Journal: bioRxiv

Article Title: Exercise improved P2Y12-regulated microglial dynamics during stroke via endocannabinoid signaling

doi: 10.1101/2023.04.24.538065

Figure Lengend Snippet: Microglia interacted with neurons, inhibited neuronal calcium hyperactivity, and promoted neuronal mitochondria. A. Left panel: Representative images showing GCaMP6s signal changes in neurons imaged at 24 h after transient middle cerebral artery occlusion. Right panel: Representative images about the relative immunofluorescent intensity changes of GCaMP6s-dTomato. B. Comparisons of the amplitudes and frequencies of GCaMP6s signal changes in the vehicle, PE, and PE+PSB0739 groups. C. Representative images of the neuronal mitochondria and the microglia-neuron membrane junction captured using transmission electron microscope (TEM). Upper panel: images captured using 1200X objective, red box labeled the magnified site showed in the below panel. below panel: images captured using 11000X objective, red arrows indicates the neuronal mitochondria, purple box in PE group indicates the microglia-neuron junction. D. Comparisons of the relative expression of TOM20 among the vehicle, PE, and PE+PSB0739 groups. E. Comparisons of the relative expression of iNOS among the vehicle, PE, and PE+PSB0739 groups. F. Comparisons of the relative expression of TNF α and IL-10 among the vehicle, PE, and PE+PSB0739 groups. Each dataset is expressed as the mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. n = 6 mice.

Article Snippet: The sections were then incubated overnight with primary antibody [1:100 Mouse Anti-Neun, MAB377, Millipore, Massachusetts, USA; 1:100 Chicken Anti-Neun, ab134014, Cambridge, England; 1:400 Rbbit anti-ionized calcium binding adapter molecule 1 (Iba1), Wako, Tokyo, Japan; 1:100 Mouse anti-P2Y12, ab233760, Cambridge, England; 1:100 Rabbit anti-TOMM20, ab186735, Cambridge, England; 1:100 Rabbit anti-Cannabinoid Receptor I, ab259323, Cambridge, England; 1:100 Mouse anti-cannabinoid receptor 2, H00001269-M01, Novus, Colorado, USA; 1:100 Rabit Anti-Synaptophysin, ab32127, Cambridge, England], and then with a secondary antibody at room temperature for 1 h. Apoptosis was detected using a TUNEL Kit (TUNEL Apoptosis Detection Kit; KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol.

Techniques: Membrane, Transmission Assay, Microscopy, Labeling, Expressing

Journal: bioRxiv

Article Title: Exercise improved P2Y12-regulated microglial dynamics during stroke via endocannabinoid signaling

doi: 10.1101/2023.04.24.538065

Figure Lengend Snippet: URB597 mimicked the improvement of microglial-neuron interactions induced by physical exercise, while AM630 decreased the protection of PE. A. Representative images showing GCaMP6s signal changes in neurons imaged at 24 h after transient middle cerebral artery occlusion.. B. Representative images about the immunofluorescent intensity changes of GCaMP6s-dTomato. C. Comparisons of the amplitudes and frequencies of the GCaMP6s signal changes among the vehicle, PE, URB597 and PE+AM630 groups. D. Representative images of the neuronal mitochondria and the microglia-neuron membrane junction captured by transmission electron microscope (TEM). Upper panel: images captured by1200X objective, red box labeled the magnified site showed in the below panel. below panel: images captured using 11000X objective, red arrows indicates the neuronal mitochondria and purple box in PE and URB597 group labeled the microglia-neuron junction. E. Comparison of TOM20 expression among the vehicle, PE, URB597 and PE+AM630 groups (Each dataset is expressed as the mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. n = 6 mice)

Article Snippet: The sections were then incubated overnight with primary antibody [1:100 Mouse Anti-Neun, MAB377, Millipore, Massachusetts, USA; 1:100 Chicken Anti-Neun, ab134014, Cambridge, England; 1:400 Rbbit anti-ionized calcium binding adapter molecule 1 (Iba1), Wako, Tokyo, Japan; 1:100 Mouse anti-P2Y12, ab233760, Cambridge, England; 1:100 Rabbit anti-TOMM20, ab186735, Cambridge, England; 1:100 Rabbit anti-Cannabinoid Receptor I, ab259323, Cambridge, England; 1:100 Mouse anti-cannabinoid receptor 2, H00001269-M01, Novus, Colorado, USA; 1:100 Rabit Anti-Synaptophysin, ab32127, Cambridge, England], and then with a secondary antibody at room temperature for 1 h. Apoptosis was detected using a TUNEL Kit (TUNEL Apoptosis Detection Kit; KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol.

Techniques: Membrane, Transmission Assay, Microscopy, Labeling, Comparison, Expressing