Extended Data Fig. 6: Optimized longitudinal three-photon imaging does not increase glial, neuronal, or vascular reactivity.
a) Related to Fig. 3. Coronal brain section from the imaged (right) and contralateral (left) posterior parietal cortex (PPC) of a mouse that was perfused 24 hrs. following 10 weeks of chronic 3P imaging. b) Coronal brain section from the imaged (right) and contralateral (left) posterior parietal cortex (PPC) of a mouse that received supra-threshold excitation across the cortex and white matter to generate laser-induced positive control tissue (laser injury, right). c) High-resolution confocal images of the contralateral (left), longitudinal imaging field (middle) and laser-induced injury (right) regions in the deep cortical layers of the PPC, stained for transgenic Mobp-EGFP expression (mature oligodendrocytes), Iba-1 (microglia), GFAP (reactive astrocytes), 8-hydroxyguanosine (8-OHG, neuronal RNA oxidation), and CD13/Lectin-49 (pericytes, vasculature, respectively). All images were taken with identical power settings, processed with a 100-pixel rolling ball background subtraction and 0.7 pixel gaussian blur, and brightness/contrast correction was applied identically across channels. The bottom row shows an example of the trainable WEKA segmentation to measure pericytes (green) and vascular coverage (magenta, see Methods). Note the Lectin-positive microglia-like cells present in the laser injury vasculature image (white arrowheads). d) No difference in the density of Mobp-EGFP OLs between the contralateral and imaged regions in healthy mice. e) No difference in the density of Mobp-EGFP OLs between the contralateral and imaged regions in laser-induced injury tissue. f) The ratio of the EGFP mean intensity for the imaged:contralateral (contra.) hemispheres was ~1 for healthy mice and did not change significantly in the positive control tissue. g) No difference in the density of Iba-1 microglia (MG) between the contralateral and imaged regions in healthy mice. h) Density of Iba-1 MG is significantly increased on the ipsilateral side of laser-induced injury tissue (p = 0.016). i) The ratio of the imaged:contralateral Iba-1 mean intensity was ~1 for healthy mice and significantly increased in positive controls (p = 0.026). j) No difference in the density of GFAP-positive reactive astrocytes (rAstros) between the contralateral and imaged regions in healthy mice. k) Density of GFAP+ rAstros is significantly increased on the damaged side in laser-induced injury tissue (p = 0.017). l) The ratio of the imaged:contralateral (contra.) GFAP mean intensity was ~1 for healthy mice and was significantly increased in positive controls (p = 0.037). m) No difference in the density of 8-OHG-positive neurons after auto-thresholding (see Methods) between the contralateral and imaged regions in healthy mice. n) No difference in the density of 8-OHG-positive neurons between contralateral and imaged regions in laser-induced injury tissue. o) No difference in the imaged:contralateral ratio of 8-OHG mean intensities between healthy vs. laser-damaged mice. p) No difference in vessel coverage (% positive pixels) between the contralateral and imaged hemispheres of healthy multi-month longitudinal 3P imaging mice. q) Increase in vessel coverage (%) on the ipsilateral hemisphere of laser-induced injury positive control mice (p = 0.022). r) No difference in the density of pericytes between the contra. and ipsi. hemispheres of healthy long-term 3P mice. s) No difference in the density of pericytes between the contra. and ipsi. hemispheres of laser-induced injury mice. *p < 0.05, **p < 0.01, n.s., not significant; n = 5 mice (healthy longitudinal), 5 mice (laser-induced injury), 2 sections, 4 hemispheres per condition. Statistical comparisons in d, e, g-h, j-k, m-n, and p-s were made with paired, two-sided Student’s t-tests (parametric) or two-sided Wilcoxon signed-rank test (nonparametric). Statistical comparisons in f,i,l,o were made with unpaired two-sided Student’s t-tests for equal/unequal variances (parametric) or two-sided Wilcoxon rank sum test (nonparametric). Box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.
