Surgical techniques and outcome assessment of a novel vascularized orthotopic rodent whole eye transplantation model

Abstract

Currently there are no surgical solutions to restore vision in the irreversibly blind. Whole eye transplantation (WET), is an appealing surgical approach for restoration, replacement, and reconstruction of nonfunctioning eyes. Development of a reliable animal model to test the integrity and functionality of the transplanted eye is an essential step towards clinical whole eye transplantation. This study presents a feasible vascularized orthotopic eye transplantation preclinical rat model to study the structural and functional outcomes of whole eye transplantation. Syngeneic orthotopic transplants were performed in rats, involving anastomoses between carotid arteries, external jugular veins, and optic nerve coaptations of donors and recipients. The transplanted and recipient native eyes were assessed by ocular exam under anesthesia, optical coherence tomography (OCT), histology, magnetic resonance imaging and electroretinography. A 100% surgical survival rate of recipients with maintained long-term health demonstrated this to be a reliable and reproducible model. Assessment from clinical examination under anesthesia revealed that segments of native eyes appeared normal throughout the duration of the study, but transplanted eyes presented mild chemosis of the eye lids, mild ciliary flush of the conjunctiva, cornea neovascularization, mild engorgement of the vessels in the iris, and mild opacities in the lens in some animals. Most of these findings improved over time after transplantation. Doppler optical coherence tomography corroborated the presence of blood flow in transplanted retinas. There was no significant difference in measured IOP between native and transplanted eyes. Both histology and OCT scans demonstrated increased central corneal thickness and decreased total retinal thickness in transplanted eyes. Transplanted eyes exhibit minimal scotopic and photopic ERG responses. To date, no other vascularized orthotopic rodent WET transplantation models have been described in the literature. As functional visual return remains the ultimate goal, this model provides a foundation for future translational strategies and is ideal for testing immunomodulatory, neuroprotective, and neuroregenerative approaches either individually or in combination, as required for total human eye allotransplantation (THEA) to become a clinical reality.

Fig 1. Experimental Design.

Syngeneic whole eye transplantation was performed by using 23 pairs of age-matched male Lewis (RT1) rats to generate n = 4-6/group/time point for structural and functional assessment. Additional animals (dashed arrow) were used if ocular conditions prevented further analysis by optical coherence tomography and electroretinography. These WET animals were not used for ocular examination under anesthesia and histologic analyses.

Fig 2. WET Donor Flap Preparation.

A) An elliptical incision is made with a linear extension for exposure of the carotid artery and external jugular vein. B) Skin around the eye and auricle is included. C) Part of the temporal bone is left with the flap to protect the pterygopalatine and ophthalmic arteries and part of the trigeminal nerve posterior to the orbital content is preserved to protect the pterygopalatine artery running beneath its membrane. D) Schematic of donor flap.

Fig 3. WET Recipient Procedure.

A) Vascular anastomoses. B) Coaptation of the optic nerve. C) Lateral and D) frontal view of recipient at PO week 4. Note the comparable color in the eyes and ears between both sides, indicative of restoration of blood flow. E) Schematic of optic nerve coaptation, venous cuff anastomosis, and insetting of the flap. General topography of the nerve was maintained during coaptation of the nerve.

Fig 4. Representative example of EUA by clinically trained ophthalmologist.

Notes reflect EUA for transplanted eye of a single recipient animal examined at 1, 3, and 7 weeks PO following WET. Fundoscopic examination evaluated viability and structural integrity. Slit lamp examination was used to assess the eyelids, conjunctiva, cornea, anterior chamber, iris, lens, and fundus. All rats examined under slit lamp had a vascularly intact graft with a patent vascular network. For the animal in Figure 4, there was mild chemosis and hemorrhage in the conjunctiva, mild perilimbal injections, mostly transparent cornea with some degree of peripheral neovascularization, mild-to-moderate cataracts, and red reflex elicited for all fundus exams. These ocular findings improved or remained the same during the follow-up period (see ST1). PEE = Punctate epithelial erosions. Nl = Normal.

Fig 5. Optical Coherence Tomography (OCT) b-scans of the cornea and retina from A) age-matched naïve, native, and transplanted eyes.

Representative images demonstrate gross maintenance of ocular structures in transplanted eyes from PO 1-7 weeks following WET. Measurements of SD-OCT scans for B) central corneal thickness (CCT) and C) total retinal thickness (TRT) are plotted for native and transplanted eyes relative to naïve measurements. Following WET, transplant eye CCT was thicker relative to native or naïve eyes, with TRT thinning of the transplanted retina that was particularly apparent within the anterior hyperreflective layer.

Fig 6. Histological changes in retina and cornea after WET.

Representative examples of hematoxylin and eosin (H&E) stained A) corneas of naïve and recipient native (OS) and transplanted (OD) eyes at PO weeks 3 and 7 show corneal thickening in transplanted eyes compared to naïve eyes. B) retinas from naïve and recipient native (OS) and transplanted (OD) eyes at PO weeks 3 and 7 show retinal thinning in both native and transplanted eyes compared to naïve eyes. C) Representative image of retinal layers with labels. Histological samples were measured to assess the average D) central corneal thickness (CCT) and E) total retinal thickness (TRT) for native and transplanted eyes after WET and was plotted relative to values from age and sex-matched naïve eyes. Additionally, measurements from identified retinal layers including: the retinal Nerve Fiber Layer (NFL), Ganglion Cell Layer (GCL or NFL-GCL as the two were difficult to distinguish), Inner Plexiform Layer (IPL), Inner Nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL) and Photoreceptor Layer (PL) plotted relative to naïve values at PO weeks 3 and 7 for native and transplanted eyes.

Fig 7. Gadolinium-enhanced imaging (Gd-enhanced MRI) can be used to demonstrate restoration of aqueous humor dynamics after WET.

Gd-enhanced MRI of aqueous humor dynamics T1-weighted images at A) 0-10 min and B) 60-70 min after systemic Gd contrast agent administration, showing obvious Gd enhancement in the anterior chamber (AC) but not the vitreous or lens in both eyes at PO week 3. C) Average time courses of % Gd enhancement in anterior chamber and vitreous of both eyes at PO week 3. D-F) Quantitative comparisons of D) initial rate of gadolinium increase E) peak % gadolinium signal enhancement and F) time to peak in the AC and vitreous (V) of native (OS) eye and transplanted (OD) eye at PO week 3 (two-tailed paired t-tests, P < 0.01). G) Anatomical T2-weighted images of the intraorbital optic nerves (arrows) projected from the native (OS) eye and transplanted (OD) eye at PO week 3 after WET. Note the presence of the optic nerve sheath surrounding the optic nerves of both eyes. H) Fractional anisotropy map of the prechiasmatic optic nerves (arrows) in the recipient rat using diffusion tensor MRI. Lower fractional anisotropy was observed in the optic nerve projected from the transplanted (OD) eye indicative of compromised white matter integrity.

Fig 8. Transplanted eyes exhibit minimal scotopic and photopic ERG responses to stimuli after WET.

A) Representative display of all scotopic responses from native and transplant eyes recorded from one dark-adapted rat stimulated with increasing light intensity ranging from 0.0001 – 200.0 cd * s * m^-2 revealing present but attenuated rod response from photoreceptor of transplanted (OD) eye vs contralateral native (OS) eye at PO week 7. B) Representative a-wave and b-wave amplitude and C) implicit time/latency under scotopic conditions for both the native and transplanted eyes. See SF4 for mixed-model analysis of animals (n = 6). D) Photopic (cone-driven) ERG measured from the only animal (1/6) that responded bilaterally to light stimulus ranging from 0.0001 – 16.0 cd * s * m^-2. E) Summary of a-wave and b-wave amplitude and F) implicit time under photopic conditions for both the native (OS) eye and the transplanted (OD) eye from one of six animals that displayed measurable ERG response to light stimulus ranging from 0.3 – 16 cd * s * m^-2. During a recording, dark-adapted eyes are exposed to standardized stimuli and the resulting signal is displayed showing the time course of the signal’s amplitude/ voltage. The retinal responses are recorded simultaneously. Signals are measured in microvolts. G) Schematic of a-wave and b-wave measurements under scotopic conditions. A-wave amplitude is measured from the baseline to the first trough (solid arrow). B-wave amplitude is measured from the a-wave trough to the following positive peak (dashed arrow).