C. Linn is supported by an NIH grant (NIH NEI EY022795).

All procedures using animal subjects have been in accordance with the standards of the Institute of Animal Care and Use Committee (IACUC) at Western Michigan University.
1. Animals
<ol>
	<li>Use male and female rats 3 months of age in this study.</li>
	<li>Keep animals in a 12 hr light/dark cycle with free access to food and water.</li>
</ol>
2. Preparation of KAX Cocktail for Animal Anesthesia
<ol>
	<li>Dissolve 50 mg of xylazine (20 mg/ml) in 5 ml ketamine (100 mg/ml) with 1 ml acepromazine (10 mg/ml) and 3 ml of distilled water. Mix thoroughly.</li>
	<li>Sterilize with a syringe filter and store this solution into a 10 ml serum bottle.</li>
</ol>
3. KAX Injection
<ol>
	<li>Weigh animal (g) and return to cage until ready for injection.</li>
	<li>Inject 0.1 ml&#160;KAX/100 g animal body weight intraperitoneally, using a 1 ml insulin syringe with a 28 G&#160;needle.</li>
	<li>Allow for animal to become unconscious. Check reflexes by pinching the feet and tail.</li>
	<li>Keep all animals safely in lab for the duration of surgery.</li>
	<li>Post-surgery, replace animals into their cages and keep comfortable in RT&#160;until consciousness is regained. Only return animals to the animal facility when the animals awaken and resume normal behavior.</li>
</ol>
4. Preparation for Surgery and Microneedle Assembly
<ol>
	<li>Make a sterile 2 M NaCl solution.</li>
	<li>Use a microelectrode puller (Figure 1C) to pull one 0.86 mm inner diameter heavy polished standard and thin walled borosilicate tube into two finely tapered glass microneedles (Figure 1D, Figure 1E).</li>
	<li>Backfill one microneedle from the previous step with 2 M saline using a backfilling syringe needle and a 1 ml syringe (Figure 1B) . Tap out air bubbles from the tip of the electrode.</li>
	<li>Fill a second 1 ml syringe with 2 M NaCl. Connect an 18 G&#160;needle and then attach a length (approximately 10 inches) of polyethylene tubing (Figure 1A). Use the syringe plunger to fill the polyethylene tubing with saline through the needle.</li>
	<li>When both the microneedle and tubing are filled with saline, carefully connect the two. Eliminate any air in the connection between them (Figure 2).</li>
	<li>Finely bevel the tip of the microneedle by scraping it very lightly against the grain of a course paper towel.</li>
	<li>Check the resistance of the microneedle by gently pushing the plunger on the syringe until a fine stream of liquid can be seen on the paper towel. The stream of liquid should be no wider than 0.5 mm.</li>
</ol>
5. Preparation of Animal
<ol>
	<li>Apply 1 - 2 drops topical anesthetic to cornea (Proparacaine Hydrochloride Ophthalmic Solution, USP, 0.5%). Wait until no ocular reflex occurs.</li>
	<li>Trim whiskers with scissors.</li>
	<li>Saturate a cotton tip applicator with betadine solution and swab area around the experimental eye.</li>
	<li>Using a microscope, attach a hemostat to clamp the bottom eyelid to bulge the eye, expose the episcleral vein and restrict eye movement. (Figure 3, arrowhead)</li>
</ol>
6. Glaucoma-inducing Saline Injection
<ol>
	<li>When the microneedle assembly and the animal are prepared, begin injections.</li>
	<li>When the animal is confirmed to be unresponsive to feet/tail pinch, carefully pierce the episcleral vein with the microneedle by coming at a low angle between 10 and 20 degrees to the vein (Figure 3, white arrow). A successful puncture into the vein is apparent when blood enters the tip of the microneedle (Figure 3, black arrow).</li>
	<li>Slowly and manually inject approximately 50 &#181;l saline into the vein. This should take approximately 10 sec. The veins will blanch white as the salt circulates through the vasculature (Figure 4, arrowhead). Some regions may maintain a blood red appearance (Figure 4, arrow).
	<ol>
		<li>Perform a second injection into the vein, opposite to the site of the first, to ensure thorough retinal damage to the complete retinal ganglion cell layer. 
		Note: Within minutes, one should see a distinct cloudy appearance through the iris of the eye as the salt circulates through the vascular system.</li>
	</ol>
	</li>
	<li>Leave the opposite eye untreated for use as an internal control.</li>
</ol>
7. Animal Recovery
<ol>
	<li>Remove the hemostat.</li>
	<li>Use a cotton applicator to apply triple antibiotic ointment (Bacitracin zinc, neomycin sulfate, polymysin B sulfate)&#160; to the site clamped by the hemostat and to injection sites. Tissue damage around the eye does not occur using the hemostat.</li>
	<li>&#62;Place anesthetized animals in their cages on a circulating warm water blanket to prevent hypothermia.&#160;Keep animals under observation until consciousness and normal behavior are regained. Transport awake animals back to the animal colony. Animals remain in the colony until the time of sacrifice.</li>
</ol>
8. Animal Sacrifice and Retina Removal
<ol>
	<li>One month following the procedure to induce glaucoma, animals are euthanized by CO2 asphyxiation and secondary thoracic puncture.&#160;
	<ol>
		<li>Place the animal in the chamber and put the lid on securely.</li>
		<li>Open the CO2 and gas regulator valves to allow 20% volume/min CO2 displacement of oxygen in the chamber.</li>
		<li>Allow four to 5 min for the animal to expire.</li>
		<li>Turn off both valves.</li>
		<li>Remove animal from the chamber and perform a secondary thoracic puncture with a sterile scalpel.</li>
	</ol>
	</li>
	<li>After euthanasia, use a scalpel to cut the connective tissue in the orbital cavity surrounding the eye, being careful not to cut into the eyeball itself.</li>
	<li>Carefully use curved edge scissors to cut the optic nerve and any remaining tissue to extract the intact eyeball. Place extracted eyeball in a sterile 60 mm x 15 mm disposable petri dish containing fresh PBS.</li>
	<li>Make an eyecup from the eyeball. To do this, make a small incision with the scalpel just posterior to the border between the iris and the sclera. Follow this incision around the circumference of the eye with small spring scissors to remove the corneal hemisphere from the eyeball. The hemisphere connected to the optic nerve remains.</li>
	<li>Find the very thin pink/beige retina inside the eyecup from the euthanized animal. Hold the pigmented layer of the retina with blunted forceps to stabilize the eyecup. Use another pair of closed forceps to very gently tease the whole intact retina off of the back of the eye. Avoid pinching, pulling, or tugging the retina directly.</li>
	<li>Use small spring scissors to cut the area where the optic nerve is still attached to the retina.</li>
	<li>Be sure to cut away any residual pigment epithelium or scleral tissue from the retina.</li>
	<li>Using a transfer pipette, very gently transfer the isolated retina to a clean sylgard coated 35 mm x 10 mm petri dish containing fresh PBS.</li>
</ol>
9. Whole-Mount Retina Preparation
<ol>
	<li>Once in the sylgard dish, use forceps and one cactus needle to pin the retina in place. Keep the retinal ganglion cell layer facing up and optic nerve down. The retina&#39;s hemispherical shape is notable even after fixation. The curvature of the retina will curl toward the ceiling when the retinal ganglion cell layer is in the desired orientation.</li>
	<li>Use small scissors to cut the retina into four quadrants, making the shape of a four leaf clover radiating from the optic nerve head.</li>
	<li>Pin the quadrants of the retina with additional cactus needles to make the retina as flat as possible without stretching (Figure 5).</li>
	<li>Fix the pinned retinas in the sylgard dish with 3 ml of 4% paraformaldehyde O/N&#160;at RT.</li>
</ol>
10. Antibody Staining of Retina
Note: Stain fixed retinas with primary and secondary antibodies for viewing neurons in the retina (Figure 6).
<ol>
	<li>Rinse fixed, flat-mounted retinas three times for 2 min each in PBS.</li>
	<li>Permeabilize the retinas with 1% Triton X-100 with 1% fetal bovine serum in PBS for 60 min.</li>
	<li>Rinse retinas three times, 2 min each, in PBS.</li>
	<li>Rinse twice with 0.1% Triton X-100 in PBS, 5 min per wash.</li>
	<li>Rinse twice with PBS, 5 min per wash.</li>
	<li>Incubate with 1% Triton X-100 and 1% fetal bovine serum in PBS at RT for 45 min.</li>
	<li>Rinse twice with 0.1% Triton X-100 in PBS, 5 min per wash.</li>
	<li>Rinse twice with PBS, 5 min per wash.</li>
	<li>Incubate each retina in 3 ml 1% fetal bovine serum in PBS with purified mouse anti-rat CD90/mouse CD90.1 (1:300 dilution) O/N at RT.</li>
	<li>Rinse retinas once with 0.1% Triton X-100 in PBS for 5 min.</li>
	<li>Rinse twice with PBS, 5 min per wash.</li>
	<li>Incubate each retina in 3 ml PBS (no FBS) with secondary Alexa Fluor 594 goat anti-mouse IgG (1:300) O/N at RT.</li>
	<li>Wash retinas with PBS liberally.</li>
	<li>Using a dissecting microscope, carefully remove cactus needles from the retina.</li>
	<li>Gently transfer retinas onto microscope slides with a transfer pipette. Be sure to maintain orientation with retinal ganglion cell layer facing to the ceiling. The retina&#39;s hemispherical shape is notable even after fixation. The curvature of the retina will curl toward the ceiling when the retinal ganglion cell layer is in the desired orientation.</li>
	<li>Absorb any excess PBS with KimWipe or other such absorbent material. Be careful not to absorb the retina.</li>
	<li>Add 5 drops of &#189; glycerol and &#189; PBS by weight as a mounting media.</li>
	<li>Cover retina with coverslip, avoiding air bubbles.</li>
	<li>Secure coverslip using clear nail polish, glue, or other adhesive.</li>
</ol>

The authors have no conflicting or competing interests to disclose.

This protocol describes a method of inducing glaucoma-like conditions in an in vivo rat model. This procedure uses an injection of hypertonic saline to induce scarring in the trabecular meshwork 29, 32. Developing scar tissue occludes the outflow of aqueous humor which increases pressure in the anterior chamber. With decreased outflow and pressure build up, the lens suspended by elastic ligaments pushes back into the vitreous chamber. Vitreous humor then applies pressure onto the retina damaging the f...

Glaucoma is a group of eye diseases affecting neurons in the retina, specifically, the retinal ganglion cells1-2. The axons of these cells converge to become the optic nerve carrying visual information to the brain where vision is perceived. Damage to RGCs and their axons therefore causes visual defects.
The primary characteristics associated with glaucoma disorders are RGC degeneration and death, increased intraocular pressure (IOP), and optic disk cupping and atrophy. These features lead to visual field loss or complete, irreversible blindness. Currently, glaucoma has caused blindness in 70 million people worldwide 3. As such, it is the world&#39;s third largest cause of blindness 4.
The exact mechanism of RGC death in glaucoma remains unknown. Much research has been done to unlock the mystery. It is known, however, that the primary risk factor of glaucoma is an increase in intraocular pressure due to irregular circulation of aqueous humor (AH) in the anterior chamber of the eye. AH acts as a transparent and colorless replacement for blood in the avascular anterior chamber of the eye. It nourishes the surrounding cells, removes secreted waste products from metabolic processes, transports neurotransmitters, and permits the circulation of drugs and inflammatory cells within the eye during pathological states 1.
The maintenance of aqueous humor circulation involves the ciliary body and the trabecular meshwork. Aqueous humor is produced by the ciliary body. It then flows into the anterior chamber to maintain the overall health of the ocular tissue. 75 - 80% of aqueous humor outflow is actively secreted through non-pigmentary ciliary epithelium when the fluid is filtered through three layers of spongy tissue in the ciliary muscle. The fluid exits through the trabecular meshwork and through Schlemm&#39;s Canal which empties into the blood system 5.The remaining 20 - 25% of outflow bypasses the trabecular meshwork and is passively secreted by ultrafiltration and diffusion through the uveo-scleral pathway. This pathway appears to be relatively independent of intraocular pressure 1.
When aqueous humor production and outflow are out of balance, pressure builds within the eye. As stated, this increase in intraocular pressure is the primary risk factor in the development of glaucoma. Such pressure causes damage to the intricate layers of neurons in the retina at the back of the eye. Damage to the retinal ganglion cell axons of the optic nerve causes the brain to no longer receive accurate visual information. As a result, the perception of vision is lost and complete blindness can occur.
To date, there is no cure for glaucoma. Different treatment methods exist that primarily aim to reduce intraocular pressure. These include topical medication classes such as beta1-adrenergic receptor blockers, or topical prostaglandin analogues. Beta blockers reduce the intraocular pressure by decreasing the production of aqueous humor 7. Prostaglandins function to reduce IOP by increasing the outflow of aqueous humor 8-14. Alpha adrenergic agonists and carbonic anhydrase inhibitors are also used as secondary methods of treatment. Alpha adrenergic agonists increase outflow through the uveoscleral pathway 15-17. Carbonic anhydrase inhibitors reduce the production of AH by enzymatic inhibition 18. Much more invasive procedures are also being used to treat glaucoma. Laser trabeculoplasty is used to increase the outflow of aqueous humor 19. Another surgical therapy, called trabeculectomy, creates an alternative drainage site to filter AH when the traditional trabecular pathway is blocked 20-21.
These treatment options have been known to effectively reduce IOP. However, up to 40% of glaucoma patients show normal IOP levels indicating a need for more complete therapeutic methods.22,23 Additionally, retinal ganglion cell death seen in glaucoma is irreversible once it begins and current treatments do not stop the progression of the disease 24-28. This has highlighted the need for effective neuroprotective therapies that target the survival of the neurons themselves. Development of glaucoma models is crucial for this development.
In this study we are demonstrating a method of inducing glaucoma-like effects in adult Long Evans rats using a modified procedure originally outlined by Morrison29. In this procedure, injections of 2 M hypertonic saline into the episcleral venous plexus induces glaucoma-like conditions by scarring tissue to reduce aqueous humor outflow in the trabecular meshwork leading to an increase in intraocular pressure and a significant loss of RGCs within one month of the procedure 30-31. Glaucoma-inducing procedures, such as the one described here, may be the key to unlocking new developments in glaucoma treatments.

<table border="0" cellpadding="0" cellspacing="0" width="982">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:299px;">Xylazine hydrochloride, Minimum 99%</td>
			<td style="width:156px;">Sigma, Life Science</td>
			<td style="width:200px;">X1251-1G</td>
			<td style="width:327px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ketamine hydrochloride injection, USP, 100mg/mL&#160;</td>
			<td>Putney, Inc</td>
			<td>NDC 26637-411-01</td>
			<td>10 mL bottle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Acepromazine Maleate, 10mg/mL</td>
			<td>Phoenix Pharmaceutical, Inc</td>
			<td>NDC 57319-447-04, 670008L-03-0408</td>
			<td>50 mL bottle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Serum bottle, 10 mL</td>
			<td>VWR</td>
			<td>16171319</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">1 mL insulin syringe&#160;</td>
			<td>VWR</td>
			<td>BD329410</td>
			<td>28 gauge needle&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Sodium chloride</td>
			<td>Sigma&#160;</td>
			<td>S7653</td>
			<td>2 M Solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microelectrode Puller&#160;</td>
			<td>Narishige Group</td>
			<td>PP-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Heavy Polished Standard and Thin Walled Borosilicate Tubing&#160;</td>
			<td>Sutter Instruments</td>
			<td>B150-86-10HP</td>
			<td>without filament, 0.86 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microfil syringe needle for filling micropipettes</td>
			<td>World Precision Instruments, Inc</td>
			<td>MF28G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">18 gauge Luer-Lock needle</td>
			<td>Fisher Scientific</td>
			<td>1130421</td>
			<td>Syringe needle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Flexible Polyethylene Tubing</td>
			<td>Fisher Scientific</td>
			<td>22046941</td>
			<td>0.034 inch diameter, approximately 10 inches&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Proparacaine Hydrochloride Opthalmic Solution, USP, 0.5%</td>
			<td>Akorn, Inc</td>
			<td>NDC 17478-263-12</td>
			<td>15 mL&#160; sterile bottle&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Curved Scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microscope</td>
			<td>Leica&#160;</td>
			<td>StereoZoom 4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Hemostat Clamp&#160;</td>
			<td>Fine Science Tools</td>
			<td>1310912</td>
			<td>curved edge</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Triple Antibiotic Ointment&#160;</td>
			<td>Fisher Scientific</td>
			<td>NC0664481</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Scalpel handle</td>
			<td>Fine Science Tools&#160;</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Scalpel blade # 11</td>
			<td>Fine Science Tools&#160;</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">60 mm x 15 mm Disposable Petri Dish</td>
			<td>VWR</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Phosphate Buffered Saline 10x Concentrate</td>
			<td>Sigma, Life Science&#160;</td>
			<td>P7059-1L</td>
			<td>1x dilution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Spring Scissors</td>
			<td>Fine Science Tools&#160;</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Forceps (2), Dumont # 5</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">3 mL Transfer Pipets, polyethylene, non sterile</td>
			<td>BD Biosciences</td>
			<td>357524 or 52947-948</td>
			<td>1 and 2 mL graduations</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">35 mm x 10 mm Easy Grip Petri Dish&#160;</td>
			<td>BD Biosciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Sylgard 184</td>
			<td>VWR</td>
			<td>102092-312</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Cactus Needles</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Paraformaldehyde</td>
			<td>EMD Millipore&#160;</td>
			<td>PX0055-3 or 818715.0100</td>
			<td>Made into a 4% solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T9284-100 mL</td>
			<td>Made into both a 1% and 0.1% solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Fetal Bovine Serum&#160;</td>
			<td>Atlanta Biological</td>
			<td>S11150</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Purified Mouse Anti-Rat CD90/mouse CD90.1</td>
			<td>BD Pharmingen</td>
			<td>Cat 554892</td>
			<td>1:300 dilution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Alexa Fluor 594 goat anti-mouse&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>A11005</td>
			<td>1:300 dilution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microscope Slides</td>
			<td>Corning&#160;</td>
			<td>2948-75x25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Glycerol&#160;</td>
			<td>Sigma&#160;</td>
			<td>G5516-100 mL&#160;</td>
			<td>50% glycerol to 50% PBS, by weight&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Coverglass&#160;</td>
			<td>Corning&#160;</td>
			<td>2975-225</td>
			<td>Thickness 1 22 x 50 mm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Confocal Microscope</td>
			<td>Nikon&#160;</td>
			<td>C2 Eclipse Ti</td>
			<td></td>
		</tr>
	</tbody>
</table>

glaucoma-inducing procedure in an in vivo rat model and whole-mount retina preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

This section illustrates the apparatus components and procedure used to induce glaucoma-like conditions in an in vivo rat glaucoma model. We show the individual tools and equipment used to perform a hypertonic saline injection which causes an increase in intraocular pressure. We show the injection into the episcleral venous plexus with its characteristic blanching effect and the cloudy appearance of the anterior chamber that results. We also describe the process of retina removal...

Watch this Scientific Journal Video about Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation at JoVE.com

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

glaucoma-inducing-procedure-an-vivo-rat-model-whole-mount-retina

Research

JoVE Journal

Medicine

12.9K Views.  Western Michigan University. Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis. 

Video: Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.