The authors would like to acknowledge Dr. Steve Heap (BVSc, CertVOphthal) for his assistance in establishing this protocol and performing the injections described by Murray et&#160;al.15. The authors also acknowledge funding from CureKids New Zealand, the Canterbury Medical Research Foundation, Neurogene Inc, and the Batten Disease Support and Research Association.

All experimental protocols were approved by the Lincoln University Animal Ethics committee and are in line with the US National Institutes of Health guidelines for the care and use of animals in research and the New Zealand Animal Welfare Act (1999). Borderdale sheep were diagnosed at birth14 and maintained at Lincoln University research farms. Three 3-month-old homozygous (CLN5-/-) ewes received a single IVT injection to the left eye, with the untreated right eye acting as an internal control. Electroretinography and pathology data were compared to historical healthy and affected control data. The viral vector used in this study was a self-complementary adeno-associated virus serotype 9, containing the chicken beta action (CBh) promoter and codon-optimized ovine CLN5 (scAAV9/CBh-oCLN5opt). The viral vector was&#160;provided by the University of North Carolina Vector Core, NC, USA.
1. Presurgery
<ol>
	<li>Autoclave the surgical kit (Figure 1).</li>
	<li>Fast the sheep for 24 h before the surgery.</li>
	<li>Record live weights prior to surgery.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63823/63823fig01.jpg" /> 
Figure 1: Intravitreal surgery kit. Instruments required for IVT surgery include&#160;(1) a speculum to hold the eyelids open and (2) a pair of curved-nose forceps to grasp the bulbar conjunctiva and rotate the eye. (3) A straight nose hemostat is also included as an alternative instrument to grip the bulbar conjunctiva and hold the eye in place if it has rolled back into the eye orbit. This kit is autoclaved prior to surgery. <a href="https://www.jove.com/files/ftp_upload/63823/63823fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Surgical procedure
<ol>
	<li>Restrain the animal and, using electronic clippers, shave the wool from one side of the neck over the jugular vein.</li>
	<li>Occlude the jugular vein by applying pressure at the base of the jugular groove and visualize the raised vein.</li>
	<li>Draw up the appropriate amount of diazepam (0.3 mg/kg) and ketamine (7.5 mg/kg) into a sterile syringe and attach a sterile 20 G needle. Insert the needle into the jugular vein and gently draw back on the plunger to ensure blood enters the hub and the needle is inside the vein. Once confirmed, induce through intravenous (jugular) administration.</li>
	<li>Immediately after induction, place the animal in dorsal recumbency, extend the neck, and hold the tongue up and forward, using a laryngoscope to visualize the larynx. Perform endotracheal intubation by gently inserting an endotracheal tube (size 6.0-9.0 depending on the size of the sheep) between the vocal cords when the animal exhales. Inflate the endotracheal cuff immediately and secure the tube with a tie around the lower jaw. Confirm air flow through the tube.</li>
	<li>Transfer the sheep to the surgical table and place it in lateral recumbency.</li>
	<li>Immediately connect the endotracheal tube to the hoses of the anesthetic machine for delivery of isoflurane in 100% oxygen. Initially commence with 3%-4% isoflurane and then reduce to 2%-3% for maintenance. Observe the spontaneous ventilation of the sheep.</li>
	<li>Monitor heart (pulse) rate, respiratory rate, oxygen saturation, end-tidal CO2 levels, and rectal body temperature throughout the procedure. See Table 1 for physiological values for these parameters in anesthetized sheep (variable, but use as guidance).</li>
	<li>Place a large, sterile, square drape on a surgical operating cart, followed by the sterile instruments.</li>
	<li>Position a sterile, fenestrated surgical drape over the eye to be injected.</li>
	<li>Aseptically disinfect the eye using a sterile 20 mL syringe to irrigate the eye with 1-5% povidone-iodine solution.</li>
	<li>Apply 1-2 drops Alcaine 0.5% W/V ophthalmic solution, as a local anesthetic, to the eye.</li>
	<li>Fit a Nopa Barraquer-Colibri eye speculum (10 mm) to the eyelids to hold the eye open.</li>
	<li>Grasp the bulbar conjunctiva on the dorsolateral aspect of the eye with forceps, and rotate the eye globe ventromedially.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Conscious</td>
			<td>Anesthetized</td>
			<td>Recommended critical intervention point</td>
		</tr>
		<tr>
			<td>Heart rate (beats/min)</td>
			<td>50-80 (rest) to 280 (active)</td>
			<td>50-80</td>
			<td>&#60;50, &#62;100</td>
		</tr>
		<tr>
			<td>Respiratory rate (breaths/min)</td>
			<td>15-40 (rest) to 350 (overheated)</td>
			<td>&#160;10-30</td>
			<td>&#60;8, &#62;40</td>
		</tr>
		<tr>
			<td>Oxygen saturation (mm Hg)</td>
			<td>95-100</td>
			<td>98-100</td>
			<td>&#60;90</td>
		</tr>
		<tr>
			<td>End-tidal CO2 (mm Hg)</td>
			<td>35-45</td>
			<td>35-45</td>
			<td>&#62;55</td>
		</tr>
		<tr>
			<td>Body temperature (&#730;C)</td>
			<td>38.5-39.5</td>
			<td>38.5-39.5</td>
			<td>&#60;36, &#62;40</td>
		</tr>
	</tbody>
</table>
Table 1: Physiological values of parameters to be monitored in anesthetized sheep.
3. Viral preparation
<ol>
	<li>Store AAV vector aliquots at &#8722;80 &#730;C until use.</li>
	<li>On the day of surgery, thaw the required number of vials for IVT delivery on ice.</li>
	<li>Immediately prior to administration, vortex the viral vector aliquot and centrifuge at 400 &#215; g for 10 s to collect the contents.</li>
	<li>Dilute each viral vector aliquot in sterile-filtered 1x phosphate-buffered saline (PBS) to the desired dose in a final volume of 100 &#181;L. Prepare vector dilutions in a sterile 1.5 mL low-protein-binding microcentrifuge tube using sterile filter pipette tips. Dispose of all consumables that have been in contact with the viral vector in disinfectant solution (see the Table of Materials). 
	NOTE: In the original publication15 the dose of the therapeutic agent (AAV9.CLN5) was 1.9 x 1010 viral genomes. The recommended dosage will vary depending on the therapeutic agent being administered; therefore, a dosage has not been included in the standard protocol presented here.</li>
	<li>Draw the full 100 &#181;L of the AAV vector preparation into a sterile, low-dead space 1 mL syringe with a permanently attached 28 G x 1/2 in needle for immediate injection. Ensure the length of time from preparation to injection is less than 2 min.</li>
</ol>
4. Viral administration
<ol>
	<li>Insert the needle approximately 7 mm posterior to the sclera on the lateral aspect of the eye and angled posteriorly to avoid the lens (Figure 2 and Figure 3). Administer the single injection of 100 &#181;L as a bolus as close to the retina as possible without disturbing the retinal surface.</li>
	<li>Rinse the eye with approximately 10-15 mL of 1-5% povidone-iodine solution followed by 10 mL of saline before removal of the speculum and drape.</li>
	<li>Turn the sheep over and repeat with the other eye if required.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63823/63823fig02.jpg" /> 
Figure 2: Ventromedial rotation of the eye globe. (A) Grasp the bulbar conjunctiva with nontoothed forceps and (B) rotate ventromedially (i.e., down and towards the snout) to expose the dorsolateral surface of the eye for injection. Abbreviations: V = ventral, D = dorsal, M = medial, L = lateral. <a href="https://www.jove.com/files/ftp_upload/63823/63823fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63823/63823fig03.jpg" /> 
Figure 3: Injection location and depth. The needle is injected on the dorsolateral aspect of the eye globe, and the full length of the needle shaft (0.5 in/12.7 mm) is inserted into the eye. Note the angle of the needle toward the posterior of the eye to avoid the lens and inject as close to the retina as possible. <a href="https://www.jove.com/files/ftp_upload/63823/63823fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Postoperative management
<ol>
	<li>On completion of the procedure, stop isoflurane gas inhalation anesthesia, flush the line with 100% oxygen, disconnect the hose from the endotracheal tube, and transfer the sheep to the recovery room.</li>
	<li>Place the sheep in sternal recumbency, with legs tucked underneath, and monitor until full recovery. Ensure the animal&#39;s mouth is clear of any obstructions.</li>
	<li>When the swallowing reflex is observed, partially deflate the cuff of the endotracheal tube and gently remove the tube from the mouth.</li>
	<li>Administer an intramuscular nonsteroidal anti-inflammatory into the biceps femoris muscle of the hind limb, subcutaneous antibiotics on the side of the neck or behind the shoulder, and 0.5% chloramphenicol eye drops to the surface of the eye globe.</li>
	<li>Provide water and food (lucerne pellets and chaff) once the sheep can stand unassisted.</li>
	<li>Administer 0.5% chloramphenicol eye drops 2-3 per day for 7 days post surgery.</li>
	<li>Keep the sheep indoors overnight before returning to the outdoor paddock approximately 24 h post surgery.</li>
	<li>Record rectal temperatures daily for 3 weeks. Monitor for any changes in pulse or respiratory rate, food consumption, neurobehavior, body temperature, weight, posture, eye health, and clinical signs of ill-health. Seek appropriate veterinary treatment if there are any indications of adverse events.</li>
</ol>
6.&#160;Assessing efficacy in vivo
<ol>
	<li>If the goal of the IVT injection is to preserve vision, monitor efficacy in vivo by methods such as maze testing or electroretinography (ERG) to assess retinal cell function or optical coherence tomography (OCT) to assess retinal structure. 
	&#8203;NOTE: These efficacy measures have been well described following IVT gene therapy11,15,16.</li>
</ol>
7. Postmortem tissue analysis
<ol>
	<li>Perform sheep euthanasia by an approved method at an appropriate endpoint following intravitreal injection surgery. 
	NOTE: Suggested euthanasia methods, such as intravenous veterinary euthanasia drugs or a penetrating captive bolt to the cervical spine followed by rapid exsanguination, are detailed elsewhere15,16.</li>
	<li>Harvest sheep eye globes using surgical sharp/blunt curved scissors. Cut the lateral and medial canthus to increase the eye socket opening and then systematically cut through the conjunctival folds, connective tissue, muscles, and optic nerve to free the eye globe from the socket.</li>
	<li>Immersion-fix intact, enucleated eye globes in 10% formalin for 2 h, followed by postfixation in Bouin&#39;s solution for 4 h, making a small (0.5 cm) cut in the sclera to allow sufficient perfusion. Alternatively, immersion-fix the eye globes in Davidson&#39;s solution for 48 h.</li>
	<li>Process sections of eye tissue via routine paraffin wax embedding and sectioning at 3-5 &#181;m. 
	NOTE: Staining procedures for hematoxylin and eosin (H&#38;E) staining and immunohistochemical analysis have been described previously15,16.</li>
	<li>Assess the efficacy in postmortem tissue by measures such as total retinal thickness, retinal layer thickness, counts of outer nuclear layer cellular rows, and immunohistochemical staining for retinal cell types, retinal glia, or proteins of interest. 
	NOTE: For protocols for these analyses, see previous publications15,16.</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+targeting+the+inner+retina+rescues+the+retinal+phenotype+in+a+mouse+model+of+CLN3+Batten+disease.">Gene therapy targeting the inner retina rescues the retinal phenotype in a mouse model of CLN3 Batten disease.</a> Human Gene Therapy. 31 (13-14), 709-718 (2020).</li><li>Kleine Holthaus, S. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+brain-directed+gene+therapy+rescues+a+mouse+model+of+neurodegenerative+CLN6+Batten+disease.">Neonatal brain-directed gene therapy rescues a mouse model of neurodegenerative CLN6 Batten disease.</a> Human Molecular Genetics. 28 (23), 3867-3879 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

Intravitreal injections are one of the most common surgical procedures in human ophthalmology and have proven effective in delivering AAV-mediated gene therapies to the retina of sheep. We had previously demonstrated the efficacy of AAV9.CLN5 gene therapy delivered intravitreally in attenuating retinal dysfunction and degeneration in sheep with CLN5 NCL15. It is hoped that the translation of this route of administration to human NCL patients will also prove beneficial.
...

Several methods can be used to deliver therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. Each route of administration involves overcoming barriers such as the blood-retina barrier or the inner and outer limiting membranes and has varying rates of efficacy depending on the drug being delivered and the specific retinal target1,2.
IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that occupies the posterior chamber of the eye. The primary function of the vitreous humor is to maintain the shape of the eye globe and keep ocular tissues, such as the lens and retina, in place. The vitreous humor is composed largely of water, with small amounts of collagen, hyaluronic acid, and other noncollagenous proteins3. IVT injection is a simple and common procedure used routinely to treat a wide&#160;range of ocular conditions, including age-related macular degeneration, diabetic macular edema, diabetic retinopathy, retinal vein occlusion, and several inherited retinal dystrophies4,5.
Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of fatal lysosomal storage diseases that cause severe degeneration of the brain and retina. There are currently 13 known variants of NCL resulting from mutations in different genes (CLN1-8, CLN10-14) that predominantly affect children but have varying ages of onset and disease severity6. The NCLs share common progressive symptoms, including cognitive and motor decline, seizures, and loss of vision. There is no cure for NCL; however, brain-directed enzyme-replacement therapy is currently in clinical trials for CLN2 disease7,8, and AAV-mediated gene therapy has shown great promise in preclinical studies, with a clinical trial for CLN5 gene therapy expected to begin in 20229,10.
Many other species develop naturally occurring forms of NCL, including cats, dogs, sheep, and cows. Two ovine models of NCL are currently under active study in New Zealand: a CLN5 disease model in Borderdale sheep and a CLN6 disease model in South Hampshire sheep. Affected sheep exhibit many of the clinical and pathological features of the human disease, including retinal atrophy and loss of vision10,11. Although brain-directed CLN5 gene therapy in sheep with CLN5 disease can prevent or halt brain atrophy and clinical decline, the treated sheep do still lose their vision9. This highlighted the need to treat the retina to preserve vision and maintain a better quality of life, leading to the establishment of a protocol for ocular gene therapy in sheep.
The sheep eye represents a good model of the human eye due to its similarity in eye globe dimensions, vitreous volume, and retinal structure10,12,13. This paper details the surgical protocol for IVT delivery of a small volume (&#8804;100 &#181;L) of therapeutic viral vector to the ovine eye.

<table><tbody><tr><td>1 mL low dead-space safety syringe with permanently attached 0.5 inch needle</td><td>Fisher Scientific, Auckland, New Zealand</td><td>05-561-28</td><td>Covidien Monoject Tuberculin Safety syringe or similar</td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Sigma Aldrich</td><td>HS4323</td><td>Autoclave tubes to sterilise prior to use</td></tr><tr><td>Anesthesia machine with gas bench and monitor&amp;nbsp;</td><td>Hyvet Anesthesia, Christchurch, New Zealand</td><td></td><td></td></tr><tr><td>Antibiotic eye drops&amp;nbsp;</td><td>Teva Pharma Ltd, Auckland, New Zealand</td><td></td><td>Commercial name: Chlorafast (0.5% chloramphenicol)</td></tr><tr><td>BrightMount plus anti-fade mounting medium</td><td>Abcam, Cambridge, United Kingdom</td><td>ab103748</td><td></td></tr><tr><td>DAPI (4&amp;prime; ,6-diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich, St. Louis, Missouri, United States</td><td>10236276001</td><td></td></tr><tr><td>Diazepam sedative</td><td>Ilium, Troy Laboratories Pty Ltd, Tauranga, New Zealand</td><td></td><td>5 mg/mL</td></tr><tr><td>Endotracheal tubes</td><td>Flexicare Medical Ltd, Mountain Ash, United Kingdom</td><td></td><td>Standard, cuffed. Sizes 7, 7.5, or 8 depending on sheep size</td></tr><tr><td>Eye speculum</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>KP151/14</td><td>Nopa Barraquer-Colibri (10 mm)</td></tr><tr><td>Fenestrated surgical drape</td><td>Amtech Medical Ltd, Whanganui, New Zealand</td><td>DI583</td><td>Or similar&amp;nbsp;</td></tr><tr><td>Filter Tips</td><td>Interlab, Auckland, New Zealand</td><td></td><td>10, 200, and 1,000 &amp;micro;L&amp;nbsp;</td></tr><tr><td>Formaldehyde solution (37%)</td><td>Fisher Scientific, Auckland, New Zealand</td><td>AJA809-2.5PL</td><td>Make up to 10% in distilled water with 0.9% NaCl</td></tr><tr><td>Goat anti-rabbit Alexa Fluor 594</td><td>Invitrogen Carlsbad, CA, USA</td><td>&amp;nbsp;A-11012</td><td>Use at a dilution of 1:500</td></tr><tr><td>Isoflurane anesthetic</td><td>Attane, Bayer Animal Health, Auckland, New Zealand</td><td></td><td></td></tr><tr><td>Ketamine HCl anesthetic/analgesic</td><td>PhoenixPharm Distributors Ltd, Auckland, New Zealand</td><td></td><td>100 mg/mL</td></tr><tr><td>Laryngoscope (veterinary)</td><td>KaWe Medical, Denmark</td><td></td><td>Miller C blade, size 2</td></tr><tr><td>Needles&amp;nbsp;</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>302025</td><td>BD Hypodermic Needles, or similar</td></tr><tr><td>Non-steroidal anti-inflammatory</td><td>Boehringer Ingelheim (NZ) Ltd, Auckland, New Zealand</td><td>49402/008</td><td>Commercial name: Metacam 20 (20 mg/mL meloxicam)</td></tr><tr><td>Non-toothed forceps</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>AB864/16</td><td>Or similar&amp;nbsp;</td></tr><tr><td>Non-toothed hemostat</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>AA150/12</td><td>Or similar&amp;nbsp;</td></tr><tr><td>Normal goat serum</td><td>Thermo Fisher Scientific, Christchurch, New Zealand</td><td>16210072</td><td></td></tr><tr><td>Oxygen (medical)</td><td>BOC Gas, Christchurch, New Zealand</td><td></td><td>D2 cylinder, gas code 180</td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>Thermo Fisher Scientific, Christchurch, New Zealand</td><td>10010023</td><td>Sterile, filtered</td></tr><tr><td>Povidone-Iodine solution</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>005835</td><td>Commercial name: Betadine (10% povidone-iodine)</td></tr><tr><td>Rabbit anti-cow glial fibrillary acidic protein (GFAP)</td><td>Dako, Glostrup, Denmark</td><td>Z0334</td><td>Use at a dilution of 1:2,500</td></tr><tr><td>Self-complementary adeno-associated virus serotype 9, containing the chicken beta action (CBh) promoter and codon-optimized ovine CLN5</td><td>University of North Carolina Vector Core, NC, USA.</td><td>scAAV9/CBh-oCLN5opt</td><td></td></tr><tr><td>Sodium Chloride 0.9% IV Solution</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>AHB1322</td><td>Commercial name: Saline solution&amp;nbsp;</td></tr><tr><td>Subcutaneous antibiotics</td><td>Intervet Schering Plough Animal Health Ltd, Wellington, New Zealand</td><td></td><td>Commercial name: Duplocillin LA (150,000 IU/mL procaine penicillin and 115,000 IU/mL benzathine penicillin)</td></tr><tr><td>Surgical sharp blunt curved scissors&amp;nbsp;</td><td>Capes Medical Ltd, Tauranga, New Zealand</td><td>SSSHBLC130</td><td></td></tr><tr><td>Terumo Syringe Luer Lock</td><td>Amtech Medical Ltd, Whanganui, New Zealand</td><td>SH159/SH160</td><td>Sterile syringes; 10 mL for drawing up induction drugs, 20 mL for drawing up saline</td></tr><tr><td>Virkon Disinfectant Powder</td><td>EBOS Group Ltd, Christchurch, NZ</td><td>28461115</td><td></td></tr></tbody></table>

intravitreal injections in the ovine eye

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. 
We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

The efficacy of IVT delivery of a CLN5 gene therapy vector in attenuating retinal dysfunction and degeneration in sheep with CLN5 NCL has previously been demonstrated by this research group15. Affected sheep received a single 100 &#181;L IVT injection of CLN5 packaged in an AAV serotype 9 (AAV9) vector (AAV9.CLN5) into one eye, with the contralateral eye serving as an untreated internal control. Vision was assessed monthly from the age at injection (3 months) to end-stage disease (18 months). Post...

Watch this Scientific Journal Video about Intravitreal Injections in the Ovine Eye at JoVE.com

Intravitreal Injections in the Ovine Eye

Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or ...

Research

JoVE Journal

Medicine

3.1K Views.  Lincoln University. Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

Video: Intravitreal Injections in the Ovine Eye

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Biology

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 &#181;L. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 &#181;L of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 &#181;L sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 &#176;C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Immunology and Infection

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats

Therapeutic delivery to the posterior segment of the eye, including the retina and optic nerve, is complicated by the presence of blood-brain and blood-retinal barriers. Small animal models, such as rats, are utilized for studying various ocular pathologies. While therapeutic delivery to the posterior eye is challenging, achieving it is essential for treating ocular disorders, many of which require validation in small animal models for translational relevance. Therefore, two posterior therapeutic delivery techniques are presented: intravitreal injection (IVI) and retrobulbar injection (RBI) for use in adult rats. Additionally, a method for the en bloc removal of the eyes and optic nerves is introduced for various histological and molecular analysis techniques. The dissection protocol enables full observation of the neuro-visual system while minimizing post-mortem injury to retinal and optic nerve tissues. Successful delivery of the therapeutic cyclosporine to the retina and optic nerve was achieved, with detectable concentrations observed twenty-four hours after injection using both IVI and RBI. Furthermore, en bloc retina and nerve samples were successfully extracted for full eye histological tissue analysis, facilitating comprehensive observation of the retina and the wider neuro-visual system.

This study presents a methodology for the delivery of therapeutics into the retina and optic nerves of the adult rat. Additionally, a unique tissue retrieval method is introduced for a top-down en bloc collection of the optic nerve and retina in an adult rat.

Therapeutic delivery to the posterior segment of the eye, including the retina and optic nerve, is complicated by the presence of blood-brain and ...

Neuroscience

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

Optimized Rat Intracameral Injection Technique for Reduced Tissue Damage

Intracameral Injection in Rats with Low Risk of Adverse Effects

Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility.
Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at&#160;an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows&#160;visualization&#160;by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection.
In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.

Author Spotlight: A Novel Protocol for Intracameral Injections to Enhance Precision in Rodent Ophthalmology

This protocol describes a technique for intracameral injection in rats using a central corneal incision and a long tunnel into the anterior chamber. This injection method minimizes the risk of inducing inadvertent tissue damage and thereby improves precision and reproducibility.

Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is ...

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model

Source: John, S., et al. <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021)
This video demonstrates an in vivo imaging technique for detecting and quantifying eye inflammation in a mouse model of post-infectious uveitis. The technique, known as optical coherence tomography (OCT), generates high-resolution cross-sectional images of the eye for assessing disease progression.

Obrazowanie optycznej koherentnej tomografii do oceny zapalenia błony naczyniowej oka w modelu mysim

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