responsible for ischemic strokes, a leading cause of mortality and morbidity, and retinal vascular diseases that lead to vision loss1,2. To model neurovascular disease, we employ a mouse model of retinal vein occlusion (RVO). This model is noninvasive and utilizes similar in vivo imaging techniques to those used to examine people with retinal vascular disease in a clinical setting. The use of this model thus increases the translational potential of studies utilizing this model. As with all mouse models, it is critical to maximize reproducibility of the model.
Retinal vascular diseases are a major cause of vision loss in people under the age of 70. RVO is the second most common retinal vascular disease after diabetic retinopathy3. Clinical features characteristic of RVO include ischemic injury, retinal edema, and vision loss as a consequence of neuronal loss3,4. Mouse models of RVO using laser photocoagulation of major vessels have been developed and refined to replicate key clinical pathologies observed in human RVO5,6,7. Advancements in ophthalmic imaging also allow for replication of noninvasive diagnostic tools used in humans, namely, fluorescein angiography (FA) and optical coherence tomography (OCT)6. Fluorescein Angiography allows for the observation of leakage due to the breakdown of the blood-retinal barrier (BRB) as well as blood flow dynamics in the retina, including sites of occlusion, using the injection of fluorescein, a small fluorescent dye8,9. OCT imaging allows for the acquisition of high-resolution cross-sectional images of the retina and the study of the thickness and organization of retinal layers10. Analysis of FA images has historically been largely qualitative, which limits the potential for direct and reproducible comparison between studies. Recently, a number of methods have been developed for the quantification of layer thickness in OCT imaging, though there is currently no standardized analysis protocol and the site of OCT image acquisition varies11. In order to properly leverage these tools, standardized, quantitative, and replicable data analysis methodology are needed. In this paper, we present three such vascular readouts used to evaluate pathological damage in a mouse model of RVO-fluorescein leakage, OCT layer thickness, and disorganization of retinal layers.

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			<td>AK-Fluor 10%</td>
			<td>Akorn</td>
			<td>NDC: 17478-253-10</td>
			<td>light-sensitive</td>
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			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>NADA #141-199</td>
			<td>keep at 4 &#176;C</td>
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			<td>Alcon</td>
			<td>00658 06401</td>
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			<td>NIH</td>
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			<td>InSight 2D</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>OCT analysis software</td>
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			<td>Ketamine Hydrochloride</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-0702-1</td>
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			<td>Phenylephrine</td>
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			<td>Phoenix Micron IV</td>
			<td>Phoenix Technology Group</td>
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			<td>Retinal imaging microscope</td>
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			<td>Phoenix Micron Meridian Module</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>Laser photocoagulator software</td>
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			<td>Phoenix Micron Optical Coherence Tomography Module</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>OCT imaging software</td>
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			<td>Phoenix Micron StreamPix Module</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>Fundus imaging and acquisition targeting</td>
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			<td>Photoshop</td>
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			<td>Tropicamide</td>
			<td>Akorn</td>
			<td>NDC: 174478-102-12</td>
			<td></td>
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			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDCL 59399-110-20</td>
			<td></td>
		</tr>
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in vivo vascular injury readouts in mouse retina to promote reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

These analysis methods allow for the quantification of retinal pathology captured by FA and OCT imaging. The experiments from which the representative data is extracted used C57BL/6J male mice who either served as uninjured controls or underwent the RVO procedure and received either Pen1-XBir3 treatment eyedrops or Pen1-Saline vehicle eyedrops. The RVO injury model involved the laser irradiation (532 nm) of the major veins in each eye of an anesthetized mouse following a tail-vein injection of rose bengal, a photoactivat...

Watch this Scientific Journal Video about In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility at JoVE.com

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

This protocol allows the user to quantitatively measure clinically analogous markers of damage in mouse retinal imaging, which bolsters the translatability of subsequent findings. These methods allow us to streamline analysis and give us the ability to reliably compare imaging between experimental animals. While these methods were developed to study a mouse model overview, they can easily extend to research on any retinal diseases that use the same imaging techniques.Turn the retinal imaging microscope light box, the optical coherence tomography machine, and the heated mouse platform on. Turn the computer on and open the imaging program. Add one drop of phenylephrine and tropicamide to each eye.Inject 100 microliters of 1%fluorescein intraperitoneal. Accommodate the mouse on the platform. Adjust the height and angle of the platform until the view of the retinal fundus is clear and focused.Take a picture of the fundus. Open the imaging and optical coherence tomography software. In the optical coherence tomography program adjust nudge to 10.Take an optical coherence tomography image, add 75 micrometers distal from the burn. Repeat for the other three quadrants of the retina. Switch the camera to a 488 nanometer filter.Increase the camera gain to 5. Take a picture of the fundus exactly 5 minutes after fluorescein injection. Open the fluorescein image on the image processing software.Duplicate the image. Using a selection tool, carefully trace the major vessels. Ignore any vessels branching out from these vessels.In the first image, delete the selection, leaving only the vessel. Save this masked image. Move the selection to the second image, invert the selection and delete, isolating the background.Save this masked image. Open the background image and measure the integrated density. Open the vessels image, select the outline of the vessels, and then measure the mean intensity.Divide the integrated density of the background by the mean intensity of the vessels, generating the leakage ratio for the eye. Record this leakage ratio for each eye in an experimental cohort. To further control for background, normalize experimental eyes to the mean leakage ratio of uninjured control eyes.Open the optical coherence tomography image in the image processing software. Trace the borders of the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, photoreceptor layer, and RPE layer. Export the files as CSV.Measure the mean thickness of each layer and repeat for each eye in the experimental cohort. Open the optical coherence tomography image in image J.Using the line tool, measure the distance where the upper border of the outer plexiform layer is indistinct. Measure horizontally, maintaining the latitude where the disorganization begins.Calculate the sum of the disorganized distances in the image. Divide the length of disorganization by the total length of the retina to obtain the ratio of disorganization. Repeat the measurement and calculation for optical coherence tomography images from the other three quadrants of the retina.Take the mean of the ratios of disorganization from the four optical coherence tomography images. This number represents the average disorganization for the whole retina. Repeat for each eye in the experimental cohort.The masked images used for the calculation of ratio of leakage for each retina image can be compared to others and analyzed, separating the major vasculature from other areas of the retina. Fluorescein quantification allows for the comparison of injury severity and treatment efficacy as well as the study of changes in leakage over the injury time course. A delineation of the layers of the retina in an OCT image is observed.The quantification of thickness for each retinal layer shows that the initial edematous response has a more profound effect on the internal retinal layers. From an analysis of a time course of retinal vein occlusion damage, the initial inflammatory swelling of retinal layers, and the eventual degenerative thinning can be observed. The inner nuclear layer experiences a much greater response to the initial injury, but the inner plexiform layer demonstrates more severe thinning after the initial edema has been stabilized and returns to baseline.The disorganization of inner retinal layer manifests as a disappearance of the upper boundary of the outer plexiform layer, blending the outer plexiform and inner nuclear layers together. The retinal disorganization of two experimental groups were compared to investigate the efficacy of an inhibitor in mitigating retinal damage. Image quality is vitally important to analysis quality.When acquiring retinal images, take the time to ensure that the fundus and retinal layers are as clear and focused as possible. These non-invasive methods can be used longitudinally and in conjunction with biochemical and immunohistochemical study of tissue to create more multifaceted and detailed profiles of disease. This technique enables the reliable quantification of in Vivo retinal imaging data in models of neurovascular disease so that data can be more readily translated to human disease.

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Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 ...