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

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 ± 13.6 mm³ vs. 83.8 ± 12.8 mm³ 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 ± 6.7 mm³ 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.