Purpose

Approximately 39 million people worldwide are suffering from complete blindness due in part to the inability of the visual system to regenerate after injury¹. Recently, we introduced a whole eye transplant (WET) animal model^2,3, which gives the opportunity to provide viable retinal ganglion cells and an intact optical system to patients with irreversible vision loss. Using optical coherence tomography and gadolinium-enhanced MRI, we showed that our WET model allows ocular blood circulation, and preserves aqueous humor dynamics as well as blood-ocular and aqueous-vitreous barriers in the transplanted eye^4,5. In this study, we aimed to further develop an in vivo imaging model system to determine the functionality of the transplanted and recipient visual pathways following WET. Manganese-enhanced MRI (MEMRI) has been used to monitor anterograde transport in optic nerve injuries^6,7,8 and axon regeneration to the visual system^9,10,11. Here, we used MEMRI to determine the level of anterograde manganese transport along the visual pathways following WET.

Methods

Animal preparation: Seven adult Lewis rats received orthotopic WET on the right side only, as previously described^2,4,5. In brief, the donor flap contained the external ear, a section of temporal bone, skin of the eye lid, all ocular tissues and the intraorbital optic nerve. A similar tissue section was removed in the recipients, with the optic nerve being cut at the base of the globe, and the donor graft was transplanted into the recipient with anastomoses between the carotid arteries and external jugular veins (Fig 1). Intraocular pressure (IOP) was measured before MEMRI using the TonoLab tonometer. MEMRI protocols: Three weeks after WET, animals received bilateral intravitreal injections of 1.5µL of 100mM manganese chloride (MnCl₂) solution. Before and 1 day after MnCl₂ injections, all animals were scanned using fast spin-echo sequences in a 9.4-Tesla/31-cm Varian/Agilent scanner, 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: FOV= 32x32x32mm³, matrix resolution=192x192x192, TR/TE=200/10.7ms, ETL=8. 2D: FOV= 26x26mm², matrix resolution=192x192, slice thickness=1mm, slice gap=0.5mm, number of slices=7, TR/TE=600/7.9ms, ETL=8. 2D slices were oriented orthogonal to the prechiasmatic optic nerves (ON). Data analysis: The patterns of manganese enhancement were evaluated qualitatively on 3D MEMRI after axial maximum intensity projection. Quantitatively, regions-of interests (ROI) were drawn manually on the intraorbital ON using 3D scans, and on the prechiasmatic ON, lateral geniculate nucleus (LGN) and superior colliculus (SC) using 2D scans in both hemispheres. An additional ROI was drawn on a nearby saline phantom. The signal intensity for each ROI was measured using ImageJ, and ON, LGN, and SC values were normalized to the phantom. Two-way ANOVA was performed followed by post-hoc multiple comparisons correction tests using GraphPad PRISM.

Results

There was no significant IOP difference between naïve and transplanted eyes (p=0.09)(Fig 2). Along the visual pathway projected from the naïve, left eye, significant signal increase was observed post-Mn injection in the left intraorbital and prechiasmatic ON (Figs 3-5), and in the right LGN and SC compared to pre-Mn injection (Figs 3-5)(p<0.0001). Along the visual pathway projected from the transplanted, right eye, significant signal increase was observed post-Mn injection in the right donor intraorbital ON compared to pre-Mn injection (p<0.001)(Figs 3 and 5C). No apparent signal difference was observed between naïve and transplanted intraorbital ON post-Mn injection (p=0.26)(Figs 5A and 5C). No apparent signal intensity difference was observed between pre- and post-Mn injection in the right recipient prechiasmatic ON, left LGN or left SC (p>0.05)(Figs 3-5). Note also the stronger signal enhancement in the transplanted eye relative to the naïve eye (Fig 3).

Discussion

The significant signal enhancement in the right intraorbital ON indicates the presence of anterograde manganese transport in the donor ON at 3 weeks after WET. Such enhancement was not statistically different from the left naïve ON, suggestive of comparable extents of anterograde transport between donor and naïve ON. No enhancement was detected in the right prechiasmatic ON, left SC or left LGN of the recipient’s brain, suggesting a discontinuation of anterograde manganese transport across the nerve coaptation site after ON transection and apposition. Such observation coincided with the less manganese clearance from the transplanted eye.

Conclusions

With comparable IOP between transplanted and naïve eyes, MEMRI suggested the presence of anterograde manganese transport in the donor ON to a comparable extent as the opposite naïve ON at 3 weeks after WET. Future MEMRI studies will examine neuroregenerative approaches^12,13 for connecting between the transplanted eye and the recipient’s brain, so as to regain the neuronal structure and function of the current WET model.

Acknowledgements

This work was supported by the The Office of the Assistant Secretary of Defense for Health Affairs under Award No. W81XWH-14-1-0421, VA Pittsburgh Healthcare Administation, National Institutes of Health P30-EY008098 (Bethesda, Maryland); Eye and Ear Foundation (Pittsburgh, Pennsylvania); and Research to Prevent Blindness (New York, New York).

References

[1] World Health Organization, www.who.int/blindness/data_maps. Accessed October 5th, 2016.
[2] Washington, KM., et al. Viability, structural integrity, and aqueous humor dynamics are established in an orthotopic whole eye transplant model. Investigative Ophthalmology & Visual Science. 2015; 56 (7), 4757.
[3] Couzin-Frankel, J. Second Sight. Science. March 2015; 347(6227):1194-7.
[4] Van der Merwe Y, et al. In vivo evaluation of ocular physiology and structural integrity of the optic nerve upon whole eye transplantation using gadolinium-enhanced MRI and diffusion tensor imaging. ISMRM 2015 Annual Meeting Conference Proceedings, Toronto, Canada, May 2015.
[5] Washington, KM., et al. Studies of structure and function in whole eye transplantation. Investigative Ophthalmology & Visual Science. 2016; 57 (12).
[6] Thuen, M., et al. Manganese-enhanced MRI of the optic visual pathway and optic nerve injury in adult rats. Journal of magnetic resonance imaging. Oct 2005; 22(4):492-500.
[7] Chan, KC., et al. Evaluation of the retina and optic nerve in a rat model of chronic glaucoma using in vivo manganese-enhanced magnetic resonance imaging. Neuroimage. April 2008; 40(3):1166-1174.
[8] Chan, KC., et al. In vivo retinotopic mapping of superior colliculus using manganese-enhanced magnetic resonance imaging. Neuroimage. January 2011; 54(1):389-395.
[9] Sandvig A., et al. Axonal tracing of the normal and regenerating visual pathway of mouse, rat, frog, and fish using manganese-enhanced MRI (MEMRI). Journal of magnetic resonance imaging. Sep 2011; 34(3):670-5.
[10] Thuen, M., et al. Combination of Mn(2+)-enhanced and diffusion tensor MR imaging gives complimentary information about injury and regeneration in the adult rat optic nerve. Journal of magnetic resonance imaging. Jan 2009; 29(1):39-51. [11] Liang, Y., et al. CNS regeneration after chronic injury using a self-assembled nanomaterial and MEMRI for real-time in vivo monitoring. Nanomedicine: Nanotechnology, biology, and medicine. June 2011; 7(3):351-359.
[12] Moore, DL., and Goldberg JL. Four steps to optic regeneration. Journal of Neuro-ophthalmology. Dec 2010;30(4):347-360.
[13] Benowitz, LI., et al. Reaching the brain: Advances in optic nerve regeneration. Experimental neurology. Dec 2015; (15)30141-2.