Stereotaxic AAV-CamKIIα-SENP1-mCherry-3FLAG injection

For SENP1 expression in neurons, mouse SENP1 cDNA was inserted into AAV vector, pAAV-CaMKIIα-MCS-mCherry-3FLAG (Obio Technology, Shanghai, China), under the control of CaMKIIα promoter and fused to mCherry-3FLAG. Vector construction and AAV production were performed by Obio Technology. The virus titer was 3.69 × 10¹² genome copies/ml. For virus infection, 8-week-old male C57BL/6 mice were anesthetized and placed in a stereotaxic frame (RWD Life Science, San Diego, CA, USA). Stereotactic intracerebral injections of AAV into the primary somatosensory cortex, barrel field and secondary somatosensory cortex were performed, using the following coordinates (in mm from bregma) according to the mouse brain atlas: AP=+0.00 mm; L=+3.70 mm; DV=–2.50 mm. A total of 1.5 μl of viral preparation was injected by microelectrodes connected with a microinjector pump (KDS 310; KD Scientific, Holliston, MA, USA) at a rate of 0.1 μl/min. Microelectrodes were left in place for a further 10 min after each infusion to allow complete diffusion of AAV from the tip. Mice were allowed to recover for 4 weeks before conducting stroke model and the injection sites were examined at the end of the experiments. Brain slices from animals injected with the viruses were examined directly using fluorescence microscopy.