We are grateful for Dr. Xinyu Li's help in preparing the manuscript. S.W. was supported by a Startup fund from Tulane University, President's Research Council New Investigator Award from UT Southwestern Medical Center, NIH Grant EY021862, a career development award from the Research to Prevent Blindness foundation, and a Bright Focus Award in Age Related Macular Degeneration Research.

Note: The following protocol and representative results use the HDAC inhibitor SAHA as an example compound. However, this protocol is by no means limited to the use of SAHA, and is recommended as a general method to test the effects of soluble compounds on corneal neovascularization. Minor modifications will need to be made for degree of dilution as well as frequency and duration of application. Additionally, compounds that are easily soluble in water will be able to be administered in the absence of DMSO.
Ethical Statement: All animal experiments should only be performed in compliance with national law and institutional regulations. This protocol was approved for use by Tulane University Institutional Animal Care and Use Committee.&#160;
1. Preparation of Materials (in Order of Use)
<ol>
	<li>Cut a piece of cellulose filter paper (11 &#956;m) into 2 mm circles.</li>
	<li>Prepare a 1 M solution of NaOH by dissolving 20 g of NaOH in 500 ml distilled H2O. Store at room temperature. CAUTION: NaOH solutions are corrosive and can cause alkali-burns.</li>
	<li>Prepare an anesthetic cocktail by diluting 10 ml of 100 mg/ml ketamine and 2.5 ml of 20 mg/ml xylazine in 37.5 ml of 1x PBS for a final concentration of 20 mg/ml ketamine and 4 mg/ml xylazine.</li>
	<li>Prepare a 0.5% solution of proparacaine hydrochloride (for topical analgesia) by dissolving 500 mg of proparacaine hydrochloride in 100 ml of filtered PBS.</li>
	<li>Prepare 1x PBS by dissolving 8 g NaCl, 0.2 g of KCl, 1.44 g Na2HPO4, and 0.23 g of NaH2PO4 in 900 ml of distilled H2O. Adjust to 7.4 pH. Bring the solution to a final volume of 1,000 ml with distilled H2O and filter to ensure sterility.</li>
	<li>Prepare 1,000x stock solutions of SAHA at ~100 mM in dimethyl sulfoxide (DMSO) by dissolving 26.4 mg of SAHA in 1 ml of DMSO. Dilute to a 1x (~10 &#956;M) solution in filtered PBS for each use. Store the stock solution at -20 &#176;C for up to one month. CAUTION: DMSO is a known toxin and mutagen.</li>
	<li>Prepare a 4% solution of paraformaldehyde (PFA) for fixation. Under a chemical hood, dissolve 4 g of PFA in 90 ml of PBS. Bring the mixture to 65 &#176;C and titrate to a pH of 7.4. Once the PFA has dissolved, bring the solution to a final volume of 100 ml. Store at 4 &#176;C for up to one month. CAUTION: PFA is a known to be allergenic, carcinogenic, and toxic.</li>
	<li>Prepare 1x blocking buffer by diluting 5 ml of goat serum and 500 ul of Triton X-100 into 94.5 ml of PBS.</li>
	<li>Prepare 1x washing buffer by diluting 500 ul of Triton X-100 into 99.5 ml of PBS.</li>
</ol>
2. Alkali-Burn Injury &#38; Compound Treatment
<ol>
	<li>Soak a round piece of filter paper, ~2 mm in diameter, in a solution of 1 M NaOH.</li>
	<li>Anesthetize the mouse with an injection of 100 mg/kg ketamine and 5 mg/kg xylazine, which is ~100 &#956;l of the anesthetic cocktail from step 1.3 per 25 g mouse. Anesthesia should take ~1-2 min to set in. Depth of anesthesia may be determined by gently pinching the toe or tail of the animal, if anesthesia is sufficient there should be no response. Note: Care should be taken to ensure the mouse does not succumb to anesthesia induced hypothermia.</li>
	<li>Using an eyedropper, topically apply a drop of 0.5% proparacaine hydrochloride to the corneal surface for local analgesia.</li>
	<li>Using sterile forceps, pick up a piece of NaOH soaked filter paper. If you notice excess NaOH clinging to or dripping from the filter paper, briefly tap the soaked filter paper on a dry piece of filter paper to absorb the excess.&#160;Place the piece of NAOH soaked filter paper on the central cornea. Leave it for 30 sec to generate an acute alkali-burn of ~2 x 2 mm2 in area. A surgical microscope is helpful in properly placing the filter paper. Note: Only one eye of the mouse should be injured with the other serving as a control.</li>
	<li>Remove the filter paper. Using a 10 ml syringe, gently flush the eye with 10 ml of 1x PBS twice to wash away residual 1 M NaOH.</li>
	<li>Immediately apply a drop of 1x SAHA working solution (or a vehicle control consisting of PBS diluted DMSO containing no SAHA) topically to the cornea. Repeat application 3x/day for 14 days. Note: during this period a topical antibiotic ointment is not recommended as it may interfere with the development of the injury and the delivery of the compound. Use a liquid antibiotic solution, such as 3% Gentamicin solution, instead.</li>
	<li>Proceed directly to Clinical Assessment (Protocol 3 below) or sacrifice the mouse and enucleate the eyes for corneal flat mount (Protocol 4 below) or conventional paraffin/frozen histology.</li>
</ol>
3. Clinical Assessment
<ol>
	<li>Perform a daily examination of the mice in a blinded fashion under a surgical microscope and score corneal NV based on corneal opacity, NV, and vessel size. Use at least two observers and record a final score that is the average of the two.
	<ol>
		<li>Score corneal opacity on a scale of 0-4. 0 = completely clear; 1 = slightly hazy, iris and pupil easily visible; 2 = slightly opaque, iris and pupil still detectable; 3 = opaque, pupils hardly detectable; and 4 = completely opaque with no view of the pupil.</li>
		<li>Score NV on a scale of 0-3. 0 = no neovessels; 1 = neovessels at the corneal limbus; 2 = neovessels spanning the corneal limbus and approaching the corneal center; 3 = neovessels spanning the corneal center.</li>
		<li>Score vessel size on a scale of 0-3. 0 = no neovessels; 1 = neovessels detectable under surgical microscope; 2 = neovessels easily seen under surgical microscope; 3 = neovessels easily seen without the microscope.</li>
	</ol>
	</li>
	<li>Using a digital camera, take representative images of the eye at 7 days and 14 days.</li>
</ol>
4. Corneal Staining and Flat Mounts
<ol>
	<li>Fix enucleated eye in 4% PFA for at least 1 hr at 4 &#176;C.</li>
	<li>Transfer the eye to 1x PBS and use forceps to carefully remove excess tissue.</li>
	<li>With the aid of a surgical microscope, use a needle (18 gauge) or micro-knife to perforate the pericorneal region of the eye (note: this should release fluid from the eye).
	<ol>
		<li>From the perforation made in section 4.3.1 use a pair of surgical scissors to cut the anterior portion of the eye (cornea) from the posterior portion.</li>
	</ol>
	</li>
	<li>Transfer the cornea back into 4% PFA for fixation overnight at 4 &#176;C.</li>
	<li>Discard the 4% PFA (CAUTION: PFA is hazardous and must be disposed of in accordance with institutional regulations) and rinse the cornea three times with 1x PBS to remove any residual PFA.</li>
	<li>Incubate in 1x blocking buffer for at least 2 hr at room temperature to permeabilize the tissue and prevent nonspecific binding of the primary antibody.
	<ol>
		<li>Apply primary antibody in 1x blocking buffer, at a 100-500 fold dilution. (e.g. rat anti-mouse-PECAM-1 for detecting blood vessels at 1:100, rabbit anti-mouse-LYVE-1 for detecting lymphatic vessels at 1:500, and/or rat anti-mouse-F4/80 for detecting macrophages at 1:100). Incubate at 4 &#176;C overnight.</li>
		<li>Wash six times with 1x washing buffer for 1 hr each time at room temperature to fully remove unbound primary antibody.</li>
		<li>Apply a secondary antibody in 1x blocking buffer at a 500-1,000 fold dilution. (e.g. a 488 nm fluorescent tagged goat anti-rat-IgG at 1:800 or a 594 nm fluorescent tagged goat anti-rabbit-IgG at 1:800). Incubate at 4 &#176;C overnight.</li>
		<li>Wash three times with 1x PBS for 1 hr each at room temperature to fully remove unbound secondary antibody.</li>
	</ol>
	</li>
	<li>Transfer the cornea into fresh 1x PBS and, with the aid of a surgical microscope, carefully make four incisions from periphery towards the center. This should divide the cornea into four quadrants of approximately equal size (the resulting shape should look rather like a butterfly) and allow it to lie flat on a slide.</li>
	<li>Mount with a mounting medium appropriate for fluorescent imaging. For best results, samples should be imaged immediately and digital photographs taken, but may be stored for several weeks protected from light at 4 &#176;C.</li>
</ol>

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The authors have nothing to disclose.

The protocol presented here results in reproducible levels of hemangiogenesis, lymphangiogenesis, and inflammation, making it an ideal system to study these three (interrelated) processes. While this method produces centralized corneal NV, several methods that have been developed to cause more directed NV, namely suturing of the cornea17 and implanted growth-factor expressing pellets18, might also be of interest. Our protocol is designed for use in the adult mouse, providing an&#160;easy to use anim...

Corneal blindness is the fourth most common cause of blindness, responsible for approximately 4% of all cases1. Corneal neovascularization (NV) plays a significant role in many of these pathologies, including herpetic keratitis (the leading infectious cause of blindness in the West) and trachoma (the leading cause of infectious blindness worldwide)2. Current therapies include steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), anti-VEGF therapies, and cyclosporin A as well as conventional or laser surgical techniques3. However, the severely debilitating nature of corneal NV based pathologies, the paucity of surgical facilities capable of treating corneal NV, and the lack of a strongly performing pharmacological option led a recent expert roundtable to conclude that, despite the extant therapies, the fundamental medical need presented by these pathologies remains unmet4.
The human cornea consists of 5 layers, 3 cellular layers (epithelial, stromal and endothelium) and 2 interface (Bowman membrane and Descemet membrane). It functions as a mechanical barrier and refractive surface for the eye. Its transparent nature is the consequence of a delicate balance of its components and is integral to its proper function5. Normally avascular, the cornea receives blood from microvessels running along its outer edge which are fed from the ciliary and ophthalmic arteries. Corneal NV occurs when a stimulus promotes angiogenesis of these vessels allowing them to grow towards the center of the cornea and thus limit vision6. Corneal angiogenesis includes hemangiogenesis and lymphangiogenesis, which result in the ingrowth of blood vessels and lymphatic vessels from the limbal vascular arcade towards the center of the cornea. This leads to a breakdown of corneal &#34;angiogenic privilege&#34;, an increase in corneal opacity and fibrosis, disruption of the corneal layers, and edema7. The precise triggers of corneal NV are numerous, ranging from a response to infectious disease such as trachoma to a chemically induced state caused traditional medicines, industrial chemicals, or even chemical warfare agents.
The molecular mechanisms of this process are not, as yet, fully characterized; however, a few key players have been identified. Under normal conditions the cornea possesses a unique &#8216;angiogenic privilege&#39; maintained by a redundant array of anti-angiogenic factors (such as soluble VEGF-R1)8. However, in response to an external stimulus (such as an injury), there will be a local upregulation of pro-angiogenic factors (e.g. VEGF-A). This tips the balance of pro and anti- angiogenic factors that underlies the cornea&#39;s angiogenic privilege, and leads to hemangiogenesis, lymphangeogenesis, and inflammation, therefore causing corneal pathology and even blindness9.
Given the unmet medical need of this highly debilitating pathology, it is of interest to the field to have a reliable animal model of corneal NV. Here we present such a model: controlled alkali-burn injury. Various eye-injury models based on using filter paper rings have been used since 1970s10. In 1989, a group of Harvard Medical School ophthalmologists characterized a standard model of a central corneal alkali-burn injury in rabbit based on soaking a piece of circular filter paper with sodium hydroxide (NaOH) and applying it to the cornea at a specific range of concentrations11. Since then, this technique has been adapted for use in the mouse12-14. Recently, the Wang lab studied the therapeutic effects of the histone deacetylase (HDAC) inhibitor SAHA in the pathogenesis of corneal NV using a mouse corneal alkali-burn injury model15. The methodology of the mouse corneal alkali-burn injury model presented here was built mainly on the prior work of two other papers14,16.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 mL Syringe</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">309659</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18 Guage Needle</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">305918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">306575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25 Guage Needle</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">305916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-F4/80 (rat anti-mouse)</td>
			<td style="width:96px;">AbD Serotech</td>
			<td>MCA497RT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-LYVE-1 (rabbit anti-mouse)</td>
			<td style="width:96px;">Abcam</td>
			<td style="width:119px;">ab14917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-PECAM-1 (rat anti-mouse)</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">553370</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-IgG Alexa488 (goat anti-rat)</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">A11006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-IgG Alexa594 (goat anti-rabbit)</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">A11012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:96px;">Tucsen</td>
			<td style="width:119px;">TCC 5.0 ICE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">12-548-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">D4540-1L</td>
			<td>Caution: Mutagenic, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps (Blunt), Iris</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps (Sharp), Dumont #4</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">500340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">P217-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Solution</td>
			<td style="width:96px;">MedVet</td>
			<td>RXKETAMINE</td>
			<td>Controlled substance, proper license required for use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Source for Microscope</td>
			<td style="width:96px;">AmScope</td>
			<td style="width:119px;">LED-14M-YA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope (Stereo 7X-45X)</td>
			<td style="width:96px;">AmScope</td>
			<td style="width:119px;">SM-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mounting Medium, Vectashield</td>
			<td style="width:96px;">Vector</td>
			<td style="width:119px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S397-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S318-1</td>
			<td>Caution: Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:96px;"></td>
			<td style="width:119px;">P6148-500G</td>
			<td>Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proparacaine Hydrochloride</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">P4554-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors (5mm blade), Vanas</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat Serum</td>
			<td style="width:96px;">MPBio</td>
			<td align="right" style="width:119px;">92939249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope Slides</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">T8787-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Whatman Grade 1 Filter Paper</td>
			<td style="width:96px;">Whatman</td>
			<td style="width:119px;">1001-6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine Solution</td>
			<td style="width:96px;">MedVet</td>
			<td style="width:119px;">RXANASED-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

an alkali-burn injury model of corneal neovascularization in the mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

After alkali-burn injury, corneal NV occurs in a predictable, time-dependent fashion. Figure 1 demonstrates the stark difference both in neovascularization and corneal opacity between an untreated animal (Figure 1A) and an animal treated with the HDAC inhibitor SAHA (Figure 1B) at the 7 day time point.
Figures 2A and&#160;2B demonstrate a corneal flat mount of an untreated control eye with primary PECAM-1 and ...

Watch this Scientific Journal Video about An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse at JoVE.com

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

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Research

JoVE Journal

Medicine

16.4K Views.  Tulane University. Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Video: An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...