Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.
Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.
Age-related macular degeneration (AMD) is the most common cause of visual impairment in adults aged 50 or older in industrially developed countries, as it causes loss of central vision. About 15% of these patients suffer from the "wet" form of the disease, in which neovascularization originates from the choroid and disrupts retinal function1. This variant can be treated by a highly effective therapy with repeated intra-vitreal injections of antiangiogenic drugs2. However, the vast majority of patients (~85%) suffer from the dry form, which is characterized by extracellular deposits (e.g., drusen) under the retinal pigment epithelium (RPE). These deposits cause RPE dysfunction leading to retinal atrophy in the macula. Given the lack of any curative therapeutic options, AMD evolved into an intensively developing research field, where many different curative therapeutic approaches are being tested. Surgical RPE replacement is one attractive future possibility to defeat this debilitating disease3.
Autologous subretinal RPE transplantation replaces dysfunctional or lost RPE in macula, and has the potential to restore its physiological function4-9. This surgical technique had a breakthrough with the development of RPE differentiation protocols from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), giving the scientist an unlimited cell source of RPE for transplantation10. RPE transplantation is now recognized as an attractive first-in-human application for stem cell derived therapeutics. The eye offers excellent surgical access and sophisticated in vivo monitoring tools11-13.
To transplant the RPE, one way is with a minimally invasive delivery using a cell suspension, alternatively, to better preserve RPE characteristics and transplant function, artificial carrier substrates (scaffolds) for RPE replacement are being considered4,14,15. Large animal models are required for preclinical validation, yet detailed technical information on animal handling and surgical technique is missing to date16-23.
We and others11,24 despite some evidence to the contrary25, suggest the use of a rigid yet elastic carrier substrate as it provides safer handling, preserves monolayer integrity and functionality. Over time we have tested several custom-designed instruments and ancillary techniques for the implantation of cell-carrier supported RPE transplants into the subretinal space (SRS). We utilized intraoperative video recordings, in vivo scanning laser ophthalmoscopy combined with spectral domain optical coherence tomography (SLO/ SD-OCT), and histology to evaluate the implantation success14,26,27. Here we provide our current recommendation for subretinal RPE implants in rabbits, which were tested in 5 different rabbit strains, 7 cell carrier materials and 4 RPE cell sources in over 150 procedures.
Ethics of animal handling in ophthalmic research: We obtained approval from the Ethics committee of the Medical Faculty, University of Bonn, and adhere to the guidelines stated by The Association for Research in Vision and Ophthalmology (ARVO). Moreover, all procedures were approved by the state regulatory authorities of North Rhine-Westphalia. Animals were held indoors in a specialized facility in an air-conditioned room with temperatures between 18 – 20 °C, exposure to regular daylight, in standardized individual cages with free access to food and water.
Note: To ensure the animals operative affinity, an animal health score sheet is followed which includes the following definitive animal exclusion criteria: 20% weight loss compared to weight on admission; apparent cyanosis of the animal; animal shivers, has cramps or cannot move in coordination; ataxia/ paresthesia, e.g., paralyses; apathy; extreme auto mutilation (skin wounds, severed limbs).
1. Instrument Sterilization
2. Instrument Preparation
3. Preparation of Anesthesia and Positioning of the Animal
4. Vitrectomy
5. Loading Shooter
Note: The work described herein does not fall under the tenets of the Declaration of Helsinki; it did not involve human patients. Here, standard RPE cells were isolated from fetal human eyes, cultured and differentiated on uncoated 10-µm-thick polyester (PET) inserts according to our previously published protocol14. A permission to work with the human fetal material was obtained from the ethics committee of the University of Bonn. Alternatively, hES-RPE were shipped from the Skottman lab (manuscript in prep.), where they were cultured according to the technique described by Vaajasaari et al.32; for these cells a permission has been obtained from the R. Koch Institute, Berlin, Germany.
6. Implantation
7. Ending Operation
8. Post-operative Animal Care
9. SLO/SD-OCT Guidance
The results from the described method for subretinal implantation are shown in Table 2. Engraftment under the retina had a success rate of ca. 61% when a core vitrectomy was performed and rose up to 76%, when posterior vitreous detachment was induced. These numbers include ca. 21% of animals who died either intraoperatively or in the first 3 postoperative days. This technique can be used to implant two scaffolds on different retinal areas in one eye simultaneously.
The rabbits underwent postoperative in vivo follow up using SD-OCT and histologic processing as described by Stanzel et al.27 (Figure 2). Fig. 2A shows scanning laser ophthalmoscope infrared reflectance image of implanted cultured RPE on a polyester membrane (PET) after an uncomplicated transplantation. The halo around implant corresponds to photoreceptor atrophy. Fig. 2B shows corresponding SD-OCT, notice retinal, mainly outer nuclear layer (ONL) thinning, hyper reflective band on SD-OCT above implant, while the neural retina adjacent to the implant shows near-normal reflection bands. These results suggest an atraumatic delivery. Fig. 2C shows Hematoxiline/Eosin (H/E) stain of the implant which shows subretinal scarring and ONL atrophy around the retinotomy site likely as a result of iatrogenic manipulation, a contiguous yet irregular pigmented layer over PET. Bruch's membrane underneath the implant also appeared to be contiguous, and the choriocapillaris contains some scattered erythrocytes. These morphologic result are comparable to the SD-OCT and strengthen the thesis of an atraumatic delivery.
Figure 1: Loading Shooter with Human iPSC-RPE Cultured on PET Cell Carrier. A) Shows punching out an implant using custom made needle. B) Bean-shaped implant cut from cell culture. C) Positioning of implant before loading. D) Loading of shooter with implant. Please click here to view a larger version of this figure.
Figure 2: Cultured Human hES-RPE on PET Cell Carrier 4 Weeks in Rabbit Subretinal Space. A) Shows SLO infrared reflectance image, the green line demarcates the cross-section shown in Fig. 2B. B) Corresponding SD-OCT. C) H/E stain, see text or26,27 for details. Please click here to view a larger version of this figure.
Parameter | Used settings |
Vitrectomy | 6,000 cuts/min |
Vacuum | 200 mmHg |
Rise time | 1 sec |
Air | 24 mmHg |
Irrigation | 24 cmH2O |
Diathermy | 30% |
Table 1: Parameter Setup of the Vitrectomy Machine.
Vitrectomy | Implant | Rabbits operated | Successful implant | Failed implant | Death | Success rate% |
Core vitrectomy | PET | 30 | 19 | 4 | 7 | 63.33 |
PET + RPE | 70 | 42 | 12 | 16 | 60 | |
PVD, ± plasmin w/PPV | PET | 28 | 21 | 2 | 5 | 75 |
PET + RPE | 22 | 17 | 2 | 3 | 77.27 |
Table 2: Summary of the Last 150 Operations Including Method and Implant Type.
Using a rabbit model, a safe and reproducible method is presented for transvitreal delivery of cultured RPE on cell carriers into the subretinal space with a custom-designed shooter instrument. The described method offers a short/optimized surgical technique for easy learning, as it involves standard techniques in vitrectomy with subretinal maneuvers. Outcome is greatly facilitated by a clean vitreoretinal interface, and intraocular infusion that avoids fluid turbulence over the implantation site, inducing bleb retinal detachment (bRD) at low IOP, preventing retina and sclera damage through dryness, and appropriate positioning of the rabbit.
We caution however, as several intra-operative complications may occur any time, hindering the success of the implantation, for instance intra ocular bleedings, anesthesia fading off during vital steps such as the implantation, collapsing of bRD due to instrument manipulation or ocular hypotony, rabbit death due to excessive doses of anesthesia, low blood pressure during long operation causing hypoxic brain damage, or hyperthermia. Yet these complications decrease with time as they are quickly tackled and resolved by increasing experience of the surgical team.
Some complications could be reduced by following a few simple, yet crucial steps. Lubricant should be added every 5 – 10 min to prevent corneal, scleral and conjunctival damage during the operation, and to maintain a clear intraocular media, as dried/blackened sclera may be a cause of wound dehiscence, which in turn leads to ocular hypotony and/ or intraoperative leakage from sclerotomies. Heparin should be added to prevent the formation of a fibrin film that makes particularly subretinal implantation challenging and simultaneously adding epinephrine to reduce bleeding under heparin16. Too long heparin/ epinephrine exposure times (>1 hour) should be avoided to prevent corneal edema by endothelial decompensation33, hypertensive crisis or intraoperative fatality. Meticulous vitreous removal should be performed at instrument (entry) port to avoid retinal and/ or choroidal detachments. Intraocular instruments should be pointed towards posterior pole to avoid lens touch (causes iatrogenic cataract formation) or (entry site) retinal damage. An intraocular side-port infusion cannula should be used, as it attenuates the jet stream around the implantation area, thus preventing uncontrolled tearing of the retinotomy, and collapse of the bRD. bRD induction in the midline (vertical axis from optic nerve) or close to optic medullar fibers should be avoided to prevent extensive iatrogenic retinal detachments. Finally, last but not least bRD should be induced at low IOP, to avoid subretinal BSS injection using excessive flow rates which may lead to a retinal damage (e.g., by stretching).
Many study variables such as cell carrier variants, fetal, adult or stem cell derived RPE cell sources, choices for immunosuppressants, etc., can be explored 14,26,27,34. Further improvement such as serum-free RPE culture methods, characterization of xenoRPE in subretinal space, removal of the host RPE layer14 or strategies for implant anchorage are current work in progress.
To date the described techniques have been used on 5 different rabbit strains, including chinchilla bastard, Chinchilla bastard/KBL hybrids, New Zealand White/Red Cross, New Zealand White (albino) and Dutch belted. Both male and female rabbits were operated on, with rabbits at least 1.5 kg or 2 months of age (depending on species). Most surgeries were on pigmented rabbits (chinchilla bastard or chinchilla bastard hybrids) with weights between 2.5 – 3 kg.
All of the rabbit strains we have had the opportunity to work with seem to have some peculiarities. Given the exclusive availability of pigmented rabbits of the chinchilla bastard strain in Germany in 2009-13, we have collected the most experience with these animals. Unfortunately it is no longer available, since breeding has been discontinued, but compares very well to New Zealand White/Red Cross except for the more advantageous thicker sclera and larger eye volumes in the latter. Chinchilla bastard hybrids have significant intraoperative fibrin formation and require heparin/epinephrine use as outlined above to ensure successful subretinal maneuvers. This protocol has also been performed in non-pigmented albino rabbits (New Zealand White), however particularly bRD creation and subretinal implantation is more challenging given reduced contrast appreciation. The feasibility of inducing a posterior vitreous detachment did not seem rabbit strain dependent in our hands.
Transvitreal subretinal delivery is likely the future surgical strategy of choice given it is the most common route nowadays clinically to access the retina. As a result many other groups have presented such techniques for cultured RPE on carrier supports in animal work11,15,23,35. Aramant et al.36 have an instrument, which places rather than pushes their hydrogel-encapsulated soft implant to its subretinal target site. The design of Thumann et al. utilizes a hollow spatula, which releases the carrier-supported graft by floating it off through fluid injection 19. Both former strategies require subretinal insertion of the instrument, which in our view is more prone to complications, when compared to an epiretinally appositioned instrument. Montezuma et al.22 described a subretinal inserter instrument for the delivery of subretinal chip implants in pigs but no further work has been published since to the best of our knowledge. We have been able to extend the described technique with some modification to pig.
Our preferred cell carriers are 10 micron thick polyester terephthalate (PET) membranes. From a surgical perspective, this material has favorable stiffness and elasticity parameters, in addition to its broad versatility during cell culture experiments. We found similar experiences with expanded tetrafluoroethylene (ePTFE) 37 or nanofiber membranes electrospun from PET, poly-lactic/capronolactic acid (PLCL) or poly–lactic-co-glycolic acid (PLGA), as well as composite nanofiber (PLGA or PET) and ultrathin PET 26. When PET membranes are used with our metallic shooter instrument, they do have an occasional tendency to exhibit electrostatic charge, which challenges their ejection from the shooter 27. Ultrathin polyimide membranes could in our hands not be implanted in the subretinal space with the protocol outlined above (manuscript in preparation).
Marmor et al. have systematically studied spontaneous resorption of subretinal fluid in iatrogenic localized retinal detachments38-41. Even following manipulation in the subretinal space these were found to be reabsorbed by postoperative day 4 in uneventful surgeries. Laser retinopexy is not performed to secure the edges of the retinotomy. Although counterintuitive when compared to human surgery, air/ gas tamponade is not required. Unless meticulous removal of peripheral vitreous can be achieved, particularly in the superior quadrant, this may in fact result in giant retinal tears originating from the retinotomy site. It is only recommended to perform fluid air exchange with subsequent 20% SF6 gas tamponade to salvage intraoperative iatrogenic retinal detachments or in case a particular implant position needs to be secured.
Although the mechanically induced ablation of the neural retina can cause RPE and photoreceptor damage in rabbits42,43, its extent varies greatly (even with regular BSS) depending on factors such as IOP, syringe type used, injection volume with thereby induced retinal stretching, etc. We have also tested the often recommended Ca/Mg-free BSS facilitated detachment 42-44, but found that it causes intraoperative lens opacification (particularly with elevated temperature), and significantly delays or even impairs retinal re-attachment 27. Slow subretinal injection of 20 – 30 µl volume of regular BSS with a 100 µl syringe is therefore recommended; injection needle movements should be minimal so the retinotomy seals around it and prevent Bruch's membrane damage. Some of the iatrogenic damage may be resolved by RPE wound healing, and the observed relative preservation of ONL thickness after reattachment, suggests that the RPE/photoreceptor complex can tolerate this impairment, as also described by others45.
Cell-based therapeutics or retinal prosthetics require preclinical animal testing prior to regulatory approval and commencing human safety studies. The former vary from country to country. The rabbit model described here may serve as a cost-efficient and less challenging platform for establishing or even carrying out all requirements by regulatory authorities. Moreover, it may subsequently serve for training of surgeons in eventual multicenter clinical trials or further improvements of the technique along the way.
The authors have nothing to disclose.
Supported by Rüdiger Foundation grants in 2008 & 2010 (BVS), BONFOR/Gerok Scholarship O-137.0015 (BVS), BONFOR/Gerok Scholarship O-137.0019 (FT), Deutsche Forschungsgemeinschaft/ DFG (BVS) STA 1135/2-1, Chinese Scholarship Council No. 2008627116 (ZL) and an unrestricted grant by Geuder AG, Heidelberg (Fig. 2). Members of H. Skottman's laboratory, University of Tampere Finland are gratefully acknowledged for providing hES derived RPE shown in Fig 2.
s30 ultrasonic cleaning unit | Elmasonic | 100 4631 | 2.75L |
DE-23 autoclave | Systec | C 2209 | 23L |
Syringe | BD | 300013 300995 301285 300294 300330 |
1ml x3 2ml x3 5ml x1 10ml x1 20ml x1 |
Needle | BD | 305196 305136 |
18G x 1 27G x5 |
Scalpel | Feather | 2975#20 | blade#20 x3 |
Surgical drape | HARTMANN LOHMANN & RAUSCHER |
277 502 25 440 |
60×40 cm x2 12×17 cm |
Ocular sticks | LOHMANN & RAUSCHER | 16 516 | 66×5 mm |
Twister gauze sponges | HARTMANN | 481 274 | x2 |
Closure strips | HARTMANN | 540 686 | x4 |
Opmi Visu CS Microscope | Zeiss | N/a | incl. fundus imaging system BIOM II |
Chandelier endoillumination | Geuder | G-S03503 + G-S03504 |
25G incl. trocar |
Light machine | Geuder | G-26033 | Xenotron III |
Vitrectomy machine | Geuder | G-60000 | MegaTRON S4 S4/ HPS |
Vitrector | Geuder | G-46301 | MACH2 vitreous cutter 23G |
Venturi cassette | Geuder | G-60700 | |
Sideport-infusion cannula | Geuder | custom | 1x23G |
3-0 silk suture | ETHICON | V546G | x1 |
Caliper | Geuder | G-19135 | x2 |
Vannas scissors | Geuder | G-19777 | x1 |
Sclerotomie blade | Ziemer | 21-2301 | 1x23G 1x20G |
7-0 silk suture | ETHICON | EH6162H | x1 |
Needle holder | Geuder | G-32320 | x2 |
Iris forceps | Geuder | G-18910 | x1 |
Colibri forceps | Geuder | G-18950 | x1 |
Extendible subretinal injection needle | DORC | 1270.EXT | 41G |
VR scissor | Geuder | G-36542 | 25G |
Grieshaber forceps holder | Alcon | 712.00.41 | 23G |
Curved scissor forceps tips | Alcon | 723.52 | 23G |
Implant loading station | Dow Corning | 3097358-1004 | SYLGARD 184 Silicone Elastomer Kit |
blunt oval implant trephine | Geuder | custom-made | 2.4 x 1.1 mm |
Shooter dummy | Geuder | G-32227 | x1 |
Shooter | Geuder | G-S03443 | x1 |
Flute needle | DORC | 1281.SD | 20G (Vacuum) |
Manual microliter syringe | Hamilton | 24535 | 100µl |
Tissue culture plates | Greiner bio-one | 664160 | 100 x 20 mm |
Spectralis Multi-Modality Imaging System |
Heidelberg 工程学 |
N/a | Spectralis HRA + OCT |
Drugs and solutions | |||
Name | Company | Active agent | Comments |
Mucadont-IS | Merz Hygiene | virucidal instrument disinfectant | 2L |
Mucocit T | Merz Hygiene | Aldehyde-free instrument disinfectant | 2L |
Ketamin 10% | WDT | Ketamine | 10ml (100mg/ml) |
Domitor | Orion Pharma | Medetomidine hydrochloride | 10ml (1mg/ml) |
Antisedan | Orion Pharma | Atipamezole hydrochloride | 10ml (5mg/ml) |
Neosynephrin POS 10% | URSAPHARM | Phenylephrine HCl | 10ml |
Mydriacyl | Alcon | Tropicamid | 10ml (5mg/ml) |
Methocel 2% | Omni Vision | hydroxypropyl methylcellulose | 10g |
PURI CLEAR | ZEISS | Balance salt solution (BSS) | 500ml |
Glucose 5% | B.Braun | Glucose 5% solution | 100ml |
Heparin-Natrium-25 000 | Ratiopharm | Heparin | 5ml (2500 unit/ml) |
Suprarenin | SANOFI | Epinephrine | 1ml (1mg/ml) |
Triamcinolone | University of Bonn pharmacy | preservative-free Triamcinolone | 1ml (40mg/ml) |
Isoptomax eye ointment | Alcon | dexamethasone 1 mg/g neomycin sulfate 3,500 IU/g polymyxin B sulfate 6,000 IU/g |
10ml |
Betaisodona | Mundipharma | Povidon-Iod | 30ml (1g/10ml) |
Optive | ALLERGAN | sodium carboxymethylcellulose glycerol | 10ml |