3-Dimensional visualization and quantification of optic nerve degeneration after traumatic brain injury in CUBIC-cleared tissue

Abstract

Purpose : As demonstrated by our group and others, closed-head, repeated, mild traumatic brain injury (r-mTBI) results in a characteristic localized inflammatory activity in the visual system of wild type and hTau mice. Tissue clearing approaches such as CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis; Nature Protocols 2015, 10:1709–1727) provide visualization and quantification of such inflammatory activity that is potentially less artefact-inducing and more sensitive than traditional sectioning methods. In the present study, CUBIC was used to quantitatively analyze differences in cellularity of whole mouse optic nerves of sham mice and mice that had undergone r-mTBI.

Methods : Adult hTau mice (n=9) were subjected to injury under anesthesia according to an established protocol. Optic nerve tissue was extracted, fixed and processed according to a modified CUBIC-protocol 3 and 6 months after r-mTBI or anesthesia only (r-sham). Levels of cellularity in the intracranial portion of each optic nerve were observed using tiled mosaics of consecutive z-slices from confocal microscopy spanning the length of the nerve and staining with DAPI at a horizontal plane corresponding to mid-depth. Cellularity was quantified using ImageJ and compared to cellularity determined by optic nerve H&E staining (n=19) in hTau mice at comparable time points.

Results : Increased cellularity in the optic nerves of r-mTBI mice compared to r-sham was observed at both 3 and 6-month time points after injury. Pathological changes associated with r-mTBI were comparable with those observed in H&E-stained r-mTBI optic nerves and showed a distinct, spindle-like 3-dimensional arrangement of increased cellularity. A greater number of cell nuclei were detected in r-mTBI optic nerves that had undergone CUBIC clearing (124.4%, p<0.05), likely due to the detection of additional fluorescence coming from areas of increased cellularity above and below the plane of imaging that was not present in the r-sham group.

Conclusions : Use of CUBIC-cleared optic nerve tissue resulted in the detection of a greater number of cells than had previously been detected in sectioned optic nerves using traditional methods. These differences should be taken into consideration when comparing and quantifying results using traditional embedding and sectioning vs. tissue clearing (e.g. CUBIC) methods.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.