The study design complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [20]. Male C57BL/6 mice, ranging in weight from 20 to 25 g, were used for this investigation. All Specific Pathogen Free (SPF)-grade mice were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, Liaoning, China) and were maintained by animal-care protocols established by the First Affiliated Hospital of Harbin Medical University. Throughout the study, the mice were housed under strictly regulated conditions with a 12/12-h photoperiod and an ambient temperature maintained at 23–25 °C. Under anesthesia, the mice were kept at a constant 37 °C. Mice were randomly assigned to the following groups: (1) sham; (2) transient middle cerebral artery occlusion/reperfusion (tMCAO/R); (3) tMCAO/R + negative control (NC); (4) tMCAO/R + IL7R; and (5) tMCAO/R + IL7R + the PI3K pathway inhibitor (LY294002). A total anesthetic and analgesia were administered during every surgical procedure. The detailed grouping criteria and the mice that died or bled are already provided in the Supplementary Table 1, Experiment 1–3.

Hanheng Biological Technology (Shanghai, China) Co., Ltd. supplied the AAV serotype 9 expressing IL7R (AAV-IL7R; Titer: 1.5 $\times$ 10¹² vector genome/mL; Promoter: CMV, 87012638) used in this research. The transfection efficiency was assessed with an AAV encoding green fluorescent protein (AAV-GFP; Titer: 1.3 $\times$ 10¹² vector genome/mL, 87012636, Hanheng Biological Technology). The negative control was AAV-null (87012637, Hanheng Biological Technology), which is devoid of IL7R encoding. Each mouse was given a 2 µL intraventricular injection of the viral suspension three weeks before tMCAO induction. The lateral ventricle was injected with AAV-IL7R, AAV-GFP, or AAV-null 21 days before tMCAO induction. The experimental groups were chosen at random. The mice were prepared by shaving their heads and disinfecting them under anesthesia (1.25% avertin, JT0781, Jitian Biotechnology Co., Ltd., Beijing, China, i.p.). The mice were fasted for 4 h before anesthesia to prevent any incidents during the procedure. Core temperature was continuously monitored and maintained at 37 $\pm$ 0.5 °C. The mouse’s head was fixed in the stereotaxic instrument while the mouse was under deep anesthesia (Anesthesia was induced with 1.25% avertin [0.02 mL/g, i.p.]. When a mouse showed signs of awakening during surgery, an additional dose of 1/2 or 1/3 of the original anesthetic was administered.). The skull was exposed. A saline-dipped cotton swab was used to remove the periosteum, and bregma and lambda were identified. After this, the injection site was aseptically exposed and marked (0.5 mm posterior and 1.0 mm lateral to the bregma, at a depth of 2.5 mm) [21]. The microinjection pump was connected, the air bubbles were expelled, and the needle was inserted vertically and slowly to the target depth. AAV (0.5 µL/min) was delivered to the lateral ventricle using a microsyringe. To ensure optimal delivery, the needle was left in position for 10 min post-injection to prevent backflow. An equivalent volume of AAV-null was administered to the lateral ventricle of the tMCAO/R + NC mice. Upon completion of the infusion, the needle was gradually retracted to avoid pulling on the brain tissue. The skin was sutured. The mouse was placed on a 37 ℃ heating pad until it regained consciousness.

LY294002 (S1737, Beyotime Biological Co., Ltd., Shanghai, China) was dissolved in phosphate-buffered saline containing 25% Dimethyl sulfoxide (DMSO, ST038, Beyotime Biological Co., Ltd.) at 50 mM. After dilution, 2 µL of the solution was administered by intracerebroventricular injection to each mouse 1 h before tMCAO induction [22]. LY294002 (a concentration of 10 µM) was supplemented to C8D1A cells (CL-0506, Procell Life Science&Technology Co., Ltd., Wuhan, China) 1 h before oxygen-glucose deprivation/reoxygenation (OGD/R) treatment [23].

The middle cerebral artery (MCA) was occluded in an ischemia model using line embolism. To summarize, anesthesia was induced with 1.25% avertin (0.02 mL/g, i.p.). When a mouse showed signs of awakening during surgery, an additional dose of 1/2 or 1/3 of the original anesthetic was administered. The mouse’s vital parameters were continuously tracked to ensure successful completion of the surgery. The procedure involved shaving and sterilizing the neck in preparation for a midline incision that would expose the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). Using surgical thread, the proximal end of the CCA, the distal end of the ICA, and both ends of the ECA were ligated. To occlude the MCA successfully, a tiny incision was made between the ligatures of the ECA. A line embolism then progressed into the ICA through the carotid bifurcation until it reached a mark of approximately 1 cm. After the surgery was completed, the mouse was placed on a heating pad. The line embolism was removed, and the incision was sutured after 1 h of tMCAO. After the mouse regained consciousness, it was placed in an incubator with temperature control for a full day. The only difference in the protocol of the sham group was the absence of MCA occlusion. Reducing the wound size without exposure was the goal of keeping the neck incision small. To ensure the mouse’s comfort and rapid recovery, it was kept on a heating pad set at 37 °C from the time it was anesthetized until it regained consciousness [24]. The mice were randomly divided into each experimental group. The sham-operated mice underwent identical surgical exposure without MCA occlusion. The survival rate of the mice reached 90%, and the mice that died or bled were excluded.

To evaluate neurological impairments in the mice, the modified Longa score was used. Each mouse underwent neurological function testing, and a researcher who was blind to the aim of the experiment assessed the mice. The scoring system was as follows: 0, no abnormalities; 1, trunk turning to the ipsilateral side when the tail was grasped; 2, the contralateral forelimb showed incomplete extension; 3, turning toward the paralyzed side; 4, leaning toward the opposite side; 5, inability to walk independently, loss of consciousness, or death. If the score was between 1 and 4, the tMCAO model was deemed successful [25]. Mouse data were excluded from the study if (1) the mouse’s score was 0 or 5 at the time of euthanasia; (2) the mouse died before euthanasia; or (3) the mouse showed signs of subarachnoid or intraparenchymal hemorrhage.

The mouse was euthanized, and its brain was removed and cut into 6 coronal sections, each 1 mm thick, beginning posterior to the frontal lobe. After 30 min of dark incubation in 2% TTC (G3005, Solarbio, Beijing, China) at 37 °C, the tissue sections were fixed in 4% paraformaldehyde (SI101-01, Sevenbio, Beijing, China). We used ImageJ software (1.53k, National Institutes of Health, Bethesda, MD, USA) to measure the infarct volume after imaging the brain slices. The infarct volume measured by TTC staining was corrected for edema using the formula: corrected infarct volume = volume of the contralateral hemisphere – volume of the viable tissue in the ipsilateral hemisphere. The infarction volume (%) = (contralateral hemisphere volume – ipsilateral hemisphere viable tissue volume)/contralateral hemisphere volume $\times$ 100%.

The brains were obtained without perfusion, immediately weighed (wet weight). The tissue was dried in an oven for 12 h and re-weighed (dry weight), and the BWC percentage was calculated as: [(wet weight – dry weight)/wet weight] $\times$ 100%.

The mice’s brains were harvested 24 h after tMCAO. After the blood was completely removed by perfusion with 4 °C saline, the mice were perfused with 4% paraformaldehyde until they became stiff. Coronal sections (7 µm thick) prepared using a cryostat (NX50, Thermo Fisher Scientific, Waltham, MA, USA) underwent immunofluorescence staining. After 20 min permeabilization in 0.5% Triton X-100 (BL3087A, Biosharp, Jiangsu, China) at room temperature, sections were blocked with 10% goat serum (AR0009, BOSTER, Wuhan, China) for 1 h. Primary antibodies targeting IL7R (1:100, TD6362, Abmart, Shanghai, China), glial fibrillary acidic protein (GFAP, 1:100, MU184607, Abmart), allograft inflammatory factor 1 (IBA1, 1:1000, MU146067, Abmart), and neuron-specific nuclear protein (NEUN, 1:1000, M050661, Abmart) were applied overnight at 4 °C, followed by 1 h incubation with suitable fluorophore-conjugated secondary antibodies (ZF-0516, ZF-0512 ZSBIO, Beijing, China) at room temperature under light-protected conditions. Nuclear counterstaining was achieved via 4^′,6-Diamidino-2-phenylindole (DAPI, BL739A, Biosharp). Fluorescence images were captured using a fluorescence microscopy platform (Nikon Eclipse Ti2, Tokyo, Japan).

RNA isolation was performed using distinct protocols: TRIzol reagent (15596026CN, Invitrogen, Thermo Fisher Scientific) was used for brain tissue samples, and total RNA extraction reagent (BSC51, Biozol, Hangzhou, China) was used for C8D1A cells. After RNA isolation, genomic DNA extraction, and cDNA synthesis were conducted according to the reverse transcription kit (FSQ301, ToYoBo, Tokyo, Japan). The sequences of primers used were as follows: IL7R-F, 5^′-ACGATTACTTCAAAGGCTTCTGGAG-3^′; IL7R-R, 5^′-AATGGTGACACTTGGCAAGACAG-3^′; Bcl-2-F, 5^′-CTACGAGTGGGATGCTGGAGATG-3^′; Bcl-2-R, 5^′-GGTTGCTCTCAGGCTGGAAGG-3^′; Bax-F, 5^′-GGAGACACCTGAGCTGACCTTG-3^′; Bax-R, 5^′-GCTCCATATTGCTGTCCAGTTCAT-3^′; Caspase3-F, 5^′-GACTGGAAAGCCGAAACTCTTCAT-3^′; Caspase-3-R, 5^′-AGTCCCACTGTCTGTCTCAATG-3^′; $\beta$-actin-F, 5^′-GTGCTATGTTGCTCTAGACTTCG-3^′; and $\beta$-actin-R, 5^′-ATGCCACAGGATTCCATACC-3^′. RT-qPCR analysis was performed on the SLAN-965 system (Hongshi Medical Technology Co., Ltd., Shanghai, China) with SYBR Green Kit reagents (SM143, Sevenbio). Gene expression quantification was performed by the 2^{-Δ⁢Δ⁢Ct} calculation method.

At 24 h after ischemia/reperfusion (I/R) induction, protein samples were obtained from the right hemisphere and C8D1A cells. Protein concentration was quantified using a BCA assay kit (P0010S, Beyotime Biological Co., Ltd.). Electrophoresis was conducted on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (12.5% or 10%) loaded with 30 µg of protein per sample. After 1 h of blocking with 5% skim milk at ambient temperature, the membranes were subjected to primary antibodies overnight at 4 °C. The secondary antibodies used were goat anti-rabbit (S0001, 1:3000, Affinity, Jiangsu, China) and goat anti-mouse (1:5000, ZB-2305, ZSBIO) for 1 h. Three Tris Buffered Saline Tween (TBST) washes preceded chemiluminescent detection using the BL520A ECL substrate (Biosharp). The primary antibodies used included IL7R (1:1000, TD6362, Abmart), caspase-3 (1:1000, T40044, Abmart), Bax (1:1000, T40051, Abmart), Bcl-2 (1:1000, T40056, Abmart), PI3K (1:1000, T40115, Abmart), phosphorylated PI3K (1:1000, T40116, Abmart), Akt (1:1000, 4691, Cell Signaling Technology, Danvers, MA, USA), and phosphorylated Akt (1:1000, 4058, Cell Signaling Technology), with GAPDH (1:150,000, 60004-1, Proteintech, Wuhan, China) and $\beta$-actin (1:1000, TA-09, ZSBIO) used as loading controls. Band intensity quantification was performed with ImageJ software.

The C8D1A (CL-0506, Procell Life Science & Technology Co., Ltd., Wuhan, China ) astrocyte cell line has undergone STR identification and mycoplasma testing, which confirmed that the quality of the C8D1A cell line is correct and there is no mycoplasma contamination. It was cultured in complete medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, CM-0506, Procell Life Science&Technology Co., Ltd.) supplemented with 10% fetal bovine (CM-0506, Procell Life Science&Technology Co., Ltd.) serum and 1% penicillin/streptomycin [26]. The cell lines were authenticated using DNA short tandem repeat analysis. Cultured cells were placed in a tri-gas incubator with 5% CO₂, 1% O₂, and balanced N₂ for 3 h after being cultured in glucose-free DMEM (NaHCO₃, glutamine, sodium pyruvate, serum-free, pH: 7.2–7.4) to induce OGD/R [27]. The cells were then cultivated for 2, 6, 12, 24, or 48 h under normoxic conditions (95% air, 5% CO₂) after restoration of complete medium. The cells that were used as controls were those that were kept under anoxic conditions. This study divided the cells into 5 groups: (1) control; (2) OGD/R; (3) OGD/R + NC; (4) OGD/R + IL7R; and (5) OGD/R + IL7R + LY294002.

A lentivirus with a titer of 10⁸ TU/mL was generated by cloning the coding sequence of the target gene into the LV5 vector. Gemma Gene Co., Ltd. (Suzhou, Jiangsu, China) generated the lentiviral construct and oversaw its synthesis. To measure the success of transfection, a virus that did not express any GFP was used as a negative control [28]. Once the transfection efficiency surpassed 70%, stable cell line selection was initiated. After lentiviral transfection, stably transfected cells, also known as mixed clones, were selected with puromycin (G04010, Genepharma, Suzhou, China) as a selection factor. The cells were grown and expanded after selection. After that, RNA was taken from the overexpression group and the negative control group for RT-qPCR.

C8D1A astrocytes (4500 cells/well, 96-well plates) were subjected to transfection and OGD/R [29], then incubated with 10 µL CCK-8 (C0037, Beyotime Biological Co., Ltd.) for 1 h. Cell viability was quantified by absorbance at 450 nm on a microplate reader integrated with an optical density detection system (EPOCH, BioTek, Winnsboro, VT, USA).

The rate of neuronal cell apoptosis was measured using the Fc method. After induction of OGD/R and lentiviral transduction, the C8D1A cells were harvested. The cells were assessed by Fc from an Annexin V-APC/7-AAD Apoptosis Detection Kit (abs50008, Absin Biotechnology Co., Ltd., Shanghai, China) [30].

The C8D1A cell samples were fixed, embedded, dehydrated, and cut into thin slices (60–70 nm). An imaging TEM microscope (H-7700, Hitachi, Tokyo, Japan) was used to examine and photograph these slices.

Data analysis was conducted with GraphPad Prism version 8.0 software (GraphPad Software, San Diego, CA, USA), and the experimental results are expressed as the means $\pm$ standard deviation (SD). The data in each of the experiments were independently obtained at least three times. When the data were normally distributed and showed equal variances, one-way analysis of variance (ANOVA) was performed; otherwise, the Kruskal-Wallis test was performed, with subsequent application of either Dunnett’s or Tukey’s post hoc tests for multiple comparisons. Statistical significance was considered to be p 0.05. The following thresholds were established for significance markers: *p 0.05; **p 0.01; ***p 0.001, with ns indicating nonsignificant differences.

To explore the involvement of IL7R in CIRI, we conducted temporal expression profiling of this receptor in the right cerebral hemisphere after tMCAO. Compared with controls, IL7R expression was significantly down-regulated following I/R injury. WB analysis revealed a time-dependent reduction in the IL7R protein level, with the lowest expression observed at 24 h postinjury (Fig. 1A). A marked reduction in IL7R expression was detected at 24 h post-tMCAO (p 0.001), with expression levels starting to recover at 48 h (Fig. 1B, p 0.01). On the basis of these findings, subsequent experiments were conducted 24 h after CIRI induction. These results suggested that IL7R plays a certain role in CIRI.

Fig. 1.

IL7R protein levels are suppressed at multiple post-tMCAO time points. (A) IL7R protein levels were detected by WB (n = 7 per group). (B) The quantitative analysis of IL7R protein in different groups. Data are shown as mean $\pm$ SD. The data were analyzed by one-way ANOVA followed by Dunnett posttests. **: p 0.01; ***: p 0.001; IL7R, interleukin-7 receptor; tMCAO, transient middle cerebral artery occlusion; SD, standard deviation; ANOVA, one-way analysis of variance; tMCAO/R, transient middle cerebral artery occlusion/reperfusion.

In normal brain tissue supplied by the middle cerebral artery of the mice, we performed double IF staining of IL7R with specific markers for astrocytes (GFAP), microglia (IBA1), and neurons (NEUN) (Fig. 2A). IL7R was colocalized with astrocytes, and IL7R was expressed in astrocytes. IL7R was hardly expressed in microglia (Fig. 2B). IL7R was rarely expressed in neurons (Fig. 2C). The Pearson correlation coefficients for these three groups of data were calculated with ImageJ software (Fig. 2D); |r| $>$0.5 was taken to indicate a clear association (closer to 1 = stronger, closer to 0 = weaker). The Pearson correlation coefficient of the GFAP group was the highest, leading to the conclusion that IL7R is abundantly expressed in astrocytes but rarely expressed in microglia and neurons. In summary, IL7R is detected in astrocytes rather than in neurons or microglia in the mouse brain.

Fig. 2.

IF double–staining of IL7R, GFAP, IBA1, and NEUN in mouse brain tissue. (A) Representative images of double-IF staining showing the colocalization of IL7R (red)/GFAP (green)/DAPI (blue). (B) Representative images of double-IF staining showing the colocalization of IL7R (red)/IBA1 (green)/DAPI (blue). (C) Representative images of double-IF staining showing the colocalization of IL7R (red)/NEUN (green)/DAPI (blue). (Scale bar = 50 µm for Fig. 2A–C). (D) The Pearson correlation coefficients for these three groups (n = 3 per group). Data are shown as mean $\pm$ SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; ***: p 0.001. IF, Immunofluorescence; GFAP, glial fibrillary acidic protein; DAPI, 4^′,6-Diamidino-2-phenylindole; IBA1, allograft inflammatory factor 1; NEUN, neuron-specific nuclear protein.

AAV-IL7R was injected into the lateral ventricle of mice to induce IL7R overexpression. In mouse brain sections of the MCA supply area treated with AAV-GFP (AAV-ZsGreen, 1.3 $\times$ 10¹² vector genome/mL), green fluorescence was detected by microscopy, confirming the success of AAV-mediated transduction. We found that GFP green fluorescence was widely expressed in the brain tissue surrounding the lateral ventricle of the mice (Fig. 3A). Subsequently, colocalization of IL7R with GFAP was assessed by IF double staining. The experimental data indicated that the levels of IL7R and GFAP in the brain tissue of the mice subjected to tMCAO were significantly reduced (Fig. 3C, p 0.05). Comparable levels were maintained between the tMCAO and tMCAO + NC groups. Notably, the experimental group with IL7R overexpression demonstrated substantial increases in both IL7R and GFAP expression in the brain specimens (Fig. 3C, p 0.05). These findings confirmed the localization of IL7R in astrocytes. After tMCAO/R, the level of IL7R decreased, and IL7R was successfully overexpressed. We reached the same conclusion in the RT-qPCR data, and the results confirmed that IL7R was successfully overexpressed after pretreatment with AAV-IL7R (Fig. 3D, p 0.001). Furthermore, immunofluorescence triple staining was conducted to verify that IL7R was expressed on the membrane of astrocytes after overexpression of IL7R (Supplementary Fig. 1).

Fig. 3.

Verification of successful overexpression of IL7R in tMCAO/R mouse brain tissue. (A) Representative IF image of AAV-GFP-transfected brain sections from mice. (Scale bar = 50 µm). (B) Representative images of double-IF staining showing the colocalization of IL7R (red)/GFAP (green)/DAPI (blue). (Scale bar = 50 µm). (C) Statistical results of IF staining for IL7R in the groups (n = 3 per group). (D) Relative mRNA levels of IL7R in the sham, tMCAO/R, tMCAO/R + NC, and tMCAO/R + IL7R groups (n = 3 per group). Data are shown as mean $\pm$ SD from at least three independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; *: p 0.05; **: p 0.01; ***: p 0.001. AAV, Adeno-Associated Virus; NC, negative control; GFP, Green Fluorescent Protein.

In our previous studies, we observed minimal IL7R expression at 24 h post-tMCAO (Fig. 1A), which prompted us to harvest brain tissues at this time point. To determine whether IL7R overexpression mitigates CIRI, we conducted TTC staining (Fig. 4A). Mice treated with IL7R-AAV presented smaller cerebral infarction volumes (Fig. 4A,B, p 0.001). Neurological deficit scores were also assessed, and the results are shown in Fig. 4C. The IL7R-treated group presented lower neurological function scores (Fig. 4C, p 0.01). Additionally, we measured BWC to evaluate cerebral edema induced by tMCAO. BWC was elevated in the tMCAO group (Fig. 4D, p 0.001). However, in the mice treated with IL7R, the BWC was reduced (p 0.01).

Fig. 4.

Overexpression of IL7R enhanced cerebral protection by reducing infarct volume, BWC, and neurological deficit scores in vivo. (A) TTC staining of brain tissues after tMCAO/R. (Scale bar = 5 mm). (B) Statistical analysis of the TTC staining results (n = 6 per group). (C) Neurological deficit scores of the mice post-tMCAO/R (n = 6 per group). (D) BWCs in the cerebral hemisphere were measured using the wet and dry weight method (n = 6 per group). Data are shown as mean $\pm$ SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; **: p 0.01; ***: p 0.001. TTC, 2,3,5-Triphenyl Tetrazolium Chloride; BWC, brain water content.

Apoptosis is a critical process in CIRI. Therefore, we investigated the effects of IL7R on apoptosis. The RT-qPCR data revealed substantial elevation in the mRNA levels of caspase-3 (Fig. 5C, p 0.001) and Bax (Fig. 5B, p 0.01), whereas Bcl-2 mRNA expression was reduced in tMCAO/R mice (Fig. 5A, p 0.001). IL7R treatment reduced caspase-3 (Fig. 5C, p 0.001) and Bax (Fig. 5B, p 0.01) mRNA levels and increased Bcl-2 mRNA levels (Fig. 5A, p 0.001) after tMCAO/R. WB results confirmed these findings. The overexpression of IL7R elevated the protein level of Bcl-2 (Fig. 5D,E, p 0.001) and decreased the protein expression of Bax (Fig. 5F,G, p 0.01) and caspase-3 (Fig. 5H,I, p 0.01). These effects were reversed by the LY294002 (Fig. 5D–I). Additionally, LY294002 treatment worsened neurological deficits (Fig. 5J, p 0.01) and exacerbated brain edema (Fig. 5K, p 0.05). Together, these results suggested that IL7R reduced CIRI by activating the PI3K/Akt pathway to partially regulate apoptosis.

Fig. 5.

The overexpression of IL7R improved the cerebral-protective effects by reducing apoptosis after tMCAO in vivo. (A–C) Relative mRNA levels of (A) Bcl-2, (B) Bax, and (C) caspase-3 in the sham, tMCAO/R, tMCAO/R + NC, and tMCAO/R + IL7R groups (n = 3 per group). (D–I) WB and quantitative analysis of Bcl-2, Bax, and caspase-3 protein levels in the sham, tMCAO/R, tMCAO/R + NC, tMCAO/R + IL7R, and tMCAO/R + IL7R + LY294002 groups (n = 7 per group). (J) Neurological deficit scores of each group of mice (n = 6 per group). (K) BWC of each group of mice (n = 6 per group). Data are shown as mean $\pm$ SD from at least three independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; *: p 0.05; **: p 0.01; ***: p 0.001. WB, western blot; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; LY294002, PI3K pathway inhibitor.

IF staining for IL7R and GFAP in brain tissue revealed that IL7R and GFAP expression were reduced after tMCAO, whereas GFAP expression was increased upon IL7R overexpression. These results confirmed that IL7R was colocalized with astrocytes (Fig. 3B), leading us to select the C8D1A cell line for further in vitro validation. Cell viability was assessed in vitro at various time points during OGD. Cell viability significantly decreased as the duration of OGD increased. To achieve a cell survival rate of approximately 50%, OGD was set to 3 h (Fig. 6A). In the OGD/R model, IL7R mRNA levels were markedly down-regulated by RT-qPCR, particularly after 24 h of OGD/R (Fig. 6B, p 0.001). We applied 3 h of OGD and 24 h of reperfusion to simulate the OGD/R model in C8D1A cells. IL7R overexpression was successfully established in stable transformants. This was indicated by the green fluorescence signal, seen under a fluorescence microscope, in C8D1A cells transfected with lentivirus-green fluorescent protein (LV-GFP) (Fig. 6C). The RT-qPCR analysis confirmed the successful establishment of the IL7R overexpression model (Fig. 6D, p 0.001).

Fig. 6.

Generation of an OGD/R model and an IL7R overexpression model in mouse brain astrocytes. (A) Cell viability analysis via CCK-8 assays after various durations of OGD (1, 2, 3, 4, 5, or 6 h), followed by 24 h of reoxygenation (n = 3 per group). (B) IL7R expression was significantly downregulated in OGD/R-exposed C8D1A cells. RT-qPCR analysis confirmed the significant decreasing in IL7R mRNA levels at 24 h of OGD/R (n = 3 per group). (C) Representative IF images of LV-IL7R and GFP-transfected C8D1A cells under normal and fluorescence microscopy. (Scale bar = 50 µm). (D) Relative mRNA levels and quantitative analysis of IL7R (n = 3 per group). Data are shown as mean $\pm$ SD from at least three independent experiments. The data of B were analyzed by one-way ANOVA followed by Dunnett posttests, and the rest of the data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; **: p 0.01; ***: p 0.001. OGD/R, oxygen-glucose deprivation/reoxygenation; CCK-8, Cell Counting Kit-8; LV-IL7R, lentivirus-IL7R.

Fc assessment was employed to determine the relationship between IL7R and apoptosis in vitro. The results revealed that C8D1A cells exposed to OGD/R exhibited a pronounced elevation in apoptotic rate, indicating substantial cellular damage. However, IL7R overexpression significantly reduced apoptosis (Fig. 7A,B; p 0.001). Furthermore, OGD/R markedly reduced cell viability relative to control conditions, but it was notably improved after IL7R overexpression (Fig. 7C, p 0.001). TEM analysis revealed various morphological alterations, such as DNA fragmentation and early apoptotic body formation, in the OGD/R and OGD/R + NC groups. These morphological changes were significantly less in the OGD/R + IL7R group (Fig. 7D). Collectively, these results suggested that IL7R exerts protective effects by inhibiting apoptosis in an in vitro model of CIRI.

Fig. 7.

IL7R overexpression reduced apoptosis after OGD/R in vitro. (A,B) Fc and quantitative analysis of apoptosis rates in C8D1A cells across different groups (n = 3 per group). (C) Cell viability analysis with the CCK-8 assay (n = 3 per group). (D) Representative TEM images of C8D1A cells (Scale bar = 5.0 µm). Data are shown as mean $\pm$ SD from at least three independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; ***: p 0.001. Fc, Flow Cytometry; TEM, Transmission Electron Microscopy.

To investigate the mechanism of IL7R-mediated neuroprotection via PI3K/Akt signaling in CIRI, pharmacological inhibition was implemented using LY294002, preceding IL7R overexpression and OGD/R induction. The RT-qPCR data revealed a marked reduction in Bcl-2, in parallel with elevated Bax and caspase-3 expression, in the OGD/R model. The overexpression of IL7R counteracted these alterations, resulting in significant enhancement of the Bcl-2 mRNA level and concurrent suppression of the Bax and caspase-3 mRNAs. Conversely, pharmacological PI3K inhibition with LY294002 abolished the antiapoptotic effects mediated by IL7R overexpression (Fig. 8A–C, p 0.05), indicating the involvement of the PI3K/Akt pathway. Subsequent WB analysis corroborated these observations, revealing increased Bcl-2, phosphorylated PI3K (p-PI3K), and activated Akt (p-Akt) protein expression coupled with decreased Bax and caspase-3 levels post–OGD/R treatment in the IL7R-overexpressing groups (Fig. 8D–M). Additionally, LY294002 reversed the protective effects of IL7R overexpression, as indicated by reduced cell viability (Fig. 8N) and decreased levels of Bcl-2 (Fig. 8D,E), p-PI3K (Fig. 8J,K), and p-Akt (Fig. 8L,M) but increased Bax (Fig. 8F,G) and caspase-3 (Fig. 8H,I) protein levels (all p 0.05). Together, the data pinpoint PI3K/Akt signaling as the operative route through which IL7R suppresses apoptosis.

Fig. 8.

LY294002 reversed the ability of IL7R to inhibit apoptosis after CIRI. (A–C) Relative mRNA levels and quantitative analysis of Bcl-2, Bax, and caspase-3 (n = 3 per group). (D–M) Representative WB images and quantitative analysis of Bcl-2 (D,E), Bax (F,G), caspase-3 (H,I), p-PI3K/PI3K (J,K), and p-Akt/Akt (L,M) protein levels in C8D1A cells of different groups (n = 3 per group). (N) Cell viability of C8D1A cells in different groups (n = 5 per group). Data are shown as mean $\pm$ SD from at least three independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns: not significant; *: p 0.05; **: p 0.01; ***: p 0.001. CIRI, Cerebral ischemia-reperfusion injury; p-PI3K, phosphorylated phosphatidylinositol 3-kinase; PI3K, phosphatidylinositol 3-kinase; p-Akt, phosphorylated protein kinase B; Akt, protein kinase B.