a hypertonic saline injection technique for inducing glaucoma in rats

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

In this procedure, after anesthetizing the animal with KAX cocktail, check the reflexes by giving pinches to its feet or tail to ensure the animal is unconscious. Then, trim the whiskers with scissors. Subsequently, saturate a cotton tip applicator with betadine solution and swab the area around the experimental eye.
Under the microscope, use a hemostat to clamp the bottom eyelid in order to bulge the eye, expose the episcleral vein, and restrict the eye movement. Next, carefully pierce the episcleral vein with the micro-needle at an angle between 10 and 20 degrees to the vein. A puncture into the vein is successful when the blood is observed to enter the tip of the micro-needle.
Slowly inject 50 microliters of saline into the vein. The veins will blanch white as the salt circulates through the vasculature, but some regions may maintain a blood-red appearance.
The single most critical step in this procedure occurs during the injection of the two molar saline. It is imperative that blanching in the vasculature occurs to ensure a successful outcome. Maintaining a steady needle in a 20-degree angle are crucial for this procedure.
After that, perform a second injection into the vein to ensure thorough damage of the retinal ganglion cell layer. Within minutes, one should see a distinct cloudy appearance through the iris of the eye as the salt circulates through the vascular system.

a-hypertonic-saline-injection-technique-for-inducing-glaucoma-in-rats

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Epilepsy and Seizure Disorders

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 (Ketamine + Xylazine + Acepromazine) 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 the lab for the duration of surgery.</li>
	<li>Post-surgery, replace animals to their cages and keep comfortable in room temperature (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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Xylazine hydrochloride, Minimum 99%</td>
			<td>Sigma, Life Science</td>
			<td>X1251-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection, USP, 100mg/ml</td>
			<td>Putney, Inc</td>
			<td>NDC 26637-411-01</td>
			<td>10 ml bottle</td>
		</tr>
		<tr>
			<td>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>
			<td>Serum bottle, 10 ml</td>
			<td>VWR</td>
			<td>16171319</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>1 ml insulin syringe</td>
			<td>VWR</td>
			<td>BD329410</td>
			<td>28 G needle</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>2 M Solution</td>
		</tr>
		<tr>
			<td>Microelectrode Puller</td>
			<td>Narishige Group</td>
			<td>PP-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Heavy Polished Standard and Thin Walled Borosilicate Tubing</td>
			<td>Sutter Instruments</td>
			<td>B150-86-10HP</td>
			<td>without filament, 0.86 mm</td>
		</tr>
		<tr>
			<td>Microfil syringe needle for filling micropipettes</td>
			<td>World Precision Instruments, Inc</td>
			<td>MF28G</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge Luer-Lock needle</td>
			<td>Fisher Scientific</td>
			<td>1130421</td>
			<td>Syringe needle</td>
		</tr>
		<tr>
			<td>Flexible Polyethylene Tubing</td>
			<td>Fisher Scientific</td>
			<td>22046941</td>
			<td>0.034 inch diameter, approximately 10 inches</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride Opthalmic Solution, USP, 0.5%</td>
			<td>Akorn, Inc</td>
			<td>NDC 17478-263-12</td>
			<td>15 ml&#160; sterile bottle</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>StereoZoom 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat Clamp</td>
			<td>Fine Science Tools</td>
			<td>1310912</td>
			<td>curved edge</td>
		</tr>
		<tr>
			<td>Triple Antibiotic Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0664481</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade #11</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm x 15 mm Disposable Petri Dish</td>
			<td>VWR</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 10x Concentrate</td>
			<td>Sigma, Life Science</td>
			<td>P7059-1L</td>
			<td>1x dilution</td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (2), Dumont # 5</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>VWR</td>
			<td>102092-312</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Gossman, C. A. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53831">Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation.</a> J. Vis. Exp. (2016)
This video demonstrates glaucoma induction in an anesthetized rat by injecting hypertonic saline into the episcleral vein. The saline enters the trabecular meshwork via Schlemm&#39;s canal, causing tissue scarring, which blocks aqueous humor outflow and raises intraocular pressure. The elevated pressure damages retinal ganglion cells, leading to glaucoma.

<img alt="Figure 1" src="/files/ftp_upload/22971/22971_Figure1.jpg" /> 
Figure 1. Microneedle Components. (A)&#160;Syringe with polyethylene tubing used for saline injection.&#160;(B)&#160;Backfill syringe used to backfill the borosilicate microneedle.&#160;(C)&#160;Narishige electrode puller used to make borosilicate microneedles.&#160;(D)&#160;Borosilicate glass electrodes before and after being pulled in the elect...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take an anesthetized rat and apply an anesthetic to its eye to numb reflexes.
Trim whiskers and disinfect the area around the eye to prevent contamination.
Under a microscope, clamp the lower eyelid to bulge the eye, exposing the episcleral vein.
This vein connects to the anterior chamber via Schlemm&#39;s canal and trabecular meshwork for the outflow of aqueous humor.
Insert a microneedle with hypertonic saline into the exposed vein.
Inject the saline, displacing blood. Perform a second injection opposite the first for a uniform effect across the eye.
The saline enters the trabecular meshwork.
The high salt concentration causes osmotic stress, leading to cell death and tissue scarring. This scarring blocks aqueous humor outflow through the meshwork, increasing intraocular pressure.
The elevated pressure damages the retinal ganglion cells, which are neurons carrying visual information to the brain. The damage leads to glaucoma, a condition that causes vision loss.

17 次观看. 一种高渗盐水注射技术诱导大鼠青光眼

视频: 一种高渗盐水注射技术诱导大鼠青光眼 - 实验

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

科研

JoVE Journal

医学

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

通过隔离检查视觉诱发潜力评估患者早期开放角青光眼

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

神经科学

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

成績單

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats