A total of 48 healthy C57BL/6 mice (half male and half male), weighing 20-25 g. A total of 96 Caspase gene knockout mice (48 Caspase-1, 48 Caspase-3, half male and half female), weighing 20-25 g, were purchased from Cyagen Biosciences Co. Ltd. (Guangzhou, China; license number: SCXK (Su) 2018-0003). The experimental procedure followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1986). The mice were fed standard rodent chow and allowed free access to water. It was approved by the Animal Ethics and Welfare Committee of Southern Medical University of China (approval No. 2021-005). The temperature of the animal house was maintained at a room temperature between 20℃ and 22℃ and relative humidity of 65–70%. The C57 mice and the Caspase knockout mice were randomized into sham (sham control) group, I/R (model) group, and EA (I/R + EA) group (n = 16 per group).
Establishing cerebral I/R injury mouse model
The middle cerebral artery of the mice was blocked according to the modified Zea-Longa’ s method to induce cerebral I/R model. After anesthetizing the mice with 10% chloral hydrate (1 ml/kg) by intraperitoneal injection, the left common carotid artery, left internal carotid artery, external carotid artery, and vagus nerve were carefully exposed through the midline incision under the surgical microscope. A nylon wire of about 11 ± 0.5 mm was introduced into the internal carotid artery to occlude the middle cerebral artery until slight resistance was observed during insertion. After 30 minutes of occlusion, the blood supply of the ischemic area was restored by slowly drawing the thread, and the reperfusion was achieved. Mice in the sham group underwent the same procedures as described previously but without arterial occlusion. At 2 hours after the operation, the 5-point Zea-Longa’ s criteria were used for evaluation: 1–3 points indicated successful model establishment, and the corresponding mice were included in the study; the mice with scores 0 or 4 were excluded.
The mice in the EA group underwent EA stimulation 1.5 hours after I/R injury. Stainless steel acupuncture needles (diameter: 0.16×13 mm2; Suzhou Universal Acupuncture Medical Devices Co., Ltd., Suzhou, China) were inserted 2–3 mm into LU5, LI4, ST36, and SP6 acupoints of the paralyzed limb. Selection of acupoints and EA stimulation were made according to “experimental acupuncture” edited by Li. The location of the selected acupoints was as follows: as for LU5, in the depression of the outer end of the transverse cubital crease, an acupuncture needle was inserted perpendicularly to a depth of 3 mm. As for LI4, located between 1st metacarpal bone and 2nd metacarpal bone, an acupuncture needle was inserted perpendicularly to a depth of 1 mm; as for ST36, located at 5 mm below fibular head at outer lateral posterior knee and puncture, an acupuncture needle was inserted perpendicularly to a depth of 7 mm; as for SP6, located at the tip of the inner ankle of the posterior limb, a needle was inserted upward 10 mm and perpendicularly to a depth of 5 mm; The acupoints were stimulated for 20 minutes with a dilatational wave of frequency 5/10 Hz and intensity 2 mA using an EA instrument (Model KWD-808I, Suzhou Universal Acupuncture Medical Devices Co., Ltd.).
Neurological deficit scores
At 24 hours after the operation (before tissue sampling), the neurological deficit score of mice was evaluated by the 5-point Zea-Longa’ s method. The neurological deficit scores were defined as follows: score 0, no obvious defect; score 1, failure to fully extend the right forepaw; score 2, circling to the contralateral side; score 3, falling to the opposite side; and score 4, not spontaneously walking or loss of consciousness.
The samples were collected 24 hours after I/R injury. After excessive anesthesia (10% chloral hydrate, 10 ml/kg), rapid perfusion with 0.9% normal saline (NaCl) and 4% paraformaldehyde in phosphate-buffered saline for 3–5 minutes eliminated the influence of blood factors. Then the whole brain was dissected out of the cranial cavity immediately. In each group, 4 fresh brain tissues were taken for TTC staining, 6 were placed in 4% paraformaldehyde solution for TUNEL staining, and 6 were stored in - 80℃ refrigerator for Western blot analysis.
Triphenyl tetrazolium chloride (TTC) Staining
Fresh brain tissue was stored at - 20℃ for 20 min and then sectioned every 2 mm in the coronal plane. The slices were placed in 2% TTC phosphate buffer, incubated in a 37℃ water bath for 30 minutes in the dark. Turn the slices evenly every 10 minutes to make the slices even in contact with the TTC staining solution. After staining, the infarct was stained white, and the normal brain tissue was stained red. Use the AlphaEaseFC analyzer software (AlphaInnotech, San Leandro, CA, USA) to measure the infarct size and calculate the brain infarct volume percentage (BIVP).
TdT-mediated dUTP Nick-End Labeling (TUNEL) assay
Brain tissues were routinely dehydrated, paraffin-embedded, and sliced. The sections were incubated with protein K at RT for 15 min, washed in phosphate-buffered saline (PBS) (Ruigu Biotechnology Co., Ltd., Shanghai, China) for 3 times (5 min/per time). Membrane lysis solution was added, incubated at room temperature for 10 min, and washed in PBS 3 times (5 min/per time). Reagent 1 (TdT) and 2 (dUTP) from the TUNEL kit (Kaiji Biotechnology Co., Ltd., Nanjing, China) were obtained, mixed at a ratio of 1:9, added to cover the tissues, and then incubated in a 37℃ water bath (Kaiwei Biotechnology Co., Ltd., Guangzhou, China) for 60 minutes. The sections were washed in PBS 3 times (5 min/per time), DAPI was used for staining the dried section. The section was incubated for 10 min at RT in the dark and was rinsed in PBS 3 times (5 min/per time). Morphological changes in the cerebral tissues were observed under a microscope (Jiangnan Optical Instrument Group, Nanjing, China), and the images were collected for analysis (AlphaEaseFC). Two 20x fields of view in the hippocampal CA1 area were randomly selected for photography. Apoptosis index (AI) was calculated according to the following formula: number of apoptotic cells/ total cells × 100%.
Western blot assay
Hippocampus tissues were homogenized in Radio Immunoprecipitation Assay (RIPA) lysis buffer and centrifuged at 12,000 ×g for 5 min, then determined protein concentration in supernatants. Protein lysates were separated by 10% SDS-PAGE gels and then electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Boston, USA). The membranes were blocked with 5% nonfat dry milk for 1 hour and incubated with primary antibodies: Caspase-1 (1:1000; Servicebio, Wuhan, CNH; GB11383), Caspase-3 (1:1000; Servicebio, Wuhan, CNH; GB11767C) overnight at 4°C. The membranes were incubated with a corresponding secondary antibody (1:3000; goat anti-rabbit/mouse IgG; Servicebio, Wuhan, CNH; GB23303) in TBST for 30 minutes. The blots were developed using enhanced chemiluminescence, and the intensity of the bands was measured using the AlphaEaseFC analyzer software (AlphaInnotech, San Leandro, CA, USA). The optical density value ratio of the target band to the internal reference served as the relative expression of the target protein.
SPSS 20.0 software (IBM, Armonk, NY, USA) was used for statistical analysis. Quantitative data are expressed as the mean ± SD. If the data meet the normal distribution, one-way ANOVA is used for inter-group comparison, and LSD test is used for pairwise comparison within the group. If the data does not meet the normal distribution, Mann-Whitney nonparametric test is used for pairwise comparison, and Kruskal-Wallis test is used for multiple comparisons. A P value less than 0.05 was considered statistically significant.