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. Author manuscript; available in PMC: 2023 Apr 15.
Published in final edited form as: J Neurosci Methods. 2022 Feb 22;372:109534. doi: 10.1016/j.jneumeth.2022.109534

In vivo MRI Evaluation of Anterograde Manganese Transport along the Visual Pathway Following Whole Eye Transplantation

Chiaki Komatsu 1,#, Yolandi van der Merwe 2,#, Lin He 1,3, Anisha Kasi 4, Jeffrey R Sims 4, Maxine R Miller 1,2, Ian A Rosner 1, Neil J Khatter 5,6, An-Jey A Su 1,5, Joel S Schuman 4,7,8,9, Kia M Washington 1,2,5,10,&, Kevin C Chan 2,4,7,8,9,11,&,*
PMCID: PMC8940646  NIHMSID: NIHMS1783352  PMID: 35202613

Abstract

Background:

Since adult mammalian retinal ganglion cells cannot regenerate after injury, we have recently established a whole-eye transplantation (WET) rat model that provides an intact optical system to investigate potential surgical restoration of irreversible vision loss. However, it remains to be elucidated whether physiological axoplasmic transport exists in the transplanted visual pathway.

New Method:

We developed an in vivo imaging model system to assess WET integration using manganese-enhanced magnetic resonance imaging (MEMRI) in rats. Since Mn2+ is a calcium analogue and an active T1-positive contrast agent, the levels of anterograde manganese transport can be evaluated in the visual pathways upon intravitreal Mn2+ administration into both native and transplanted eyes.

Results:

No significant intraocular pressure difference was found between native and transplanted eyes, whereas comparable manganese enhancement was observed between native and transplanted intraorbital optic nerves, suggesting the presence of anterograde manganese transport after WET. No enhancement was detected across the coaptation site in the higher visual areas of the recipient brain.

Comparison with Existing Methods:

Existing imaging methods to assess WET focus on either the eye or local optic nerve segments without direct visualization and longitudinal quantification of physiological transport along the transplanted visual pathway, hence the development of in vivo MEMRI.

Conclusion:

Our established imaging platform indicated that essential physiological transport exists in the transplanted optic nerve after WET. As neuroregenerative approaches are being developed to connect the transplanted eye to the recipient’s brain, in vivo MEMRI is well-suited to guide strategies for successful WET integration for vision restoration.

Keywords: Anterograde transport, magnetic resonance imaging, manganese, neuroregeneration, optic nerve, whole-eye transplantation

1. Introduction

Approximately 43.3 million people worldwide are suffering from complete blindness (Blindness et al., 2021). In many cases, vision loss is irreversible because of severe ocular trauma or the inability of the retinal ganglion cells in the eye to regenerate after injury. Recently, we introduced a whole-eye transplantation (WET) small animal model, which provides the entire optical system using a vascularized orthotopic allograft to recipients (Washington et al., 2015). Successful translation of WET to the clinic requires the use of non-invasive techniques to monitor structural and functional outcomes after WET. Previously, we reported the use of several methods to evaluate these metrics (Washington et al., 2016; Washington et al., 2015). For example, optical coherence tomography (OCT) demonstrated restoration of blood flow in WET recipients and also showed preservation of both corneal and lens transparency. Additionally, gadolinium-enhanced magnetic resonance imaging (MRI) revealed functional aqueous humor dynamics and existing blood-ocular and aqueous-vitreous barriers after WET (Van der Merwe et al., 2015; Washington et al., 2016; Washington et al., 2015). Overall, these findings suggested that WET can maintain the integrity and dynamics of the transplanted eye.

Apart from the ocular structures within the globe, it is necessary to determine whether the transplanted optic nerve is physiologically functional and connected to the recipient’s brain. To date, it remains to be elucidated whether axoplasmic transport is maintained in the transplanted and recipient visual pathways for cellular and axonal communications, as well as for transfer of nutrients and waste products after WET. Fluorescent probes allow for anterograde and retrograde tracing of neurons with high specificity and resolution (Heilingoetter and Jensen, 2016). However, fluorescence imaging has limited penetration depth for assessing the visual pathway deep in the brain. Thus, neuroregeneration in the visual system is typically assessed ex vivo via histology after eye and brain tissue harvesting, rendering non-destructive and longitudinal whole-brain evaluation difficult. In contrast, in vivo MRI allows for non-invasive, three-dimensional, and quantitative assessments of the visual system across multiple time points with no depth limitation. Among the contrast-enhanced MRI techniques, manganese-enhanced MRI (MEMRI) has been used to probe the structure and function of neural circuits by enhancing neuronal tracts and detecting changes in neural activity and axon integrity (Chan et al., 2014a; Deng et al., 2019; Massaad and Pautler, 2011; Pautler et al., 1998; Silva and Bock, 2008). Divalent manganese ions (Mn2+) have several important properties that give MEMRI its unique advantages. First, Mn2+ is paramagnetic and can act as a T1-positive MRI contrast agent, enhancing tissues where Mn2+ has accumulated on T1-weighted MRI (Mendonça-Dias et al., 1983). Next, Mn2+ is similar to Ca2+ in terms of atomic size, chemistry, and valence, which allows Mn2+ to act as a Ca2+ analogue, passing through voltage-gated Ca2+ channels and entering excited cells (Takeda, 2003). This allows MEMRI to distinguish between active and non-active areas of the brain (Silva and Bock, 2008). Lastly, when Mn2+ ions enter the brain, they can move along the neuronal pathways in an anterograde direction away from the soma and toward the projection terminals of the neurons. This allows MEMRI to map the neuronal tracts non-invasively and trans-synaptically (Pautler et al., 1998). It is worth noting that MEMRI has the ability to visualize regeneration in the visual system (Liang et al., 2011; Sandvig et al., 2011; Sandvig and Sandvig, 2014; Sandvig et al., 2012; Thuen et al., 2009). For example, in a chronic injury model by optic tract transection in hamsters, MEMRI allowed for longitudinal evaluation of the extent of injury and the optimal delivery of self-assembled nanomaterials for axon regeneration, supporting its usefulness for assessing WET integration (Liang et al., 2011). In another study comparing optic nerve crush injury in rodents versus lower vertebrates including frogs and fish, MEMRI demonstrated long-term visualization and serial monitoring of spontaneous axon regeneration in the central nervous system of lower vertebrates but not mature mammals (Sandvig et al., 2011). In contrast, in rodents with optic nerve crush followed by peripheral nerve grafts or lens injury, regeneration of the mammalian retinal ganglion cell axons was promoted, and manganese enhancement was found to increase with optic nerve regeneration with immunohistochemical validation (Sandvig et al., 2011; Thuen et al., 2009). In addition, active manganese transport has been found to be proportionate to the viability, number, and electrical activity of axon fibers, which would be useful in studying regeneration after WET (Fischer et al., 2014). Taken together, MEMRI is well-positioned in evaluating the functionality of neuroregenerative approaches to connect the transplant and recipient’s visual pathways in WET by assessing the levels of anterograde manganese transport and corresponding neural activity.

In this study, we leveraged the unique properties of Mn2+ and used MEMRI to develop an in vivo imaging model system for assessing WET integration in rats. We aimed to determine if combined MEMRI and WET can allow for in vivo measurements of the levels of anterograde Mn2+ transport along the transplanted visual pathway. We hypothesized that WET preserves comparable physiological transport between the transplanted and native visual pathways. Ultimately, the established in vivo imaging model system can be useful for testing neuroregenerative approaches for WET and guiding vision restoration.

2. Material and Methods

2.1. Animal Preparation

Seven donor and 7 recipient male Lewis rats (RT1) at 10 to 13 weeks old were used in this study. Syngeneic WET was performed on the right side only under ketamine/xylazine anesthesia (Figure 1A). Preparation of the donor began with an elliptical and continuous linear incision to expose the carotid artery and external jugular vein, followed by the creation of a hemifacial donor flap consisting of all ocular tissues anterior to the optic chiasm and a section of the temporal bone and the skin of the surrounding eyelid and auricle. The flap was perfused with heparinized Lactated Ringer’s solution while remaining connected to the rat prior to sacrifice for insetting. In the recipient rat, the optic nerve was transected at the base of the globe, and exenteration of orbital contents was performed. Then, the carotid arteries and external jugular veins were anastomosed, followed by optic nerve coaptation between donor and recipient (Figure 1B). The hemifacial flap was sutured into the same anatomical position on the hemiface of the recipient. Intraocular pressure was measured before baseline MRI scans using a handheld TonoLab rebound tonometer (ICare Finland Oy, Vantaa, Finland) within 5 minutes after isoflurane gas anesthesia induction. This procedure was repeated to obtain at least 3 instrument-derived intraocular pressure values for each eye, and the average value was used for final analysis. All experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Study ID: 15074553), and investigators followed guidelines from the Association for Research in Vision and Ophthalmology’s statement for Use of Animals in Ophthalmic and Vision Research.

Figure 1.

Figure 1.

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Surgical procedures for whole-eye transplantation (WET). (A) Schematic of syngeneic whole eye transplantation. Hemifacial graft is raised from the donor inset into strain-, age- and sex-matched recipient. (B) Neurorrhaphy during WET. Left: Both the optic nerve from the donor graft and the optic nerve of the recipient rat were transected. Right: The donor and recipient optic nerves were connected by neurorrhaphy.

2.2. MEMRI protocols

Three weeks after WET, animals received baseline and post-Mn MRI scanning at one day after bilateral intravitreal injections of 1.5 μL of 100 mM manganese chloride (MnCl2) solution. All animals were scanned under isoflurane gas anesthesia (3% induction, 1.5% maintenance) using fast spin-echo sequences in a 9.4-Tesla/31-cm Varian/Agilent horizontal bore scanner (Santa Clara, CA, USA) with a volume transmit and receive coil. 3D isotropic T1-weighted MRI and 2D coronal T1-weighted MRI scans were taken with the following parameters: 3D: field-of-view = 32 × 32 × 32 mm3, matrix resolution = 192 × 192 × 192, repetition time = 200 ms, echo time = 10.7 ms, echo train length = 8, and total acquisition time = 1 h. 2D: field-of-view = 26 × 26 mm2, matrix resolution = 192 × 192, slice thickness = 1 mm, slice gap = 0.5 mm, number of slices = 7, repetition time = 600 ms, echo time = 7.9 ms, echo train length = 8 and total acquisition time = 10 min. 2D slices were oriented orthogonal to the prechiasmatic optic nerves. A saline phantom was placed near the top left corner of the head of the rat for signal normalization.

2.3. Data analysis

The patterns of manganese enhancement were evaluated qualitatively on 3D MEMRI after axial maximum intensity projection. Quantitatively, regions of interests were drawn manually on the intraorbital optic nerves using 3D scans, and on the prechiasmatic optic nerves, lateral geniculate nuclei, and superior colliculi using 2D scans in both hemispheres using ImageJ v1.47 (Wayne Rasband, NIH, USA). An additional region of interest was drawn on the nearby saline phantom. The signal intensity for each region of interest was measured using ImageJ, and the optic nerve, lateral geniculate nucleus, and superior colliculus values were normalized to the phantom in order to account for potential MRI systematic fluctuations between experimental sessions. For intraocular pressure, a two-tailed paired t-test was performed between left and right eyes, whereas for MEMRI, two-way ANOVA was performed, followed by post-hoc multiple comparisons correction tests using GraphPad Prism v5.00 (GraphPad Software Inc., La Jolla, CA, USA).

3. Results

There was no significant difference in the intraocular pressure between native (14.76 ± 1.80 mmHg) (mean ± S.D.) and transplanted eyes (20.95 ± 7.36 mmHg) at 3 weeks after WET (p=0.09) (Figure 2). Along the visual pathway projected from the native left eye, significant T1-weighted signal increase was observed post-Mn injection in the left intraorbital (1.91 ± 0.46) and prechiasmatic optic nerve (1.55 ± 0.05) (Figures 3–5) and in the right lateral geniculate nucleus (1.70 ± 0.25) and superior colliculus (2.03 ± 0.22) when compared to pre-Mn injection (intraorbital optic nerve: 1.02 ± 0.12; prechiasmatic optic nerve: 0.87 ± 0.05; lateral geniculate nucleus: 1.16 ± 0.17; superior colliculus: 1.29 ± 0.11) (Figures 3–5) (p<0.0001). Along the visual pathway projected from the transplanted right eye, significant T1-weighted signal increase was observed post-Mn injection in the right donor intraorbital optic nerve (1.72 ± 0.49) compared to pre-Mn injection (1.03 ± 0.09) (p<0.001) (Figures 3 and 5C). No apparent T1-weighted signal difference was observed between native and transplanted intraorbital optic nerves post-Mn injection (p=0.26). Stronger T1-weighted signal enhancement was also found in the transplanted eye (2.74 ± 1.09) relative to the native eye (1.68 ± 0.44) post-Mn injection (p<0.05) (Figure 3). No apparent signal intensity difference was observed between pre- and post-Mn injection in the right recipient prechiasmatic optic nerve (pre-Mn: 0.88 ± 0.07; post-Mn: 1.12 ± 0.05), left lateral geniculate nucleus (pre-Mn: 1.14 ± 0.18; post-Mn: 1.21 ± 0.16), or left superior colliculus (pre-Mn: 1.30 ± 0.13; post-Mn: 1.34 ± 0.12) (p>0.05) (Figures 3–5).

Figure 2.

Figure 2.

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(A) Intraocular pressure (IOP) at 3 weeks after WET. There was no significant difference in intraocular pressures between the left, native and right, transplanted eyes (Two-tailed, paired t-test, p=0.09). Box plots show minimum, first quartile, median, third quartile, and maximum. (B) Schematic of optic nerve segments and their projections to the visual brain nuclei in the normal adult rodent. Note that more than 90% of rodent optic nerve fibers project to the contralateral hemisphere after reaching the optic chiasm, while the remaining 5–10% of fibers project to ipsilateral hemisphere (Forrester and Peters, 1967). ONio: intraorbital optic nerve, ONpc: prechiasmatic optic nerve, OC: optic chiasm, LGN: lateral geniculate nucleus, SC: superior colliculus, VC: visual cortex. Figure 2B is adapted with permission from (Deng et al., 2019).

Figure 3. Axial maximum intensity projection of 3D manganese-enhanced MRI from 2 representative WET animals.

Figure 3.

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(Left column) Maximum intensity projection pre-Mn injection. (Right column) Maximum intensity projection post-Mn injection. WET was performed to the right eye. Signal enhancement was observed in the left intraorbital optic nerve (ONio), left prechiasmatic optic nerve (ONpc), right lateral geniculate nucleus (LGN), and right superior colliculus (SC) projected from the native, left eye. Along the visual pathway projected from the transplanted, right eye, significant T1-weighted signal increase was observed post-Mn injection in the right donor ONio. No apparent signal enhancement was observed in the right ONpc, left LGN, or left SC projected from the transplanted, right eye. OT: optic tract, , PT: pretectum, CS: coaptation site of the right, transplanted eye.

Figure 5. Normalized T1-weighted signal intensities of the intraorbital optic nerves (ON) taken from 3D manganese-enhanced MRI (MEMRI) scans (A,C), and the prechiasmatic optic nerve (PC ON), lateral geniculate nucleus (LGN), and superior colliculus (SC) taken from 2D MEMRI scans (B, D).

Figure 5.

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pre- (black) and post-Mn injection (grey). A and B show the visual pathway projected from the native, left eye, and C and D show the visual pathway projected from the transplanted, right eye. Signals were normalized to a nearby saline phantom. Post-hoc Sidak’s tests between pre- and post-Mn injection, *p<0.001, **p<0.0001. Post-hoc Sidak’s tests between left and right hemispheres, ##p<0.0001. Box plots show minimum, first quartile, median, third quartile, and maximum.

4. Discussion

In our WET animal model, the insignificant intraocular pressure difference between the native and transplanted eyes align with our prior dynamic gadolinium-enhanced ocular imaging study, indicating comparable amounts of passive contrast agents entering the anterior chamber via the blood-aqueous barrier and not aqueous-vitreous or blood-retinal barrier (Van der Merwe et al., 2015). These suggest that some aspects of ocular physiology such as aqueous humor dynamics and blood circulation remain functional after WET. The measured intraocular pressure values in native and transplanted eyes after WET comport with previous reports using tonometers in naïve Lewis rats (Bakalash et al., 2002; Mermoud et al., 1994).

In rats, the majority of retinal ganglion cells project to the superior colliculus, and likely no more than 25,000 ganglion cells project to the lateral geniculate nucleus (Linden and Perry, 1983). Using MEMRI, we found that intravitreal injection of MnCl2 into the left, native rodent eye resulted in visualization of the ipsilateral left retina, ipsilateral left optic nerve, optic chiasm, contralateral right superior colliculus, contralateral right pretectum, and contralateral right lateral geniculate nucleus by T1-weighted MRI, reflective of continuous anterograde manganese labeling along the intact visual pathway (Watanabe et al., 2001). The significant signal enhancement in the right, transplanted intraorbital optic nerve post-Mn injection compared to pre-Mn injection indicates that there is anterograde manganese transport occurring along the visual pathway before the coaptation site three weeks after WET in the current animal model. Differential preservation of axonal transport after axotomy has been demonstrated previously, where fast axonal protein transport and the fluorescent labeling of many axons were preserved in the ocular stumps of axotomized optic nerves, while slow axonal transport of tubulin and neurofilaments was negatively affected (McKerracher et al., 1990). Taken together, we report a basic protocol to test neuroregenerative or retinal preservation approaches for restoration of the transplanted intraorbital optic nerve physiology and integrating the optic nerve fibers into the WET recipient visual pathway.

The use of MEMRI to study the visual system has a strong foundation in the literature. In particular, it has been applied to visualize optic nerve damage in terms of reduced or delayed T1-weighted signal increase in experimental animal models of optic nerve crush, optic nerve transection, and chronic glaucoma (Chan et al., 2008; Chan et al., 2011; Thuen et al., 2005). While there are other in vivo methods such as diffusion tensor MRI, functional MRI, and optical or ultrasound imaging for examining the optic nerve, MEMRI has certain advantages. For example, with the use of the active manganese tracers, MEMRI has greater specificity than diffusion-tensor MRI of passive water diffusion to resolve axonal fibers projected from one eye under the complex microenvironment of the injured brain (Deng et al., 2019; Lin et al., 2001). Furthermore, in contrast to conventional blood-oxygenation-level-dependent functional MRI, MEMRI can detect active neural regions independently of hemodynamics (Yang and Li, 2020). Likewise, MEMRI has no depth limitation, as opposed to optical imaging or ultrasound biomicroscopy for accessing the optic nerve and other visual pathways deep in the brain (Yan et al., 2012). In a model of partial transection of the intraorbital optic nerve, it was found that MEMRI can map the precise retinotopic projections in the subcortical visual nuclei along the intact and injured visual pathways at submillimeter resolution (Chan et al., 2011; Chan et al., 2017). Taken together, it is expected that MEMRI can be used to provide high-resolution visualization of the health status along the visual pathways based on levels and distribution of anterograde manganese transport in the transplanted optic nerve and beyond.

In the current animal WET model, no enhancement was detected in the right prechiasmatic optic nerve, left superior colliculus, or left lateral geniculate nucleus of the recipient’s brain after intravitreal Mn2+ injection into the right, transplanted eye. These findings suggest a lack of continuity of anterograde manganese transport across the nerve coaptation site after optic nerve transection and apposition. We also observed accumulation of Mn2+ in the vitreous humor in the transplanted eye relative to the native eye. This suggests less manganese clearance due to impaired transport from the transplanted eye, similar to previous studies involving optic nerve damage (Chan et al., 2008; Chan et al., 2014b). Taken together, the in vivo MEMRI findings indicate that, as expected, our current WET model has not achieved functional reinnervation past the coaptation site. Despite this, normal axonal transport was observed in the native visual pathway, indicating that WET did not negatively impact the native contralateral ocular system substantially. Future research can focus on approaches for neuroregeneration in the visual system with the use of MEMRI to visualize and monitor this process.

WET has been successfully conducted in zebrafish, which may provide insights for neural repair that could be translated to mammals (So, 2016; Tian et al., 2016). In contrast, the optic nerves in adult mammals are unable to regenerate properly after injuries, and retinal ganglion cell death occurs. Early studies found that rats with optic nerve injury were able to regenerate retinal ganglion cells and their axons after implantation of cellular peripheral nerve grafts from predegenerate teased segment of the sciatic nerve into the vitreous body, or by transplantation of peripheral nerve autografts into the retina (Berry et al., 1996; So and Aguayo, 1985). The mechanism was suggested to be through Schwann cells that secrete trophic molecules and stimulate retinal ganglion cell axon growth in the severed optic nerve. These initial studies have led to new avenues for optic nerve regeneration and WET integration by altering regulators of neuroregeneration. For example, some degree of optic nerve regeneration has been achieved by altering factors associated with intraocular inflammation, providing exogenous neurotrophic factors, promoting the intrinsic growth capacity of mature retinal ganglion cells, or changing the extrinsic growth-inhibitory environment of the optic nerve (Benowitz et al., 2017; Chun and Cestari, 2017; Cui et al., 2003). Among the candidate drugs, tacrolimus (FK506) was suggested as an ideal starting point for WET, due to its neuroregenerative properties and immunosuppressive effects (Bourne et al., 2017; van der Merwe et al., 2017). Exploring the above possible avenues for optic nerve regeneration and preservation will be important to determine how to functionally connect the transplant and recipient’s visual pathways in WET (Diederich et al., 2012; Guo et al., 2021; Prilloff et al., 2007; Schuettauf et al., 2006; van der Merwe et al., 2019; van der Merwe et al., 2021).

There are several limitations of the current in vivo MEMRI model system for monitoring WET. First, it is debated whether signal enhancements from MEMRI may be attributed to factors other than physiological axoplasmic transport. While MnCl2 accumulation was found to increase with brain activation (Lin and Koretsky, 1997), depth of anesthesia and body temperature can also alter neuronal transport, thus strongly affecting the signal contrast during activity-induced MEMRI (Aoki et al., 2004). Thus, this study minimized the duration of isoflurane anesthesia and the WET animals returned to their cages and remained awake at normal room temperature between baseline and post-Mn injection MRI scans. Future studies may examine the Mn2+ transport rate quantitatively via longitudinal MEMRI monitoring with careful control of the anesthesia dose and type and the body conditions. Furthermore, age can affect the bioavailability of manganese ions and neuronal manganese transport, directly affecting signal enhancement on MEMRI (Minoshima and Cross, 2008; Smith et al., 2007). Thus, age effects of both the donor and recipient should be taken into account in future experiments. Another limitation of MEMRI is the relative signal changes in T1-weighted MRI, as the intensity depends on the gradient, the radiofrequency field homogeneity, and coil sensitivity. While the current study had included a nearby saline phantom for signal normalization, for more quantitative measures of manganese concentrations, one can use R1 mapping, which is less affected by signal non-uniformity than the relativity of signal comparisons in T1-weighted MRI (Chuang and Koretsky, 2006; Chuang et al., 2009). Last but not least, the toxicity of MnCl2, especially at high concentrations limits its human use (Deng et al., 2019). Thus, less toxic Mn2+ salts such as the clinically approved mangafodipir trisodium (MnDPDP) for imaging liver lesions are now being explored for human MEMRI neuroimaging research as alternative chelating contrast agents (Olsen et al., 2008; Sudarshana et al., 2019; Suto et al., 2020). Despite these current limitations, mapping axonal transport by exogenous manganese tracers is likely useful for probing regenerated fibers that integrate from the transplanted optic nerve to the recipient brain more specifically than existing endogenous contrast-based imaging methods such as diffusion tensor MRI, which would mix all intact, injured, and regenerating fibers altogether. Furthermore, mapping MEMRI also allows us to conduct high-resolution three-dimensional in vivo topological mapping of the retinocollicular and retinogeniculate pathways (Chan et al., 2011). This is important to determine if individual retinal ganglion cells from the transplanted eye were attached to the correct organization in the downstream visual structures to gain and maintain proper function.

In the future, it may be useful to combine our in vivo MEMRI imaging model system with other modalities for more comprehensive evaluation of the WET outcomes in a single setting. These modalities may include gadolinium-enhanced MRI of aqueous humor dynamics and blood-ocular barriers, optical coherence tomography of retinal morphology and physiology, diffusion tensor MRI of axonal and glial integrity, electrophysiology of retinal, optic nerve, and visual cortical functions by pattern or flash electroretinography, compound action potential recording, and visually evoked potentials (Spees et al., 2018; Wang et al., 2012), blood-oxygenation-level-dependent functional MRI of hemodynamic visual brain responses, behavioral assessments of optokinetic and optomotor reflexes, histological confirmation of in vivo imaging findings at end time points, and their inter-relationships thereof (Haenold et al., 2012; Ho et al., 2015). It is essential to continue developing this WET animal model and the combined in vivo imaging model system to guide clinical translation and to improve the functionality of outcomes in patients (Bourne et al., 2017; Davidson et al., 2016a; Davidson et al., 2016b).

5. Conclusions

This paper offers a study platform to assess the physiological transport in both the transplanted and recipient visual pathways after WET via non-invasive, non-terminal imaging. Such parameters will be important for longitudinal monitoring and the successful clinical translation of WET. Three weeks after WET, in vivo MEMRI indicates comparable levels of anterograde manganese transport in the transplanted and native intraorbital optic nerves at one day after intravitreal Mn2+ administrations to both eyes, suggestive of the presence of physiological axoplasmic transport in the transplanted visual pathway. No anterograde manganese transport is observed past the coaptation site without applying a regenerative strategy to the current WET model. Future MEMRI studies may use this in vivo imaging model system to examine neuroregenerative approaches that help integrate the transplanted eye to the recipient’s brain topologically and functionally for vision restoration.

Figure 4. Representative 2D coronal manganese-enhanced MRI at the levels of the prechiasmatic optic nerve (ONpc, top row), lateral geniculate nucleus (LGN, middle row) and the superior colliculus (SC, bottom row) pre- (left column) and post-Mn injection (right column).

Figure 4.

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Arrows indicate signal enhancement in the left ONpc, right LGN, and right SC projected from the native, left eye. No apparent signal enhancement was observed in the right ONpc, left LGN, or left SC projected from the transplanted, right eye.

Highlights:

  • Developed an in vivo imaging model system to assess whole eye transplantation (WET)

  • Insignificant intraocular pressure difference between native and transplanted eyes

  • Comparable anterograde manganese transport along native and transplanted optic nerves

  • No manganese contrast detected past coaptation site in the recipient brain

  • Manganese-enhanced MRI allows in vivo testing of neuroregeneration in WET integration

Acknowledgements:

We would like to thank all members of the whole eye transplantation consortium for their intellectual exchanges and technical support in this study. Special thanks to Bing Li, MD for assistance with generating the WET schematic.

Funding:

This work was supported by The Office of the Assistant Secretary of Defense for Health Affairs under Award No. W81XWH-14-1-0421 and W81XWH-16-1-0775, VA Pittsburgh Healthcare Administration, National Institutes of Health P30-EY008098 and R01-EY028125 (Bethesda, Maryland); Eye and Ear Foundation (Pittsburgh, Pennsylvania); and unrestricted funds from Research to Prevent Blindness (New York, New York) to University of Pittsburgh and NYU Langone Health Department of Ophthalmology.

Footnotes

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Declaration of Competing Interest:

None of the authors has conflicts of interest.

Conflicts of interest: None for all authors.

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