Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats

Summary

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.

Abstract

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.

Introduction

Delivering therapeutics to the retina and optic nerve is incredibly difficult due to the complex anatomy of the eye^1,2, specifically the presence of the blood-retinal barrier (BRB)^3,4,5. The BRB serves to protect the retina from systemic circulation invasion, but is a challenging opponent to therapeutic administration as systemic therapeutic circulation is often blocked by the BRB^6,7. Small lipophilic molecules can readily diffuse through the BRB, but larger and hydrophilic molecules have a harder time gaining access to the retina⁶. Intravitreal (IVI) and retrobulbar (RBI) injections enable the delivery of drugs to the ocular tissues, overcoming the limitations imposed by the BRB. The IVI serves as a promising compromise by administering therapeutics into the internal environment of the eye^8,9. This method requires the drug to cross through the vitreous, thus bypassing the BRB, and diffusing through the retina and choroid in order to reach the optic nerve⁷. The RBI is delivered behind the eye into the retrobulbar space¹⁰. Therapeutics can be delivered by diffusion through the tissues and glands in the retrobulbar space, affecting the optic nerve and surrounding structures without directly entering the retina, which maintains the integrity of the BRB. By delivering drugs directly or indirectly into the eye, both intravitreal and retrobulbar injections can achieve higher local concentrations of the therapeutic drug, which enhances its effectiveness compared to topical or systemic administration (oral or intravenous)². This is particularly important for treatments that require rapid action or high potency, as seen in many ocular diseases. Targeted delivery also limits the exposure of the rest of the body to the drug, which reduces the risk of off-target effects and helps to minimize potential adverse effects that can occur when medications are administered topically, orally, or intravenously¹¹.

Other periocular injections, such as subconjunctival, posterior subtenon, and subretinal, have their own benefits and limitations^2,5. Posterior subtenon injections have been observed to deliver high drug concentrations to ocular tissues; however, the subtenon injection is closer to the scleral than the orbital vasscularture^5,12. In contrast, the RBI places the therapeutic closer to the optic nerve than the posterior subtenon or subconjunctival¹³. This may mean that optic nerve pathologies favor RBI-delivered therapeutics over other periocular injection types. Posterior subtenon injections have associated risks, including strabismus, hyphema, and elevated intraocular pressure⁵. Elevated intraocular pressure is also a reported risk factor in IVI, subconjunctival, and subretinal injections². These injection types often require repetitive dosing in order to achieve the desired therapeutic effect². Other risk factors associated with subretinal injections, subconjunctival injections, and IVI include cataract formation, retinal hemorrhage, retinal detachment, and inflammation². These IVI, subretinal injections, and subconjunctival injections are more invasive than the RBI injections, as these injections are intraocular². The RBI may be considered less invasive as it places the therapeutic in the retrobulbar space, without directly entering the needle into the globe of the eye. Other less invasive therapeutic delivery strategies, such as topical administration, fall short of sufficient drug delivery, with less than 5% of the drug being retained on the ocular surface^2,5.

IVI is a prominent technique in preclinical models that is used for its ability to deliver therapeutic agents directly into the posterior segment of the eye. IVI delivers the drug directly to the vitreous humor, making it a preferred delivery technique for localized treatment¹⁴. The IVI technique allows the therapeutic to bypass the blood-retinal barrier, which is a common hindrance to drug penetration into the retina¹⁴. IVI introduces the opportunity for inflammation and damage to ocular structures, so meticulous adherence to the procedure must be employed¹⁴. To minimize retinal detachment and cataract formation, Chiu et al. describe an IVI approach that emphasizes a 45-degree bevel insertion and injection at the level of the par plana, avoiding the lens, retina, ocular muscle, and vessels¹⁵. In this technique, a 30 G needle is inserted into the nasal sclera for therapeutic delivery¹⁵. IVI is still associated with risks due to its invasive nature. Potential risks include retinal detachment, cataract formation, endophthalmitis, or hemorrhage¹⁶. The invasive nature of IVI techniques also increases intraocular pressure, as shown in an experiment on porcine eyes performed by Ikjong Park et al.¹⁶. The study shows changes in intraocular pressure during different stages of needle insertion and fluid injection. They report substantial variation in intraocular pressure during the procedure¹⁶.

RBIs have been successfully utilized in previous studies as a means of therapeutic delivery to rodents. One such study compared the effects of various prostaglandin analogs given via RBI¹⁷. Albino rats were given an RBI with a 26 G needle of 0.1 mL injectate inserted through the lateral area of the inferior fornix at a 45-degree angle¹⁷. The protocol used in this study was adapted from a previously described method in which the rats were anesthetized via intraperitoneal (IP) injection of chloralhydrate¹⁸. Another study conducted on rats compared topical drops to retrobulbar injections¹⁹. The rats were anesthetized via an IP injection of ketamine/xylazine, and the RBI was given via a 30 G needle¹⁹. In contrast to the previously discussed sedation methods, one study observing the effects of RBI on orbital fat used inhalational isoflurane to sedate the rats prior to RBI²⁰. While these studies provide insight into what anesthetics and needle specifications could be successful, the positioning and handling of the animals during the procedure are not discussed.

Various studies in mice also conduct RBIs for therapeutic delivery methods. One study compared RBI to lateral tail vein injection for successfully inducing nephrotic syndrome²¹. A second study also compared the same two injection techniques in the administration of contrast media for cardiac imaging²². The mice were anesthetized with inhalational isoflurane and injected in the medial side of the eye²². Both studies adapted their RBI method from a previously written protocol. It is important to note that this protocol named their injection as retro-orbital yet described the injection location as the retrobulbar space behind the eye. The authors of this protocol utilized inhalational isoflurane as a preferred sedation method, noting the quick activation and recovery time of the mice²³. For an RBI, the eye was partially protruded from the socket by applying pressure to the skin around the eye²³. Then, the needle was introduced at the medial canthus bevel side down at an angle of 30 degrees and was inserted until it reached the base of the eye²³. Care must be taken when applying pressure to the animal, as accidental blood flow blockage or tracheal collapse may occur²³. The injector is also blind to the needle tip upon insertion, and therefore, damaging the eye is an associated risk.²³ Damaging the eye upon therapeutic administration is a critical risk in this experiment, as causing additional injury directly undermines the results of the study. It also must be noted that the positioning and handling technique previously described was conducted on mice and did not include comments on applicability to rats.

There are many manners in which optic nerve and retina removal have been attempted. One such method explored the removal of optic nerves and eyes en bloc, preserving an intact optic chiasm²⁴. This method is the most comparable to the current study as the individual eyes and optic nerves are also preserved for en bloc removal; however, the optic chiasm is separated. Exercising caution in this procedure would be of the utmost importance due to the complexity of the procedure. In the current method, we begin the dissection via the caudal skull and work rostrally in order to provide access in a way that limits damage to the optic nerves and allows the entire nerve to remain intact. Furthermore, keeping the nerve intact and attached to the eye is crucial to the embedding process, as damage to each part of the nerve can correspond to a different pathological observation²⁴. The orientation of the optic nerve is important to consider as how it is embedded allows for different cross sections, which may be important for histological analysis.

A custom-made device known as the small animal laboratory ophthalmic operating table (SALOOT, an ophthalmic surgery platform) is comprised of a series of 3D printed materials to provide anesthesia and hold the animal in a stable position for ocular therapeutic injections. The SALOOT design allows for stability of the head and ocular structures for ophthalmic procedures, which improves the speed and reproducibility of operations while allowing for gas anesthesia delivery and scavenging of exhalation particulates. The SALOOT is a three-dimensional printed block featuring a concave reduction to hold the rat body with a narrower region at the front to hold the head of the animal into a nose cone with an isoflurane inlet. Beneath the nose cone is a small reservoir and exhaust outlet. The following methods were developed for therapeutic ocular delivery and precise ocular tissue retrieval; they were designed for studying tissues after ocular trauma, so it is crucial to delineate the effects of the trauma, injection, treatment, and dissection to avoid confounded interpretation of findings.

This article presents two ocular therapeutic injection methods, the intravitreal and retrobulbar injections, for use in adult rats. In addition, a tissue retrieval method is presented for the en bloc removal of the intact optic nerve and retina from an adult rat. These techniques enable the investigation of ocular and peri-ocular effects of induced pathology and treatment.

Protocol

All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and approved by the institutional animal care and use committee at Ohio State University. Male Sprague Dawley rats weighing ~200 g and around 2 months of age were used for this study²⁵. Details of the reagents and the equipment used are listed in the Table of Materials.

1. Intravitreal injection (IVI)

1. Attach the isoflurane and waste leads to the SALOOT attachment ports. Anesthetize the animal using standard isoflurane procedures following the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and approved IACUC protocol.
   NOTE: Alternatively, if the animal was already under anesthesia from another ophthalmic testing session (i.e., ketamine/xylazine mix; 90 mg/kg ketamine and 10 mg/kg xylazine), transfer the animal to the ophthalmic surgery platform and titrate anesthesia with isoflurane (3 oxygen: 3 isoflurane ratio) to ensure adequate depth of anesthesia is maintained.
2. Place the ophthalmic surgery platform with the anesthetized animal under a live operating microscope. Assess the anesthetic depth via pedal withdrawal reflex.
3. Place one to two drops of 0.5% tetracaine hydrochloride ophthalmic solution into the eye or eyes being operated on. Apply lubricating topical ophthalmic ointment such as hypromellose 0.3% to the contralateral eye if no manipulation is to be performed.
   1. Then, use one to two drops of 5% povidone-iodine on the ocular surface, and using a cotton swab, apply povidone-iodine to the skin surrounding the eye.
4. Use a 10 µL syringe with a 33 G needle with a style 4 tip at a length of 10 mm and angle of 15 degrees and draw up 0.9% bacteriostatic sodium chloride. Flush the syringe and needle with the saline before dipping the needle into a hot bead sterilizer.
   1. Allow it to cool before proceeding. After the needle has cooled, draw up the therapeutic of interest to the desired amount (4 µL).
      NOTE: For proof of concept studies, non-dilute tattoo ink and Evan's Blue dye in phosphate-buffered saline (PBS) were utilized. Evan's Blue powder was mixed with PBS until opaque. For therapeutic proof of concept studies, the following concentrations were utilized: ibudilast at 1 mg/mL, tauroursodeoxycholic acid (TUDCA) 5 mg/mL, cyclosporine injection, 250 mg/mL, and anakinra 100 mg/0.67 mL. Therapeutic powders were diluted in 0.9% bacteriostatic saline.
5. Using fine ophthalmic forceps with teeth, gently grasp the scleral tissue at the limbus to stabilize. There will be a faint red ring around the iris. Use this ring as a landmark, insert the needle, bevel side down, and inject it into the posterior eye.
   NOTE: Ensure to angle the needle towards the retina and only insert the needle 2/3 of the way (Figure 1). Ensure the needle does not scratch the lens, as this will lead to cataract formation.
6. After the therapeutic has been injected, slowly remove the needle. Close the eye and hold pressure for at least 10-15 s before flushing with saline.
7. Discontinue the animal from isoflurane and remove it from the ophthalmic surgery platform. Place a drop of hypromellose 0.3% in each eye and then allow the animal to recover on a heating pad.

2. Retrobulbar injection (RBI)

1. Attach the isoflurane and waste leads to the SALOOT attachment ports. Anesthetize the animal using standard isoflurane procedures.
   NOTE: Alternatively, if the animal was already under anesthesia from another ophthalmic testing session (i.e., ketamine/xylazine mix; 90 mg/kg ketamine and 10 mg/kg xylazine), transfer the animal to the ophthalmic surgery platform and titrate anesthesia with isoflurane (3 oxygen: 3 isoflurane ratio) to ensure adequate depth of anesthesia is maintained.
2. Place the ophthalmic surgery platform with the anesthetized animal under a live operating microscope. Assess the anesthetic depth via pedal withdrawal reflex.
3. Place one to two drops of 0.5% tetracaine hydrochloride ophthalmic solution into the eye or eyes being operated on. Apply lubricating topical ophthalmic ointment (hypromellose 0.3%) to the contralateral eye if no manipulation is to be performed.
   1. Then, use one to two drops of 5% povidone-iodine on the ocular surface and using an eye spear, apply povidone-iodine to the skin surrounding the eye.
4. Obtain a 0.5 mL insulin syringe with a 28 G needle. Draw up the therapeutic of interest to the desired amount (100 µL).
   NOTE: For proof of concept studies, non-dilute tattoo ink and Evan's Blue dye in phosphate-buffered saline (PBS) were utilized. Evan's Blue powder was mixed with PBS until opaque. For therapeutic proof of concept studies, the following concentrations were utilized: ibudilast at 10 mg/mL, TUDCA at 50 mg/mL, cyclosporine injection 250 mg/mL, and anakinra at 100 mg/0.67 mL. Therapeutic powders were diluted in 0.9% bacteriostatic saline.
5. Using fine ophthalmic forceps with teeth, gently grasp the lower eyelid to stabilize. Insert the needle, bevel side down, at an angle halfway between 6 and 7 o'clock along the lower orbital rim until the back of the ocular socket is felt. Pull the needle back slightly and then slowly inject the therapeutic (Figure 2).
6. Gently and slowly remove the needle. Then, close the eye and hold pressure for at least 10-15 s before flushing with saline.
7. Discontinue the animal from isoflurane and remove it from the ophthalmic surgery platform. Place a drop of hypromellose 0.3% in each eye and then allow the animal to recover on a heating pad.

3. Ocular tissue isolation dissection

1. After the animal has been euthanized via CO₂ asphyxiation or as otherwise stated in approved IACUC Protocol, place the animal in sternal recumbency. Utilize a transverse skin incision over the dorsal neck, extending to the level of the ear pinna.
2. Dissect dorsal musculature to expose the atlanto-occipital joint. Use mayo scissors to incise through the atlanto-occipital joint, separating the head from the body (Figure 3). Using blunt dissection, gently remove the skin from the dorsal cranium from the level of the nose to the dorsal neck, leaving the skin around both eyes intact.
3. Insert a pair of medium straight hemostats or needle drivers into the foramen magnum at the base of the skull. Use the hemostats to gently break through the lateral skull extending from the foramen magnum to the temporal region on both sides (Figure 3).
   1. When removing the top of the skull, keep the hemostats parallel to the dorsal side of the brain. This will ensure the brain remains intact and is not nicked in the bone removal process.
4. Using a flat spatula, gently reflect the brain rostrally to expose optic nerves. Care must be taken so that tension from the weight of the brain is not applied to the optic nerves.
5. With the nerves exposed, take a pair of medium microscissors and make a small incision across the optic chiasm, severing both nerves from the brain. At this point, the brain can be discarded or placed in paraformaldehyde in PBS (4% PFA) for 24 h at 4 °C for histological assessment.
6. Using a pair of small iris scissors and a pair of toothed ophthalmic micro forceps, carefully remove the excess tissue (i.e., eyelids, connective tissue, etc.) from around the eye. Once completed, the eye should be situated in the ocular socket but with only the extraocular muscle and glands still present.
7. Using the hemostats in conjunction with the small iris scissors, cut through the ocular orbit, taking great care not to cut into the optic nerve as it passes through the optic canal. Carefully break away the bone at the back of the ocular socket with the hemostats, and for more precise control, utilize the small dissection scissors.
8. After the bone has been removed, delicately cut around the ocular orbit with a pair of microscissors and fine forceps to remove the fat pads, glands, and extraocular muscles. Ensure to be observant of the optic nerves during this process.
9. The eye should be able to be gently reflected caudally towards the interior of the skull. At this point, use small microscissors and fine forceps to remove the connective tissue surrounding the nerve on the lower skull ridge.
10. Once this tissue has been removed, the eye and full optic nerve should be able to be lifted from the skull en bloc. Repeat these steps for the contralateral eye and nerve.
11. Further, clean the optic nerve and eye of excess tissue such as the dural sheath. If tissues do not need to be preserved for histological analysis, then flash freeze using dry ice or liquid nitrogen before being stored at -80 °C.
12. For mass spectrometry, the retina needs to be isolated from the internal region of the globe, and the optic nerve needs to be isolated. Separate the optic nerve from the globe at the closest exterior point. Place the nerve in a cryotube on ice before being transferred to -80 °C.
    1. Place the globe of the eye on a Petri dish under an operating microscope. Use a pair of curved forceps to hold the eye steady before making an incision at the limbus with a pair of small microscissors.
    2. Extend the cut to encompass the entire circumference of the globe. The globe should be bisected, and the anterior portion can be discarded.
    3. Place the posterior section interior side up, and using a pair of fine ophthalmic tweezers, remove the thin cream-colored tissue. It may appear to stick to the optic disc. If this occurs, use the microscissors to cut the retina away from the disc.
       NOTE: The retinal tissue can then be placed in a cryotube on ice before transferring to -80 °C. Tissues can then be transferred to the mass spectrometry facility for evaluation.
13. For immunohistology staining using partial perfusion, place the intact eye on a Petri dish. Use a pair of curved forceps to hold the eye steady before taking an insulin syringe with a 28 G needle and inserting it at the limbus, with the needle angled towards the retina. Repeat this step at another two points along the limbus. Puncture of the globe helps to ensure adequate perfusion and aids in appropriate fixation.
    NOTE: The globe should be fixed in 4% PFA for at least 40 min prior to transitioning to a shaker and agitating at low speed for an additional 20 min to facilitate fluid movement.
14. Once the fixation step is complete, remove the PFA and replace it with PBS. Place vials on gentle shake for 15 min, and then repeat this step for a second 15 min PBS rinse.
15. After the final PBS rinse cycle, aspirate out the PBS and replace it with 30% sucrose solution in PBS, and then refrigerate for 24 h. Following the sucrose incubation step, embed tissue in OCT for histological evaluation²⁶.

Results

Preliminary pilot experiments were performed on cadaver animals using injection dye (Evans Blue dye) and tattoo ink (Figure 2B) to optimize the placement and size of the needle for both RBI and IVI. Tattoo ink was non-dilute, and then Evans Blue powder was mixed in PBS until the fluid became opaque. We concluded the ideal RBI featured a 28 G needle inserted at an angle halfway between 6 and 7 o'clock along the lower orbital rim until the back of the ocular socket was felt. This delivered the dyes to the back of the orbit, close to the nerve, without puncturing the optic nerve. Similarly, this pilot cadaver dye trial was followed with the IVI injection, but only Evans Blue dye was used as the tattoo ink was too viscous in conjunction with the 33 G needle. Once injection protocols were determined, proof of concept in vivo therapeutic studies were performed utilizing male pigmented Long-Evans rats at 200 g (n = 18). In addition, light-adapted flash visual evoked potentials (fVEPs) were conducted utilizing male Long-Evans rats (n = 2) to determine RBI safety. Baseline fVEP values were recorded, and then 7 days after saline injection (100 µL) to the right eye (OD), fVEPs were once again conducted. Left eyes (OS) received no injection. fVEPs were conducted following pre-existing protocols, in which signals were obtained against a 30 cd/m² background utilizing a white flash (200 cd.s/m²) and an interstimulus interval of 1000 ms²⁶. fVEP amplitudes showed no significant changes between eyes or timepoints (Figure 4). Statistical analysis was performed using a two-wave ANOVA with a 95% confidence interval. Tukey's multiple comparisons tests were conducted in GraphPad Prism to determine group interactions²⁶.

To determine efficacy of ocular therapeutic administration, the following neuroprotective therapeutics of interest were obtained: ibudilast at 10 mg/mL (RBI) and 1 mg/mL (IVI), tauroursodeoxycholic acid (TUDCA) 50 mg/mL (RBI) and 5 mg/mL (IVI), cyclosporine injection, 250 mg/mL, cyclosporine topical, 0.05%, and anakinra 100 mg/0.67 mL. Cyclosporine was administered via RBI (n = 2), IVI (n = 2), and topically (n = 3). Anakinra, ibudilast, and TUDCA were administered via RBI (n = 2) and IVI (n = 2). Drugs were selected for their potential as neuroprotective therapeutics for use in a rat model of traumatic optic neuropathy. Cyclosporine was used in particular as it was highly viscous and, therefore, difficult to inject relative to other therapeutics. Tissues were obtained 24 h after injection following the previously stated method. Tissues were analyzed via mass spectrometry (TUDCA, ibudilast, and cyclosporine) or proteomic analysis (anakinra) (OSU's Pharmacoanaytic Shared Resource CORE). Control retinas and nerves were collected and assessed via Mass Spectrometry and proteomic analysis. Therapeutic-treated samples were then assessed and compared to controls to determine relative levels of drug found in each of the tissues of interest. Anakinra RBI samples were removed from analysis due to animal flinch response during injection, even under deep anesthesia. Due to this pain response, RBI Anakinra administration may not be an optimal therapeutic approach.

Cyclosporine was detected in both the retina and optic nerve 24 h later via both injection mechanisms; however, the topical delivery was not detected in either tissue type. In the IV injection group, the retina had a concentration of 383 ppb, and the optic nerve had <5 ppb. The RB injection route observed 16 ppb in the retina and 49 ppb in the optic nerve (Table 1). TUDCA was not detected in either the retina or optic nerve 24 h after injection for either injection route. Ibudilast was detected in the optic nerve tissue of only one animal after RB injection (<5 ppb). The therapeutic pilot injection study indicated both injection protocols are capable of drug delivery to the retina and optic nerve, as evidenced by cyclosporine's presence in both tissue types after both injection methods. This study also indicates cyclosporine yielded high concentrations within the target tissues of interest. One hypothesis is that ibudilast and TUDCA may have reached the retina and optic nerve, but their half-life in the ocular environment may have been too short to be detected 24 h after injection. It may be that the drugs have a neuroprotective impact at this time; however, further studies would be needed to confirm the pharmacokinetics of these drugs in both the internal (IVI) and extraocular (RBI) regions.

This pilot study also supported the protocol of ocular tissue isolation through the successful removal of the full optic nerve and eyes. Retinas and optic nerves were successfully obtained for analysis in the therapeutic delivery pilot study (Table 1). In addition, through utilizing this ocular tissue isolation dissection, eyes, and optic nerves en bloc were able to be collected for immunohistochemistry (Figure 5). En-bloc samples were collected from two male Sprague Dawley rats, as outlined above. Samples were embedded in OCT and longitudinally sectioned on a cryostat at 10 µm thickness. Samples were incubated in PBS to remove the OCT and then incubated in 1:20 normal donkey serum in PBS plus Triton-X-100 (PBT) at room temperature for 2 h. Sections were then incubated with the following antibodies: anti-β-tubulin (1:1000; MAB5564; Millipore, Burlington, MA) and anti-glial fibrillary acidic protein (GFAP; 1:50; Z0334; DAKO, Santa Clara, CA) in PBT overnight at 4°C, rinsed with PBS, and incubated in donkey anti-mouse Alexa 488 and donkey anti-rabbit Alexa 594 (1:200) in PBT overnight at 4 °C. The sections were rinsed, and mounted in a mounting media plus DAPI. The samples were imaged on a wide-field fluorescence microscope or confocal microscope using consistent settings²⁶. The intact optic nerve head was able to be visualized (Figure 5A,B) and successfully stained for the following markers of interest: β-tubulin (green), glial fibrillary acidic protein (GFAP; red), and nuclear marker DAPI (blue). This dissection method allowed for the obtainment of full optic nerve samples, as seen in Figure 5C.

Figure 1: Schematic of intravitreal (IV) injection technique. A 10 µL syringe with a 33 G needle, length of 10 mm, and angle of 15 degrees is inserted 2/3 of the way into the eye at the limbus. Please click here to view a larger version of this figure.

Figure 2: Schematic and representative image of retrobulbar (RB) injection technique. (A) Schematic image of the RB injection. A 0.5 mL insulin syringe with a 28 G needle is inserted along the lower orbital rim at an angle between 6 and 7 o'clock. The needle is advanced until the back of the ocular socket is felt and then pulled back slightly before injection. (B) Representative image of the RB injection technique using the same needle with black tattoo ink during pilot trials. Please click here to view a larger version of this figure.

Figure 3: Schematic of the major dissection points during the ocular tissue isolation. The dotted lines indicate incision points. (A) Rat displayed dorsally with atlanto-occipital joint marked. (B) Rat skull displayed dorsally with important incision marks noted. Please click here to view a larger version of this figure.

Figure 4: Flash visual evoked potentials (fVEPs) of RBI saline-treated cohorts. (A) Averaged fVEP amplitudes from saline RBI-treated right eyes (OD) and control/non-treated left eyes (OS). No significant differences were detected in the amplitude of fVEP waveforms between groups or between eyes. (B) Right eyes (OD) fVEP waveforms of saline RBI treated eyes at baseline and then at seven days after injection (n = 2 animal samples). Please click here to view a larger version of this figure.

Figure 5: Epifluorescent micrographs of the optic nerve head (A,B) and en bloc eye sample (C) of rodents collected following the Ocular Tissue Isolation Dissection. (A,B) Intact optic nerve heads were collected from the animals and were stained with markers for β-tubulin (green), glial fibrillary acidic protein (GFAP; red), and nuclear marker DAPI (blue) at 20x magnification. (C) En bloc eye sample showing the intact globe and full optic nerve. Please click here to view a larger version of this figure.

Agent	Route	Retina	Optic Nerve
Cyclosporine	IVI	383	<5
Cyclosporine	RBI	16	49
Cyclosporine	Topical	ND	ND
TUDCA	IVI	ND	ND
TUDCA	RBI	ND	ND
Ibudilast	IVI	ND	ND
Ibudilast	RBI	ND	<5
Anakinra	IVI	D	ND
Anakinra	RBI	NA	NA
ND: Not Detected
D: Detected but could not be quantified
NA: Not available

Table 1: Therapeutic pilot injections. The following therapeutic agents were investigated for their presence in the retina and optic nerve 24 h after administration: cyclosporine (topical, IVI, RB), TUDCA (RB and IVI), ibudilast (RB and IVI), and Anakinra (RB and IVI). Cyclosporine was detected in both the retina and optic nerve after both injection routes, indicating our RB and IV injection protocols are capable of ocular drug delivery to the retina and optic nerve.

Discussion

The intricate challenges associated with delivering therapeutics to the retina and optic nerve, primarily due to the impermeable barrier posed by the BRB, underscore the significance of this study^3,4. The exploration of IVI and RBI techniques not only highlights innovative approaches for overcoming these obstacles but also emphasizes the broader implications for ocular care and therapeutic development. These findings demonstrate that both IVI and RBI can facilitate targeted delivery of therapeutics directly to the affected ocular tissues. This targeted approach is vital for achieving therapeutic concentrations that are often unattainable through systemic or topical administration routes^2,11, thereby enhancing the efficacy of treatments for various ocular diseases.

The comparative analysis of these injection techniques reveals their unique benefits: IVI provides direct access to the vitreous and inner retinal layers²⁷, while RBI allows for diffusion to the optic nerve without compromising the BRB's integrity²⁸. These insights contribute to a more nuanced understanding of how best to deliver specific therapeutic agents, ultimately improving treatment outcomes for patients suffering from ocular diseases. The IVI and RBI delivery methods described here successfully delivered therapeutics, such as cyclosporine, to critical tissues, highlighting the potential for developing effective ocular therapies.

Furthermore, through this therapeutic method developmental process, inhalational isoflurane was chosen as the sedation method for this RBI and IVI protocol, as it is fast-acting both in activation and recovery. The SALOOT provides crucial support and stabilization to the rodent, while allowing for a uniform rate of inhaled anesthetic. As discussed previously, some risks associated with RBI include possible damage to the eye due to blind needle insertion, as well as blood flow blockage or tracheal collapse due to the pressure technique outlined in the retrobulbar injection protocol for mice²³. To help mitigate these risks, this protocol utilizes ophthalmic forceps to grasp the lower lid of the animal for stabilization, therefore foregoing the pressure technique and eliminating either blood flow blockage or tracheal collapse from occurring. This technique also gives better control over the animal while the needle is being inserted. The use of forceps combined with the use of the live operating microscope helps to minimize the risk of blind insertion, giving the operator better visualization of needle location. In addition, the surgery platform provides crucial head support, which keeps the skull from moving during the insertion of the needle. The surgery platform also provides an elevation of the head with respect to the rest of the body, which allows for the head to be in a more level position, and since the platform has isoflurane and oxygen hookups, the level of anesthesia is never compromised during positioning. IVI technique was adopted for its unique ability to bypass the BRB by injecting therapeutics directly into the vitreous humor. The technique introduces the opportunity for ocular damage due to its invasive nature, but the risk is minimized by carefully ensuring the needle does not scratch the lens, slowly removing the needle, and applying pressure to the eye for 10-15 s post-injection.

Successful removal and analysis of en bloc retinal and optic nerve samples was possible through this unique tissue retrieval method. Isolation of the eye and nerve en bloc was achieved, which helps with being able to assess the full visual system, as evidenced by the optic nerve head and full eye immunofluorescence staining (Figure 5). This method allows for a top-down visualization of the brain, optic nerves, and globes, which allows for easier preservation of the overall structure and integrity of our tissues.

This study has potential limitations, including the use of only one animal sex and a relatively small sample size. RB injections are also associated with the inherent limitation of therapeutic leakage. After RB injection, therapeutics can migrate from the retrobulbar space to the front of the globe. This technique aimed to minimize this inherent limitation by changing the angle of insertion and keeping the needle as close to the back of the orbit as possible. In addition, it was determined that maintaining light pressure after therapeutic administration for 10-15 s helped to keep the therapeutics from migrating out of the retrobulbar space.

These methods will be utilized in future work for therapeutic intervention in a small animal traumatic ocular injury model. The IVI methods have been expanded on for use in chicks and mice; however, the needle size and therapeutic volumes must be adjusted. For chicks, a 28-29 G insulin needle and 20 µL of therapeutic volume were found to be optimal²⁹, but with mice, it was determined a 31 G needle and 2 µL of therapeutic volume was ideal. The IVI technique changed minimally with translation to other animals. For translation of the RBI to other species, the needle size and volume would need to be adapted, but the overall technique should remain translatable as long as inherent anatomical differences between species are considered.

The insights gained from small animal models are invaluable for advancing the understanding of drug delivery mechanisms and optimizing treatment protocols. Ultimately, this research lays the groundwork for more effective therapies that could significantly enhance the quality of optical care, making strides toward preserving vision and improving outcomes in clinical practice.

Materials

Name	Company	Catalog Number	Comments
Anakinra 100 mg/0.67 mL	Sobi	NDC: 66658-0234-07
Antipamezole hydrochloride (Antisedan) 5.0 mg/mL	Zoetis	NADA #141-033 | 107204-8
Bacteriostatic sodium chloride (0.9%)	Hospira Inc.	NDC: 0409-1966-02
Cryotube	VWR	76417-258	https://us.vwr.com/store/product?keyword=76417-258
Curved forceps	Fischer Scientific	08-953F
cyclosporine injection 250 mg/mL	Perrigo	NDC: 00574-0866-10
cyclosporine topical, 0.05% (Restasis)	AbbVie (Vizient)	NDC: 00023-9163-30
Cyotube Cap	Thermo Scientific	3471BLK	https://www.fishersci.com/shop/products/screw-cap-microcentrifuge-tube-caps/14755237?searchHijack=true&searchTerm= screw-cap-microcentrifuge-tube-caps&searchType=Rapid& matchedCatNo=14755237
Evans Blue	Sigma-Aldrich	E2129-10G
Eye Spears	Fischer Scientific	NC0972725	https://www.fishersci.com/shop/products/ultracell-pva-eye-spears-100-p/NC0972725
Fine forceps	Fischer Scientific	08-953E	https://www.fishersci.com/shop/products/fisherbrand-dissecting-jewelers-microforceps-2/08953E?gclid=Cj0KCQiAkJO8BhCGARIsAM kswyiER9Kanmi3ZMgoXTr82Zg3 g44m1Q6WLftkYfb36hC7pbkwR hVAy3MaAqkLEALw_wcB&ef_id =Cj0KCQiAkJO8BhCGARIsAMks wyiER9Kanmi3ZMgoXTr82Zg3g4 4m1Q6WLftkYfb36hC7pbkwRhV Ay3MaAqkLEALw_wcB:G:s&ppc _id=PLA_goog_2086145680_81 843405274_08953E__38624700 1354_6556597232892883360& ev_chn=shop&s_kwcid=AL!4428 !3!386247001354!!!g!827721591 040!&gad_source=1
Fine ophthalmic forceps with teeth	Fisher Scientific	50-253-8287	https://www.fishersci.com/shop/products/bonn-suturing-forceps-7-5-cm/502538287
Flat spatula	Fischer Scientific	14-375-100	https://www.fishersci.com/shop/products/fisherbrand-spoonula-lab-spoon/1437510#?keyword=
Hot bead Sterilizer	Fine Science Tools	18000-45	https://www.finescience.com/en-US/Products/Instrument-Care-Accessories/Sterilization/Hot-Bead-Sterilizers
Hypromellose 0.3% (GenTeal Tears Severe Dry Eye Gel)	Alcon Laboratories Inc.		https://www.amazon.com/GenTeal-Tears-Lubricant-Ointment-Night-Time/dp/B01IN5G1L0/ref=sr_1_4?dib=eyJ2IjoiMSJ9.DxYpqjIIBNO TVuPo7jln5xeGazA_YFg0cbt3 kCyC-0ouZARw5qIHYvCM7vB R_vO30OWUEXDZhQmQfLQ9 ySld4mujpzrWjxbsEXLBs5JPhjZ eUPgPY0sHoJA46f9EYULdxiTu BQy5fVA2OB20RV09mbdW8hX 6j8-bXIYTZljPGMo5_GMq9jnJo8 3iR35c1THxEiEH2FsvSx7VXup- QK9uCkWwAYrw2v3tyLUCq2JT APPF34nsYqGnSASMgOARU_ 2lVz-kIy-QUEYHGOoIimIWwBY htz33RkFrq7YjtnC2uDbImNiudG zWJv-uUhmJngYjbBGbeWE0VX 7CGPkEokUZrCQ8AI2HeXjSMph gPhMbK88RcHJ63AyH0TiBtS2k1 Xceh-CD26_prJSNxF6Mv5-jgGf9 iLmXvVtKkkSwc-5uYLk7gZHaFC Yj73F_imbmeHYr.4vfu7h4m4Jlfy- qiqmgeAnDHlJTGYV22HJ2w_xD ir0k&dib_tag=se&keywords=Gent eal+gel&qid=1736793609&sr=8-4
ibudilast	Millipore Sigma	I0157-10MG
insulin syringe 0.5 mL with a 28 gauge Micro-Fine IV Needle	Becton, Dickinson and Company (BD)	14-826-79
Isoflurane	Covetrus	NDC: 11695-6777-2
Ketamine	Covetrus	NDC: 11695-0703-1
Long Evans Rat	Charles River Laboratories International, Inc.		https://www.criver.com/products-services/find-model/long-evans-rat?region=3611
Mayo Scissors	Electron Microscopy Sciences	72968-03
Medium microscissors	Amazon		https://www.sigmaaldrich.com/US/en/product/aldrich/z168866#product-documentation
Medium straight hemostats or needle drivers	Sigma-Aldrich	Z168866-1EA	https://www.sigmaaldrich.com/US/en/product/aldrich/z168866#product-documentation
Needle 33 G with a style 4 tip at a length of 10 mm and angle of 15 degrees	Hamilton	7803-05
paraformaldehyde 4 in phosphate-buffered saline (PBS) (4% PFA)	Thermo Fischer	J61899.AK
Petri dish	Millipore Sigma	P5606-400EA	https://www.sigmaaldrich.com/US/en/product/sigma/p5606?utm_source=google&utm_medium= cpc&utm_campaign=8674694095 &utm_content=105162454052& gad_source=1&gclid=Cj0KCQiA kJO8BhCGARIsAMkswygXXfgY ABr7EfLtf4tvuLS0E8A4SxX4XM NJQDaI80Yi4FO-iahCsPcaAp9E EALw_wcB
phosphate-buffered saline (PBS)	Sigma-Aldrich	P3813-10PAK	https://www.sigmaaldrich.com/US/en/product/sigma/p3813
Povidone-Iodine (Betadine) 5%	Alcon Laboratories Inc.	NDC: 0065-0411-30
Shaker Model 3500	VWR	89032-092
Small iris scissors	Sigma-Aldrich	Z265977-1EA	https://www.sigmaaldrich.com/US/en/product/aldrich/z265977&
small microscissors	Fisher Scientific	17-456-004	https://www.fishersci.com/shop/products/self-opening-scissors-2/17456004?keyword=true
Sprague Dawley Rat	Charles River Laboratories International, Inc.	SAS 400	https://emodels.criver.com/product/400
Sucrose	Millipore Sigma	57-50-1	https://www.sigmaaldrich.com/US/en/substance/sucrose3423057501
syringe 10 µL (Model 701 RN)	Hamilton	80330
Tattoo Ink (Intenze Tattoo Ink True Black 1 oz)	Amazon		https://www.amazon.com/Intenze-Tattoo-Ink-True-Black/dp/B01GW747L2
tauroursodeoxycholic acid (TUDCA)	Milipore Sigma	580549-1GM
Tetracaine Hydrochloride Ophthalmic Solution 0.5%	Bausch & Lomb Inc.	NDC: 68682-920-64
Xylazine (Rompun) 100 mg/mL	Dechra	NADA #047-956 |