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
<ol>
	<li>Preparation of injectable fluorescein solution. 
	NOTE: Fluorescein is very light-sensitive. Protect from light and use it shortly after preparation.
	<ol>
		<li>Dilute fluorescein to a concentration of 1% in sterile saline.</li>
	</ol>
	</li>
	<li>Preparation of Ketamine/Xylazine
	<ol>
		<li>Dilute Ketamine and Xylazine in sterile saline accordingly for the following concentration: Ketamine (80-100 mg/kg) and Xylazine (5-10 mg/kg).</li>
	</ol>
	</li>
	<li>Sterile saline
	<ol>
		<li>Prepare a 5 mL syringe with a 26 G needle with sterile saline.</li>
	</ol>
	</li>
</ol>
2. OCT and fluorescein imaging
<ol>
	<li>Turn the retinal imaging microscope lightbox, the OCT machine, and the heated mouse platform ON.</li>
	<li>Turn the computer ON and open the imaging program.</li>
	<li>Add one drop of phenylephrine and tropicamide to each eye.</li>
	<li>Inject 150 &#181;L of anesthesia (Ketamine (80-100 mg/kg) and Xylazine (5-10 mg/kg)) intraperitoneally (IP). Determine the depth of anesthesia by toe pinch and wait until the animal is unresponsive. Apply ophthalmic ointment or artificial tears to both the eyes.</li>
	<li>Accommodate the mouse on the platform.</li>
	<li>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.</li>
	<li>Open the imaging and OCT software. In the OCT program, adjust nudge to 5.</li>
	<li>Take an OCT image at 75 &#181;m distal from the burn. Repeat for the other three quadrants of the retina.</li>
	<li>Inject 100 &#181;L of 1% fluorescein IP.</li>
	<li>Switch the camera to a 488 nm filter. Increase the camera gain to 5.</li>
	<li>Take a picture of the fundus at exactly 5 min after fluorescein injection. 
	&#8203;NOTE: Avoid prolonged exposure of the eye to the camera light at maximum setting, as fluorescein can exacerbate retinal photodamage. Keep light source off until the 5 min wait time has elapsed and the mouse is ready for imaging.</li>
</ol>
3. Aftercare
<ol>
	<li>Inject 1 mL of sterile saline IP. Apply lubricant eye drops to both the eyes. Apply ophthalmic ointment or artificial tears to both the eyes.</li>
	<li>Observe the mouse as it recovers from anesthesia. Return to the cage with other animals only when fully recovered, generally after around 40 min.</li>
</ol>
4. Assessment for exclusion criteria
<ol>
	<li>Open the fundus image taken at 24 h post-procedure to assess for exclusion criteria. Exclude the eye if any of the following criteria are identified.</li>
	<li>Assess whether the image has zero occlusions
	<ol>
		<li>Evaluate the image for the number of occluded vessels. 
		NOTE: A successful occlusion usually has some purple pigmentation on or around the burn, very thin or discontinuous vessel through the burn, faint or non-existent vessel appearance outside the burn area, and retinal discoloration from hypoxia. If the entire vessel can be seen through the white burn by the laser, the vessel failed to occlude. Sometimes the vessel will appear partially obstructed, but if it looks uninterrupted outside the burn, the vessel likely didn&#39;t occlude.</li>
		<li>For ambiguous cases, use FA imaging at the same time point to evaluate occlusions. In these images, an occlusion will appear as a break in the continuity of a vessel, often with a tapering of the surrounding vessel.</li>
		<li>If zero occlusions are identified, exclude the eye from analysis, as the RVO is considered ineffective. 
		NOTE: Occlusions typically resolve by 48-72 h post-RVO, and the presence of occlusions should no longer be used as an exclusion criterion at these time points.</li>
	</ol>
	</li>
	<li>Assess the fundus and OCT images for excessive retinal detachment 
	NOTE: Subretinal fluid accumulation is common after induction of RVO, and causes separation of neural retina from RPE. Exclusionary criteria for excessive retinal detachment are defined as follows: OCT will either be completely unviewable, or some layers will appear incredibly distorted. Image quality is poor, with a loss of resolution of outer plexiform and RPE layers. The separation between the neural retina and the choroid is greater than what the OCT field of view allows. On the fundus image, the retina tone will be nearly completely white, with some purple blotching. Part of the retina may appear distorted and out of focus. This is because it has detached and is at a different focal distance than the rest of the retina.
	<ol>
		<li>If the assessment of the images from an eye determines peripheral or complete detachment of the retina, exclude the eye from the analysis.</li>
	</ol>
	</li>
	<li>Exclude images with evidence of corneal cataract 
	NOTE: A corneal cataract appears as an opaque white dot on the mouse&#39;s cornea. Cataracts typically occur due to insufficient lubrication of the eyes while the animal is anesthetized and can be largely avoided by taking care to apply eye ointment generously. Cataracts can generally be identified before imaging by inspecting the animal. Mice that have developed cataracts should be excluded from the dataset without needing to undergo the imaging process. In imaging, cataracts will obscure the retina from the camera, and the OCT will appear warped.</li>
	<li>Assess the image for excessive hemorrhage 
	&#8203;NOTE: Excessive hemorrhage can be identified as amounts of red fluid in the image, usually obscuring retinal background, vessel, and burn. These areas of red fluid will be a brighter, opaquer red than the purple splotches that are normal in successful RVO. Hemorrhages show up at the ganglion cell layer on OCT imaging and interfere with the ability to visualize other retinal layers beneath the hemorrhage.
	<ol>
		<li>If the image is determined to have an excessive hemorrhage, exclude the eye from the analysis.</li>
	</ol>
	</li>
</ol>
5. Fluorescein image processing
<ol>
	<li>Open the fluorescein image in the image processing software.</li>
	<li>Duplicate the image</li>
	<li>Using a selection tool, carefully trace the major vessels.
	<ol>
		<li>The major vessels are the thicker veins and arteries radiating out from the optic disc. Ignore any vessels branching out from these vessels.</li>
		<li>If leakage prevents the outline of the vessel from being seen near the occlusion site, trace through the leakage in the approximate location of the vessel (maintain thickness, connect the last visible point to the next visible point).</li>
	</ol>
	</li>
	<li>In the first image, delete the selection, leaving only the background. Save this masked image.</li>
	<li>Move the selection to the second image, invert the selection and delete, isolating the vessels. Save this masked image.</li>
	<li>Open the two images in ImageJ. Open the background image and measure the integrated density.</li>
	<li>Open the vessel&#39;s image, select the outline of the vessels, and then measure the mean intensity.</li>
	<li>Divide the integrated density of the background by the mean intensity of the vessels, generating the leakage ratio for the eye.</li>
	<li>Record this leakage ratio for each eye in an experimental cohort.</li>
	<li>To further control for background, normalize experimental eyes to the mean leakage ratio of uninjured control eyes. 
	&#8203;NOTE: In order to create a standardized quantification of fluorescein leakage in the FA image, this calculation uses a ratio of the background density (where the leakage will be present) with the brightness of the major vessels to create results that control for the variation in brightness from image to image and can be reliably quantified. Eyes that are undamaged have no leakage and should theoretically have ratios of zero. The ratios calculated from these undamaged control eyes, therefore, represent background noise, and this value is used to further normalize experimental values.</li>
</ol>
6. Retinal layer thickness
<ol>
	<li>Open the OCT image in the image processing software.</li>
	<li>Trace the borders of the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, photoreceptor layer, and RPE layer. Measure the mean thickness of each layer.</li>
	<li>Repeat for OCT images from the other three quadrants of the retina. Average the mean layer thicknesses across the four quadrants to obtain the mean thickness of each retinal layer for the eye.</li>
	<li>Repeat for each eye in the experimental cohort.</li>
</ol>
7. Disorganization of retinal inner layers (DRIL)
<ol>
	<li>Open the OCT image in ImageJ.</li>
	<li>Using the line tool, measure the distance where the upper border of the outer plexiform layer is indistinct. 
	NOTE: It is important to differentiate between DRIL and areas of poor layer visibility caused by imaging artifacts. Poor OCT image quality may invalidate an eye for DRIL analysis if sufficient image resolution is not possible. Images with DRIL will typically have other regions or retinal layers that are clearly resolved and organized, which can be a good indicator of sufficient image quality.
	<ol>
		<li>Measure horizontally from the latitude where the disorganization begins to the latitude where the upper border of the outer plexiform layer becomes visible again, if at all. Even if the outer plexiform layer shifts upward or downward vertically, measure perfectly horizontally.</li>
		<li>There may be multiple areas of disorganization separated by areas with no disorganization. Measure these individually and calculate the sum of the distances.</li>
	</ol>
	</li>
	<li>Divide the length of disorganization by the total length of the retina visible in each OCT image to obtain the ratio of disorganization for the image.</li>
	<li>Repeat the measurement and calculation for OCT images from the other three quadrants of the retina.</li>
	<li>Take the mean of the ratios of disorganization from the four OCT images. This number represents the average disorganization for the whole retina. Repeat for each eye in the experimental cohort.</li>
</ol>

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The authors declare that they have no competing financial interests.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AK-Fluor 10%</td>
			<td>Akorn</td>
			<td>NDC: 17478-253-10</td>
			<td>light-sensitive</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>NADA #141-199</td>
			<td>keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>GenTeal</td>
			<td>Alcon</td>
			<td>00658 06401</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>InSight 2D</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>OCT analysis software</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Akorn</td>
			<td>NDCL174478-201-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoenix Micron IV</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>Retinal imaging microscope</td>
		</tr>
		<tr>
			<td>Phoenix Micron Meridian Module</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>Laser photocoagulator software</td>
		</tr>
		<tr>
			<td>Phoenix Micron Optical Coherence Tomography Module</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>OCT imaging software</td>
		</tr>
		<tr>
			<td>Phoenix Micron StreamPix Module</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td>Fundus imaging and acquisition targeting</td>
		</tr>
		<tr>
			<td>Photoshop</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refresh</td>
			<td>Allergan</td>
			<td>94170</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide</td>
			<td>Akorn</td>
			<td>NDC: 174478-102-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDCL 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

in-vivo-vascular-injury-readouts-mouse-retina-to-promote

Research

JoVE Journal

Neuroscience

2.1K Views.  Columbia University. 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).

Video: In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

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 Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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 ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

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Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

In vivo Laser Axotomy in C. elegans

In vivo Electroporation of Developing Mouse Retina

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

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

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

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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

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