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. 'ITC-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 +/- 13.6 mm(3) vs. 83.8 12.8 mm(3) in 1 mm-thick sections (n = 9 mice, 10 slices/mouse, p = 0.88; Pearson's r= 0.91, p <0.001) and 75.1 +/- 7.6 mm3 vs. 73.6 +/- 7 mm(3) 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 (6-24h); 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. (C) 2017 Elsevier B.V. All rights reserved.