Animals and Grouping
Male Sprague-Dawley rats (eight/nine-week-old ) were purchased from the Experimental Animal Center of Nanjing University of Chinese Medicine. All rats were housed under a 12-hour light cycle in specific pathogen-free conditions and had free access to food and water. After acclimatizing for one week, rats were randomly divided into three experimental groups: the SHAM group, the MCAO group, and the MEA group. All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Cerebral ischemia-reperfusion (I/R) injury was induced by MCAO surgery described previously (Fu et al. 2014). In short, the rats were anesthetized with isoflurane, and the rectal temperature was maintained at 37 ± 0.5oC during surgery. A 3/0 monoﬁlament nylon suture was inserted into the left external carotid artery, entered the anterior cerebral artery through the internal carotid artery, and finally blocked the middle cerebral artery to induce ischemic injury. After 2 hours of middle cerebral artery occlusion, the nylon suture was removed to induce reperfusion. Rats in the sham operation group received the same surgical procedures but without suture insertion.
Rats in the MEA group were treated with EA stimulation in the "Baihui" acupoint (GV20). when the cerebral blood flow was restored, They received the first EA intervention. The second EA treatment was applied two hours before being sacrificed. GV20 is located at the line midpoint joining the tips of left and right ears. The needles were connected to Han's electroacupuncture instrument (Beijing Huawei Technologies Co. Ltd, LH402A). The stimulation current was 1 mA, and the frequency was 2/15 Hz, 20 min for each time. The SHAM and the MCAO groups were only fixed in the same way for 20 min, without EA stimulation (Fig.1).
Longa Neurologic Score was applied to assess the neurologic function: a score of 0 indicated no neurologic deficit; a score of 1 (failure to extend right forepaw fully) indicated a mild focal neurologic deficit; a score of 2 (circling to the right) indicated a moderate focal neurologic deficit; a score of 3 (falling to the right) indicated a severe focal deficiency; rats with a score of 4 did not walk spontaneously and had a depressed level of consciousness; a score of 5 suggested rats has died(Longa et al. 1989). The rats with a score from 2 to 3 were included in the experiments for follow-up research.The rats scoring outside of this range was sacrificed.
TTC staining was applied to evaluate the cerebral infarction area.Rats were sacrificed by intraperitoneal injection with excess pentobarbital. After cardiac perfusion with 0.85% saline, brains were isolated and frozen at -20oC for 20 min. Then they were cut into five sections and incubated in 2% TTC (2,3,5-triphenyl tetrazolium chloride, Sigma Chemical Co., 298-96-4) for 20 min at 37oC. The normal hemisphere exhibited red while the infarcted tissues exhibited pale. After that, the slices were immersed in 4% paraformaldehyde overnight. The sections were photographed, and the infarcted area was evaluated by Image-Pro Plus 6.0.
Hematoxylin and eosin staining (H&E staining) microscope
The ileum was fixed in 4% paraformaldehyde. The tissue was embedded in paraffin and cut into 5μm slices followed by Hematoxylin and eosin staining. The morphology damage of the small intestine was observed under a microscope (Nikon TE2000, Japan). The villous height and smooth muscle thickness in the ileum were determined using Image J software. Ten intestinal villi and the thickness were detected in each rat by pathologists blinded to the animal grouping.
The ileum tissue wax blocks were sectioned at 5μm thickness, followed by the routine immunochemistry procedure. They were incubated with flowing antibodies, ZO-1 (1:250, Invitrogen,617300), Occludin (1:250, Invitrogen, #71-150) ,Claudin-1 (1:300, Invitrogen,51-9000) at 4oC overnight. Anti-IL-1β (1:300,Abcam,ab9722),Anti-TNF-α (1:300,Abcam,ab66579).The expression of the related proteins was observed from three matched sections at least containing the small intestine from each group (n=four/group) under the microscope, and the average optical density value (integrated optical density value/area) was calculated with Image-Pro Plus software.
The ileum sample was embedded in 4% paraformaldehyde for 48 h, at 4℃. 2mm-long tissues were then dehydrated with 30% sucrose and frozen in OCT for 2 hours at -20oC. Then, they were cut into 10μm slices and treated with PBS containing 0.05%Triton (Sigma, T8787) and blocking solution (Thermo Fisher Scientific,37527). Then, slices were incubated with CD4 (1:200, BD,554843) and TCR-γ/δ (1:200,Biolegend,202605) at 4oC overnight. Then the slices were incubated with secondary antibodies at 37oC for 1 h, followed by DAPI (Absin, abs47047616) staining at room temperature (RT) for 10min. Finally, four small intestine slices were accessed per mouse and the microscope captured 3 fields for further analysis by pathologists blinded to the animal grouping.
Western Blot Analysis
After 24 h of ischemia-reperfusion, the small intestine of each group was harvested. The total protein of the tissues was extracted with RIPA buffer. Equal amounts of denatured protein samples were subjected to SDS-PAGE analysis, transferred onto a PVDF membrane and blocked with the blocking solution (5%BSA) for 1.5 h. The membrane was then incubated with primary antibodies ZO-1(1:1000, Invitrogen,617300), Occludin (1:1000, Invitrogen,#71-150) and Claudin-1 (1:1000, Invitrogen,51-9000), and β-actin (1:1000, Abcam,ab8826) under 4oC overnight and followed by a reaction with the corresponding secondary antibodies at RT. Bands were captured by Chemiluminescence imaging instrument and quantified usingImage J software.
Real-time (RT) -qPCR
Total RNA was extracted from the ischemic brain or the small intestinal using Trizol. The cDNA was produced by Revert Aid First Strand cDNA Synthesis Kit (Thermo, k1622). The two-step PCR cycling protocol was as follows: 40 cycles of 5s at 95oC and 5 s 60 oC using QuantiNova SYBR Green PCR Kit (QIAGEN,2008054). Data were analyzed by the 2-ΔΔCT method. The primers are shown in Table 1,and GAPDH was used as the housekeeping gene.
Table 1 Sequences of the primers for real-time PCR.
Primer sequence (5,- 3,)
Gene names Forward Reverse
GAPDH GGCACAGTCAAGGCTGAGAATG ATGGTGGTGAAGACGCCAGTA
TNF-α GCTACGGGCTTGTCACTC CCACGCTCTTCTGTCTACTG
IL-1β AGGTCGTCATCATCCCAC TTCAAATCTCACAGCAGCAT
CXCL1 GGGACACCCTTTAGCATCTT ACCCAAACCGAAGTCATAGC
CXCL2 GTCACCGTCAAGCTCTGG ACCCAAACCGAAGTCATAGC
50μl standard or sample was added to 96 well polystyrene microplates and incubated for 30 min at 37oC. Then 50μl HRP-Conjugate reagent was added and incubated at 37oC for 30 min. Next, 50μl chromogen solution and Tetramethylbenzidine were added in turn. The above reagents come from Nanjing Jinyibai Biotechnology Institute. Finally, the absorbance of every well was read at 450 nm.The serum sample was added to detect the IL-10,DAO and D-LAC expression. The whole small intestinal was used for evaluating IL-10 level.
Isolation of intestinal lamina propria lymphocytes Cell
Rats were sacrificed by pentobarbital overdose, a 15 cm-long piece of the terminal small intestine was excised and washed in PBS. After removal of Peyer' 's patches, mesenteric fat, and intestinal contents, the intestine was opened longitudinally, cut into 1 cm pieces, placed into 20 ml calcium- and magnesium-free DMEM with 10 mM EDTA, and then put in a shaking incubator set at 80 r.p.m and 37oC for 20 min. After incubation, tissue pieces were washed by vortexing four times with calcium-free PBS until the supernatant was clear. Next, the intestinal pieces were cut into 1mm fragments and digested in a separation solution composed of 5 mL DMEM containing Liberase TL (1 Wünsch unit/mL, Roche,05401020001) and DNase I (1 U/mL) at 37oC for 20 min with constant agitation (80 r.p.m.). After 20 min, we collected supernatants and added an equal volume of DMEM containing 10% bovine serum. The remaining tissue pieces were continued to be digested by adding 5mL new separation solution. We repeated the above steps three times for a total of 60 min. Subsequently, we separated the intestinal fragments mechanically on a 100 μm nylon net with the soft plug head, washed all the above cell suspensions twice with DMEM, and then filtered the cell suspensions with 40µm nylon cell strainer. Finally, the cells were resuspended in DPBS. Cells were stained for flow cytometry analysis.
Flow cytometry analysis
Cell suspensions were incubated for 10min with Fixable Viability Stain 780 (1:1000, BD. Horizon,565388) to clarify the dead and living cells. Then, cell suspensions were stained with CD45 (1.0 µg /100 µl, Biolegend, 202220) ,CD4 (0.25 µg /100 µl,BD,554843) and TCR-γ/δ (0.25 µg/100 µl,Biolegend ,202605) for extracellular staining. After 40 min, we used Fixation and Permeabilization buffers (Invitrogen,00-5523-00) to fix and permeabilize at 4℃ for 40min. For intracellular staining, the Foxp3 (1.0 µg /100 µl, eBioscience,17-5773-82) was used. Cells were washed and resuspended in 200 µl of PBS buffer and analyzed with a cytometer. Analysis was performed with CytExpert software.
The data were analyzed by one-way ANOVA and were expressed as mean ± SD, performing by Prism 8.0.2, Groups were compared using one-way analysis of variance (ANOVA) followed by Dunnett t3 post hoc test. P <0.05 was considered statistically significant.