Green-channel autofluorescence imaging: A novel and sensitive technique to delineate infarcts

Abstract

Background: We have found that infarcted brain regions exhibit green channel autofluorescence (GCAF). Here, we compare ex vivo GCAF-imaging with 2,3,5-triphenylteterazolium chloride (TTC)-staining.

New method: C57BL/6 mice (n=120) underwent GCAF-imaging after transient or permanent middle cerebral artery occlusion (tMCAO or pMCAO).

Comparison with existing methods: TTC-staining may not reflect subtle ischemic injury. TTC-stained tissues, when reused, are prone to processing artifacts related to prior TTC-staining. GCAF imaging requires little experimental manipulation of animals and brain tissues, and allows for more consistent measurements of infarct volume and reliable reuse of the fresh unstained tissues.

Results: Lesion volumes measured at 24-h after 1-h tMCAO by using GCAF-images were similar to those using TTC-staining: 87.6±13.6mm³ vs. 83.8±12.8mm³ in 1mm-thick sections (n=9 mice, 10 slices/mouse, p=0.88; Pearson's r=0.91, p<0.001) and 75.1±7.6mm³ vs. 73.6±6.7mm³ in 2 mm-thick sections (n=9 mice, 5 slices/mouse, p=0.99; Pearson's r=0.87, p<0.001), respectively. In serial ex vivo imaging performed at 1, 2, 3, 6, 12, and 24-h after tMCAO, GCAF-imaging correlated well with TTC-staining at all time-points. In the pMCAO model however, the correlation was strong at later time-points (6-24-h); but at time points up to 3-h, GCAF-imaging was more sensitive than TTC-staining to detect ischemic areas, as verified by histology, where ischemic damage was observed in the GCAF-positive areas of the cerebral cortex and striatum, even in the face of normal TTC-staining.

Conclusion: GCAF-imaging is a reliable alternative to TTC-staining in the qualitative and quantitative assessments of focal brain ischemia, and more sensitive for detecting early ischemic damage in pMCAO.

Keywords: 2,3,5-Triphenyl-tetrazolium chloride staining; Cerebral infarct; Green-channel autofluorescence imaging; Penumbra; Stroke imaging.