Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke

Neutrophil infiltration has been linked to worse clinical outcomes after ischemic stroke. Microglia, a key type of immune-competent cell, engage in cross-talk with the infiltrating immune cells in the inflamed brain area, yet the molecular mechanisms involved remain largely unexplored. In this study, we investigated the mechanisms of how canonical transient receptor potential 1 (TRPC1) modulated neutrophil infiltration in male mouse cerebral ischemia and reperfusion injury (CIRI) models. Our findings revealed a notable upregulation of TRPC1 in microglia within both middle cerebral artery occlusion reperfusion (MCAO/R) and in vitro oxygen-glucose deprivation/regeneration (OGD/R) model. Conditional Trpc1 knockdown in microglia markedly reduced infarct volumes and alleviated neurological deficits. Microglia conditional Trpc1 knockdown mice displayed less neutrophil infiltration in peri-infarct area. Trpc1 knockdown microglia exhibited a reduced primed proinflammatory phenotype with less secretion of CC-Chemokines ligand (CCL) 5 and CCL2 after MCAO/R. Blocking CCL5/2 significantly mitigated neutrophil infiltration in microglia/neutrophil transwell co-culture system upon OGD/R condition. Trpc1 knockdown markedly reduced store-operated calcium entry and nuclear factor of activated T-cells c1 (NFATc1) level in OGD/R treated microglia. Overexpression of Nfatc1 reversed the CCL5/2 reducing effect of Trpc1 knockdown, which is mediated by small interfering RNA in BV2 cells upon OGD/R. Our data indicate that upregulation of TRPC1 in microglia stimulates the production of CCL5/2 through the Ca 2 + / NFATc1 pathway. Upregulated CCL5/2 leads to an increase in neutrophil infiltration into the brain, thereby aggravating reperfusion injury. Our results demonstrate the importance of TRPC1 in microglia-mediated neuroinflammation and suggest a potential means for reducing CIRI induced neurological injury.


Introduction
Stroke represents one of the most common causes of death and morbidity worldwide.Acute ischemic stroke, due to inadequate blood flow to a specific brain region, account for approximately 84 % of prevalent strokes (Tsao et al., 2023).Efforts to restore blood supply, via mechanical thrombectomy and intravenous plasminogen activator infusion, often result in cerebral ischemia-reperfusion injury (CIRI) (Jurcau and Simion, 2021).CIRI cascade is driven by a myriad of physiological events mainly associated with neuroinflammation and oxidative stress, which contributes to the secondary damage following the primary ischemic insult (Lima et al., 2022;Freire et al., 2023).Among these events, neutrophils and other leukocytes migrate through the endothelial vessel wall and are attracted to the ischemic area, further aggravating injury by release proinflammatory factors and reactive oxygen species, proteases and matrix metalloproteinases (Chen et al., 2021b).Manipulating the neutrophils infiltration has emerged as a potential tactic for ameliorating CIRI outcomes, and therapeutic agents have shown promise in experimental stroke mice (Jickling et al., 2015;Nadkarni et al., 2022).Despite these advancements, our knowledge of immunomodulation remains limited.The complex molecular mechanisms regulating neutrophil infiltration after ischemic stroke are far from fully understood.
Microglia, resident immune cells in the central nervous system, play a significant role in post-ischemic stroke brain injury (Benakis et al., 2014).Microglia also engage in cross-talk with the infiltrating lymphocytes in the inflamed brain region, serving as a major source of proinflammatory chemokines that induce lymphocyte infiltration (Berchtold et al., 2020).Changes in the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) represent one of the major pathways for signal transduction in microglia (Brawek and Garaschuk, 2013).Microglia [Ca 2+ ] i is tightly regulated by diverse mechanisms and is coupled to executive functions such as release of pro-and anti-inflammatory cytokines (Färber and Kettenmann, 2006;Kettenmann et al., 2011;Lim et al., 2021).However, the identity and mechanism of the Ca 2+ regulatory apparatuses responsible for the cytokines release in microglia warrant further investigation.
Transient receptor potential (TRP) proteins constitute a vast nonvoltage-gated cation channel superfamily that can integrate multiple stimuli and transduce their activity to downstream cellular signal pathways via Ca 2+ entry (Mulier et al., 2017).Primarily functioning as nonselective Ca 2+ -permeable cation channels, TRP channels play a crucial role in various microglial cellular processes, including cytokine production, proliferation, and migration (Shirakawa and Kaneko, 2018).The canonical transient receptor potential (TRPC) channels, as the prominent nonselective Ca 2+ -permeable cation channels in this channel superfamily, play a key role in regulating inflammatory processes in the brain (Mizoguchi and Monji, 2017).The TRPC subfamily can be classified into three groups, namely TRPC1, TRPC4/5 and TRPC3/6/7 based on sequence homology (Wang et al., 2020).TRPC channels participate in regulating calcineurin (CaN) activity and nuclear factor of activated T-cells (NFAT) translocation to promote inflammatory gene expression (Hai et al., 2011;Tang et al., 2022).Nevertheless, the precise cellular and molecular mechanisms underlying the involvement of TRPCs in the response of microglia to ischemic stroke remain unclear.
In the present study, we provide evidence of TRPC1 upregulation in microglia following middle cerebral artery occlusion reperfusion (MCAO/R).Furthermore, we observe that TRPC1 increases CC-Chemokines ligand (CCL) 5 and CCL2 expression in microglia via NFATc1 pathway and promote neutrophil infiltration following MCAO/ R.These findings suggest that targeting microglial TRPC1 could be a promising therapeutic approach for ischemic stroke treatment.

Animals
Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1 flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1) CreER (Cyagen Incorporated) mice of C57BL/6 background.Adult male mice were used for the study, age matched male Trpc1 flox/flox mice were used as the Control group for in vivo experiments.The mice (8-10 weeks of age) were given tamoxifen (75 mg/kg, Sigma-Aldrich, St Louis, MO, USA) dissolved in corn oil intraperitoneally for 5 consecutive days.Mice had free access to water and food and were housed under a 12 h light/dark cycle at 25 • C. All animal experimental procedures were approved by the Animal Experiments Ethics Committee of Fourth Military Medical University.

Microglia and astrocyte culture
Primary microglia were prepared from 1 day neonatal mice.Brain cortices were dissected and placed in ice-cold Hanks balanced salt solution (HBSS) without Ca 2+ and Mg 2+ (Thermo Scientific Incorporated, Waltham, MA, USA).After removing the meninges, the cortices were ground and digested in 0.125 % trypsin (Gibco, Waltham, MA, USA) and 0.125 % Deoxyribonuclease (DNase) I (Sigma-Aldrich, St Louis, MO, USA) for 20 min at 37 • C. Then cell suspension was filtered through a 70 µm cell strainer (Corning, New York, NY, USA).Cells were then centrifuged (300 g, 5 min) and resuspended in Dulbecco's modified Eagle's medium (DMEM) F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) (Invitrogen), 1 % penicillin/streptomycin (Invitrogen), and 5 ng/mL macrophage colony stimulating factor (Sigma-Aldrich) and plated into flasks precoated with poly-Dlysine (Solarbio, Beijing, China).After mixed glial cultures reached complete confluence (10 days after culture), the flasks were placed on an orbital shaker (BSI-101, Being, Shanghai, China) and spun at 60 g for 6 h at 37 • C. The cells in the supernatant were microglia, while the adhesion cells were astrocytes.The microglia were collected and cultured for 24 h to allow adhesion.To induce Cre-loxP recombination and excise Trpc1 gene, microglia isolated from neonatal Trpc1 KD and Control mice were treated with 4-hydroxytamoxifen (0.02 mg/mL, Sigma-Aldrich) for 48 h.

Neuron culture
The cerebral cortex was isolated in a 60 mm Petri dish (Solarbio) containing ice-cold DMEM (Invitrogen).Tissues were gently pipetted and suspension was collected in a centrifuge tube.Cells were digested in 0.125 % trypsin (Gibco) for 15 min at 37 • C and after filtered with 70 µm cell strainers (Corning) and filtrates were collected.After centrifuged at 300 g for 5 min, cells were resuspended in DMEM/F-12 containing 10 % FBS (Invitrogen) and 1 % penicillin-streptomycin (Invitrogen), and then seeded on flasks precoated with poly-D-lysine (Solarbio).Cells were cultured for 4 h in a humidified incubator (LHS-250HC, Yiheng, Shanghai, China) at 37 • C. Then the medium was changed to complete medium which contained a neurobasal medium (Gibco) supplemented with B-27™ (Gibco) and 1 % penicillin-streptomycin (Invitrogen).The medium was changed by 50 % every 3 days.

Oxygen-glucose deprivation and regeneration (OGD/R)
Oxygen-glucose deprivation (OGD) followed by regeneration was performed in primary cultured cells and BV2 cells.Briefly, the culture medium was replaced by a glucose-free DMEM buffer (Invitrogen), and then cells were placed in a hypoxic humidified incubator (27310, Stem Cell, Vancouver, Canada) flushed with a gas mixture of 94 % N 2 /5 % CO 2 /1 % O 2 ) for an appropriate duration based on the cell type.For details, 1 h OGD was performed in primary cells, while 4 h OGD was applied in BV2 microglial cells as previously reported (Li et al., 2021; H. Qian et al.Cui et al., 2023).Then the medium was replaced by the previous culture medium and cells were cultured under normoxia conditions.

Real-time polymerase chain reaction (PCR)
RNA was extracted via an RNA extraction kit (Takara, Tokyo, Japan) according to the manufacturer's instrument.Reverse transcription was performed according to the instrument of a reverse transcription kit (Takara).Then, complementary DNA (cDNA) was amplified by an SYBR Premix ExTaq (Takara) on a Bio-Rad CFX96 Detection System (Bio-Rad, Carlsbad, CA, USA).Sequences of primers are listed in Table S1.DNA contamination was verified by non-template control and reversetranscriptase control, and primer specificity was determined by melt curves.Raw threshold cycle values were normalized to the glyceraldehyde-3-phosphate dehydrogenase gene using the 2 − ΔΔCt comparative method.

MCAO/R
Temporary focal cerebral ischemia was induced by MCAO/R 9 days after tamoxifen administration as previously reported (Huang et al., 2020).Mice were anesthetized via 2 % isoflurane inhalation (RWD Incorporated, Shenzhen, China).After the right common carotid artery (CCA) and external carotid artery (ECA) were isolated, a 6-0 silk suture coated with silicone (Yushun, Pingdingshan, China) was inserted from the incision near the ECA-CCA branch and advanced about 9-11 mm to block the origin of the middle cerebral artery.Reperfusion was performed 60 min later.Rectal temperature was kept at 37 • C during MCAO/R using a heating pad (Jitai, Wuxi, China).The degree of regional cerebral blood flow was monitored during the surgery using laser speckle contrast imaging (RFLSI III, RWD Incorporated).

Neurological deficit scores test
Neurological deficits were evaluated using the modified Neurological Severity Score (mNSS) test according to a previous study (Chen et al., 2001).The neurological deficits scores are graded from 0 to 18.No neurological deficit is denoted by a score of 0. Scores 1-6, 7-12, and 13-18 indicate mild, moderate, and severe neurological deficits respectively.

Foot fault test
The foot fault test was performed to determine the function of the left forelimb.Mice were placed on an 8 × 10 square grids of 2 × 2 cm (LE1305, Panlab, Barcelona, Spain).The forelimbs were placed on the wire while moving along the grid.Falling or slipping during moving between grids was recorded as foot fault for once.

Rotator test
The rotator test was performed to evaluate motor functional deficit caused by MCAO/R (Zhang et al., 2018).Briefly, mice were placed on a rotating cylinder (LE8205, Panlab) and forced to walk when the cylinder was rotated at a speed of 40 revolutions/min.The latency for each mouse to fall off the cylinder was recorded.Experiments were repeated 4 times at 10 min intervals.

Triphenyl tetrazolium chloride (TTC) staining
TTC assay was performed to determine the infarct volume as previously described (Zhang et al., 2016).Mice were anaesthetized via 2 % isoflurane inhalation (RWD Incorporated) and decapitated 24 h after reperfusion.Brains were dissected, frozen at -20 • C for 30 min and coronal sectioned at a thickness of 2 mm, and then immersed in 1.5 % TTC solution (Sigma-Aldrich) at 37 • C for 30 min.The sections were then fixed in 4 % paraformaldehyde (Sigma-Aldrich) overnight and photographed by a camera (EOS R, Canon, Tokyo, Japan) equipped with an F2.8 100 mm macro lens (Canon).The lighting was performed by two flashlights (860 II, Godox, Shenzhen, China) to ensure a stable 5500 K color temperature.Infarct volume was analyzed and calculated using Photoshop (Version CC 2019).

Evans blue staining
The impaired blood-brain barrier integrity caused by MCAO/R was assessed by Evans Blue staining (Guo et al., 2019).Evans Blue dye (2 %, 4 mL/kg, Sigma-Aldrich) was intravenously injected 22 h after reperfusion and circulated for 2 h.After being perfused with phosphate-buffered saline (PBS), the brains were dissected, frozen, and sectioned into 2-mm-thick coronal slices.The sections were then fixed in 4 % paraformaldehyde (Sigma-Aldrich) overnight and photographed by a camera (EOS R, Canon) equipped with an F2.8 100 mm macro lens (Canon).The lighting was performed by two flashlights (860 II, Godox) to ensure a stable 5500 K color temperature.To quantitatively assess the Evans Blue content in the ischemic hemispheric tissue, the ipsilateral hemisphere was separated, weighed, and homogenized in 5 mL formamide (Sigma-Aldrich) then incubated at 60 • C for 72 h and centrifuged at 250 g for 10 min.The supernatant was collected and added along with standards to 96-well plates (BKMANLab, Beijing, China).The optical density was measured at λ = 450, 570 nm by using a microplate reader (Multiskan MK3, Thermo Scientific Incorporated).Dye content in brain tissue was calculated as: Dye concentration (mL/g) × formamide volume (mL) / tissue weight (g).

Separation of peri-infarct area
The peri-infarct area was delineated based on the average infarct volume measured by TTC staining in our pilot experiments.Mice were deeply anaesthetized via 2 % isoflurane inhalation (RWD Incorporated) and decapitated 24 h after reperfusion, the brain tissue was collected and put on ice.Following the removal of the olfactory bulb and cerebellum, the left brain tissue was coronally sliced 3 mm backwards behind the top of the frontal lobe into three sections with thicknesses of 3 mm (Section 1), 4 mm (Section 2), and 3 mm (Section 3), respectively.Section 2 was taken out for further processing.A sagittal cut 1.5 mm from the midline was then made from the top to the bottom of the ipsilateral hemisphere, and a transverse diagonal cut was performed around the "1 o'clock" position.The tissue outside the "1 o'clock" position was the infarct core area.The cortical tissue between the sagittal cut and the "1 o'clock" position was classified as the peri-infarct area.
H. Qian et al.

Preparation of single-cell suspensions
The peri-infarct region of Control and Trpc1 KD mice was dissected 24 h after MCAO/R.The tissue was first enzymatic digested in HBSS (Thermo Scientific Incorporated) containing 24 Unit/mL Papain (Sigma-Aldrich) for 10 min at 37 • C, then an additional digestion step was carried out after adding 1 mg/mL collagenase D (Roche, Basel, Switzerland) and 0.1 mg/mL DNase I (Sigma-Aldrich) for 10 min at 37 • C, enzymatic reaction was ended by adding FBS (Invitrogen) at a final concentration of 10 %.The suspension was then gently triturated and filtrated over a 100 µm strainer (Corning) to obtain a single-cell suspension.

Isolation of microglia from adult brain with microbeads
Microglia were isolated from the penumbra of Trpc1 KD or Control mice 24 h after MCAO/R by using a magnetic-activated cell sorting (MACS) kit (Miltenyi Biotec., Bergisch Gladbach, Germany).Brains were removed, minced, and enzymatically digested in HBSS (Thermo Scientific Incorporated) containing 50 mg/mL collagenase D (Invitrogen), 100 μg/mL tosyl lysyl chloromethyl ketone trypsin inhibitor (Sigma-Aldrich), 0.1 mg/mL DNase I (Sigma-Aldrich) and 1 mg/mL N-2hydroxyethylpiperazine-N-ethane-sulphonicacid (Gibco) for 1 h at 25 • C.After being pushed through a 70 µm strainer, the cell pellet was resuspended in 30 % percoll solution (Univ, Shanghai, China) and centrifuged at 1200 g for 5 min to remove myelin debris.Then cells were washed in PBS and resuspended in MACS buffer (Miltenyi Biotec.).CD11b + microglia were isolated using manual MACS sorting.Cells were incubated with FcR blocking reagent (Miltenyi Biotec.)prior to labeling with CD11b antibodies (Elabscience, Wuhan, China) for magnetic isolation to prevent non-specific labeling of CD11b + microglia.The purity of microglia was verified by flow cytometry.

Multiplex chemokine immunoassay
We measured the chemokine concentrations in isolated microglia using LEGENDplex™ 13-plex proinflammatory chemokine panel (Bio-Legend, San Diego, CA, USA) allows simultaneous quantification of mouse chemokines including CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL20, CCL22, chemokine C-X-C motif ligand (CXCL) 1, CXCL5, CXCL9, CXCL10, and CXCL13.Twenty-four hours after MCAO/R, microglia were purified from the peri-infarct area of Control or Trpc1 KD mice by using the MACS method.Cells were homogenized in ice-cold PBS, concentration of protein samples was determined by using a Pierce™ bichioninic acid protein assay kits (Thermo Scientific Incorporated).Samples were then diluted in assay diluent buffer and chemokines concentration was measured.After incubation with the APC-conjugated capture beads and PE-conjugated detection reagents, the samples were analyzed on FACS Aria III and quantified using the LEGENDplex™ software (version 8.0).Chemokine concentrations of each sample were adjusted by the total protein concentrations.

Neutrophil isolation and migration assay
Splenic neutrophils were isolated from male mice 10 weeks old by using a neutrophil isolation kit (Miltenyi Biotec.).Neutrophils were cultured in RPMI-1640 medium (Gibco) supplemented with 10 % FBS (Invitrogen), 1 % penicillin/streptomycin (Invitrogen), 1 % 2-mercaptoethanol (Invitrogen) and 1 % L-glutamine (Invitrogen).A total of × 10 6 neutrophils were seeded onto a 6.5-mm, 3 µm pore size, polyester membrane transwell inserts (Corning), insert was then placed into the conditioned medium collected from the microglia.Transwell systems were incubated in 37 • C for 16 h, migration rate was quantified by counting the number of neutrophils that migrated to the lower compartment.

Small interfering RNAs (siRNA) transfection
For siRNA transfection, siRNAs targeting Trpc1 (Sense: CUA AAU CAU GGA GCG AAU AAU TT; Antisense: AUU AUU CGC UCC AUG AUU UAG TT) were synthesized by Hippo Biotechnology Co., Hangzhou, China.A scrambled siRNA, which produced similar results to the control group, was applied as a negative control.siRNA and Lipofectamine (Invitrogen) were mixed in OptiMEM (Gibco) and added to the culture medium at a final concentration of 160 nM, then the BV 2 cells were incubated for 48 h, and the medium was replaced by fresh DMEM containing 10 % FBS (Invitrogen).

Plasmid transfection
For plasmid transfection, mouse cDNA fragments encoding fulllength NFATc1 were prepared by PCR and cloned into a pcDNA3.1 vector.The plasmids encoding constructs were transfected into BV2 cells using Lipofectamine 2000 (invitrogen).Cells were seeded in culture dishes and allowed to adhere overnight.2.0 μg pcDNA3.1 and 3 μL Lipofectamine 2000 were separately diluted in serum-free OptiMEM (Gibco) to final volumes of 250 μL and incubated at 25 • C for 15 min, then 750 μL DMEM containing 10 % FBS (Invitrogen) was added and incubated at 37 • C for 5 h, finally the medium was replaced by fresh DMEM containing 10 % FBS (Invitrogen).

Measurement of [Ca 2+ ] i
For measurement of [Ca 2+ ] i , primary microglia isolated from Control or Trpc1 KD mice were exposed to OGD/R or normoxia condition.
Following 5 μM thapsigargin (Tg, Sigma-Aldich) added for 6 min, the cells were provided with a buffer containing 1.8 mM Ca 2+ .Every 1.2 s, Fura-2 was alternately excited at 340 nm and 380 nm for each 30 ms, and the emitting fluorescence intensity was recorded by an inverted epifluorescence microscope (IX51, Olympus).Ratio were calculated from the Fluorescence 340 nm and Fluorescence 380 nm.Single cell was marked as regions of interest, and a fluorescence ratio of 340 nm/380 nm versus time point was plotted.

Enzyme-linked immunosorbent assay (ELISA)
The concentration of CCL5 (KT65723, Mosak Biotechnology, Wuhan, China), CCL2 (KT98087, Mosak Biotechnology), and the activity of CaN (ml092929, Mlbio, Shanghai, China) were determined using specific ELISA kits.Microglia were homogenized in ice-cold PBS followed by centrifugation at 1000 g for 5 min at 4 • C. The supernatant was collected and used in sandwich ELISA following the protocols of the manufacturers.

Statistical analysis
Data are presented as means ± standard deviation.Normal distribution was tested by using the Shapiro-Wilk test.The choice of statistical analysis was based on the number of groups to be compared.For comparison between the two groups, a Student's t-test was performed.For comparison between more than two independent groups, one-way analysis of variance (ANOVA), followed by a Dunnett t-test was used, for analyses involving multiple influencing variables two-way ANOVA, followed by a Dunnett t-test was used, P-values < 0.05 were considered statistically significant.

CIRI induces TRPC1 upregulation in microglia
In the present study, we aimed to explore the potential role of microglia TRPCs after stroke.We isolated and cultured microglia from the brains of neonatal mice, then OGD/R injury was performed.Realtime PCR analysis showed that only Trpc1 expression was significantly upregulated upon OGD/R condition (Fig. 1A).Western blot analysis corroborated the real-time PCR results, showing that TRPC1 expression in microglia was considerably elevated after OGD/R injury, while no significant difference of TRPC1 expression was observed in astrocytes and neurons subjected to OGD/R injury (Fig. 1B).Additionally, in mice subjected to MCAO/R injury, we examined TRPC1 expression by immunofluorescence.The number of TRPC1/Iba1 + cells dramatically increased in peri-infarct area 24 h after MCAO/R (Fig. 1C).Collectively, these data suggest that TRPC1 is upregulated in the peri-infarct area after CIRI and that activated microglia are the important source of TRPC1.

Conditional genetic knockdown of Trpc1 in microglia
To determine whether TRPC1 in microglia would have an impact on stroke outcome, we generated inducible Trpc1 KD mice by crossing Trpc1 flox/flox mice with Cx3cr1 CreER mice (Fig. 2A).MCAO/R surgery was performed 9 days after tamoxifen administration.The knockdown efficacy was confirmed in isolated microglia 24 h after MCAO/R.Western blot analysis showed that expression of TRPC1 was significantly reduced in Trpc1 KD mice compared with Trpc1 flox/flox Control mice (Fig. 2B), indicating an effective conditional Trpc1 knockdown.Under physiological conditions, Trpc1 KD mice did not showed any significant growth deficit, as assessed by body weight every 2 weeks (Fig. 2C).There were no differences in either gross brain structure or the number of microglia, astrocytes and neurons compared with Control mice (Fig. 2D).Furthermore, no significant differences were observed between Trpc1 KD and Control mice in regional cerebral blood flow at baseline, ischemia as well as reperfusion period (Fig. 2E).

Microglia specific Trpc1 knockdown reduces brain injury caused by MCAO/R
To explore the potential participation of microglia-derived TRPC1 in ischemic brain injury, we induced MCAO/R injury in both Control and Trpc1 KD mice.We first tested the neurological function 24 h after induction of MCAO/R.Trpc1 KD mice exhibited less impairment in mNSS (Fig. 3A) and foot-fault test (Fig. 3B).Trpc1 KD mice also exhibited a longer latency to fall in the rotarod test (Fig. 3C).We then observed the infarct volume via TTC staining, the results indicated that Trpc1 KD mice had significantly smaller infarcts volume than Control mice 24 h after MCAO/R (Fig. 3D).Furthermore, examination of blood brain barrier permeability by Evans Blue revealed that the Trpc1 KD mice had significantly smaller Evans Blue staining area and less Evans Blue extravasation compared to Control group (Fig. 3E).Cell apoptosis was further assessed by TUNEL staining.The number of TUNEL + cells was significantly lower in peri-infarct area of Trpc1 KD mice when compared to Control group after MCAO/R (Fig. 4).Taken together, these results suggest that selective knockdown of TRPC1 in microglia plays a protective role after MCAO/R.

Microglia Trpc1 aggravates neutrophil infiltration after MCAO/R
It is known that microglia orchestrate both initiation and progression of leukocytes infiltration evoked by ischemic brain damage (Benakis et al., 2014;Chen et al., 2021a).We measured the accumulation of leukocytes in the brains of Trpc1 KD and Control mice after MCAO/R by using flow cytometry.Interestingly, results showed that there was no Fig. 1.Canonical transient receptor potential channel (TRPC) 1 expression is upregulated in microglia after stroke.A Quantitative polymerase chain reaction to assess the expression of Trpcs in primary cultured microglia underwent oxygen-glucose deprivation/regeneration (OGD/R) injury, expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) RNA and expressed as % relative to Gapdh (n = 6 per group).B TRPC1 protein levels in primary cultured microglia, neurons, and astrocytes were detected by Western blot assay upon OGD/R condition, each group (Normoxia or OGD/R) consisted of 3 bands from one sample.Each point represents the average intensity of 3 bands.Data are presented as percentages in comparison to the Normoxia group (n = 3).C Representative immunofluorescence staining for microglia marker, glial fibrillary acidic protein (GFAP, red) and TRPC1 (green) from the peri-infarct area of brain tissue after middle cerebral artery occlusion reperfusion.4',6-diamidino-2-phenylindole was used to stain the nuclei (blue).Scale bars, 100 µm.Immunofluorescence intensity of TRPC1 was quantified using ImageJ software and shown in right panel (n = 4 per group).** P < 0.01.difference between two groups in the numbers of CD4 + T cells, CD8 + T cells, B cells, macrophages, and NK cells.However, Trpc1 KD mice had decreased accumulations of neutrophil in the brain (Fig. 5A).There was no difference between two groups in the numbers of leukocyte in the spleen after MCAO/R (Fig. 5B).To confirm the decreased infiltration of neutrophils in Trpc1 KD mice after MCAO/R, we conducted immunofluorescence staining.A significant decrease in Ly6G-positive cells, indicative of neutrophils, was observed in Trpc1 KD mice compared with Control group within the peri-infarct area (Fig. 5C).This decrease persisted up to 7 days after reperfusion (Fig. 5D).The spleens of two group had comparable numbers of neutrophils after MCAO/R (Fig. 5E).When depleting neutrophils in blood, the mNSS (Fig. 6A), foot fault (Fig. 6B), latency to fall time in rotarod test (Fig. 6C) as well as the infarct volumes (Fig. 6D) were comparable between Trpc1 KD mice and Control mice after MCAO/R.These collective findings suggest that knockdown of microglia Trpc1 may lead to a reduction in the accumulation of neutrophils in the ischemic brain, potentially exerting a protective effect against CIRI.

Microglia TRPC1 aggravates neutrophil infiltration via CCL5 and CCL2 induction
Migration of neutrophils to injury tissue depends on many molecules and chemokines (Kolaczkowska and Kubes, 2013).To gain a comprehensive view of chemokines in microglia regulated by TRPC1 after MCAO/R, we adopted a multiplex chemokine immunoassay that measured proinflammatory chemokine from lysates of purified microglia at 24 h after MCAO/R in Control and Trpc1 KD mice (Fig. 7A).Results showed that Trpc1 knockdown reduced CCL5, CCL2 level (Fig. 7B), which has been reported to be involved in neutrophil infiltration after stroke (Fan et al., 2016;Geng et al., 2022).In primary cultured microglia, ELISA experiments revealed a significant increase in CCL5 and CCL2 levels upon OGD/R injury, which was attenuated by Trpc1 knockdown (Fig. 7C).Flow cytometry analysis showed that Trpc1 knockdown reduced CCL5 and CCL2 positive cells upon OGD/R condition (Fig. 7D, E).Collectively, these results indicate that microglia TRPC1 may lead to a production of CCL5 and CCL2 after MCAO/R.
To assess whether microglia TRPC1 influences neutrophils infiltration via CCL5 and CCL2 after MCAO/R.The microglia were exposed to OGD/R and then cultured for another 4 h in a Transwell system with purified neutrophils by a Transwell membrane (Fig. 7F).The number of neutrophils that had migrated into the microglia Transwell chamber was significantly decreased, followed by Trpc1 knockdown in microglia (Fig. 7G).We then added antibodies into the microglia-conditional medium to block the action of CCL5 and CCL2.Blocking of CCL5 and CCL2 increased the proportions of neutrophils in the Trpc1 KD group, notably, combining the blocking of CCL5 and CCL2 led to similar proportions of neutrophils in both the Trpc1 KD and Control groups (Fig. 7H).These data indicate that microglia TRPC1 promotes neutrophils infiltration via increasing CCL5 and CCL2 after MCAO/R.

TRPC1 upregulates CCL5 and CCL2 in microglia via NFATc1 pathway
To investigate whether TRPC1 regulates CCL5 and CCL2 in microglia under OGD/R conditions through Ca 2+ signaling, we assessed the induction of store-operated calcium entry (SOCE) in primary cultured microglia using Tg, a sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitor, by comparing the second peaks of each trace, which represents the [Ca 2+ ] i into cells (Berna-Erro et al., 2023).Results showed that the SOCE is significantly lower in OGD/R treated Trpc1 KD microglia than that in OGD/R treated Control microglia, implying that the TRPC1 channel participates in the SOCE in microglia caused by OGD/R (Fig. 8A, B).Then the microglia were treated with SKF96365 to block the TRPC-mediated Ca 2+ influx (Merritt et al., 1990), the CCL5 and CCL2 level become equal in Trpc1 KD and Control microglia (Fig. 8C).OGD/R treated Trpc1 KD microglia also displayed lower CaN, a calcium-responsive phosphatase, activity compared with OGD/R treated Control microglia (Fig. 8D), indicating TRPC1 regulates CCL5 and CCL2 likely dependent on Ca 2+ signaling.NFATc1 is directly dephosphorylated by CaN and consequently translocates to the nucleus and regulates the expression of multiple genes involved in chemokine generation (Hogan et al., 2003;Fric et al., 2012).The nuclear NFATc1 level was significantly lower in OGD/R treated Trpc1 KD microglia compared with OGD/R treated Control group (Fig. 8E).In BV2 cells, CCL5 and CCL2 level was detected by ELISA upon OGD/R condition, results showed that overexpression of Nfatc1 abolished the decreasing trend of CCL5 and CCL2 caused by siTrpc1.Taken together, these results indicate that TRPC1 regulates CCL5 and CCL2 via the Ca 2+ /CaN/NFATc1 signaling pathway in microglia upon OGD/R condition.

Discussion
Cerebral ischemia and reperfusion remain among the leading causes of brain injuries and fatalities, underscoring the critical need to uncover therapeutic targets and explore the underlying mechanisms.Neutrophil infiltration has been consistently implicated in the pathophysiology of CIRI and is associated with worse outcomes.This study investigates the involvement of microglial TRPC1 as a key molecular component exacerbating brain injury post-CIRI.
Acute ischemia and reperfusion produces an infarct core, in which the necrotic cell death is rapid and irreversible, and a peri-infarct area around the core in which the cell damage is delayed and rescuable (Yang and Liu, 2021).Therefore, the peri-infarct area is the prime region to target for ameliorating ischemic insult.In this study, we demonstrated that TRPC1 expression is upregulated in peri-infarct area after stroke.Elevated TRPC1 expression mainly co-localizes to microglia marker, Iba1.This observation prompted us to further investigate the involvement of microglial TRPC1 in the pathological processes of CIRI.
To specifically target the TRPC1 in microglia, we utilized conditional knockdown mice with inducible microglia-specific Trpc1 gene knockdown via Cre recombinase driven by the CX3CR1 promoter coupled with estrogen receptor activation.Compared with the constitutive mice, this method excludes the compensatory outcomes of other TRPCs during development.It should be noted that in addition to microglia, monocytes, dendritic cells, NK cells, and T cells also expresses CX3CR1 (Oggero et al., 2022).These myeloid cells replenish with at a rapid turnover rate, while microglia are a low turnover rate population (Ajami et al., 2007).Leveraging these distinctions, tamoxifen administration facilitated exclusive Cre-dependent gene knockdown in microglia.
In our study, we observed reduced lymphocyte infiltration in Trpc1 KD mice after CIRI, with neutrophils exhibiting the most significant decrease.Neutrophils play a critical role in the immune response following CIRI, infiltrating the ischemic region approximately 30 min after ischemia and peaking at around 24-48 h (Jickling et al., 2015;Perez-de-Puig et al., 2015).Neutrophils play a crucial part in postischemic inflammation by limiting tissue perfusion through intravascular obstruction and disrupting the integrity of the blood-brain barrier via the release of matrix metalloproteinases (Anrather and Iadecola, 2016).Considering the critical role of neuroinflammation in pathogenesis of stroke, targeting neutrophils offers an effective therapeutic for brain injury (Kelly et al., 2021).The infiltration of neutrophils into ischemic tissue is orchestrated by resident cells, which release adhesion molecules or chemokines (Kim and Luster, 2015).Among these chemokines, CCL5 and CCL2 are reported to involve in the neutrophils recruitment (Schuette-Nuetgen et al., 2012;Yu et al., 2016).In the central nervous system, microglia are an important source of CCL5 and CCL2 (Myers et al., 2009;Lanfranco et al., 2017).In this context, we found Trpc1 knockdown downregulated CCL5 and CCL2 expression in microglia upon OGD/R condition.On this basis, we reasoned that TRPC1 in microglia could affect the infiltration of neutrophils into the brain after CIRI by regulating CCL5 and CCL2 production.
TRPC1 has been shown to contribute to SOCE in several different cell types (Ambudkar, 2007).In addition to serving as a nonselective cation channel, TRPC1 confers its functions by embedding in a protein complex with cytosolic proteins (Beech, 2005;Nesin and Tsiokas, 2014).For instance, TRPC1 acts as a scaffold at the neuron surface to assemble a signaling complex independently of a Ca 2+ influx (Heo et al., 2012).To determine whether TRPC1 regulates CCL5 and CCL2 via Ca 2+ influx, we first detected the SOCE stimulated by endoplasmic reticulum SERCA inhibitor Tg, results showed that Trpc1 knockdown reduced the SOCE in microglia upon OGD/R.Furthermore, blocking TRPC-mediated Ca 2+ influx with SKF96365 reversed the regulation of CCL5 and CCL2 levels by Trpc1 knockdown, indicating that TRPC1 regulates their expression through Ca 2+ signaling.A transduction pathway is required for the TRPC-controlled Ca 2+ signaling to be transferred to the nucleus.For the [Ca 2+ ] i dependent signaling pathway, we specifically focused on the CaN signaling.The pathological role of the signaling in central nervous system diseases has been repeatedly attributed to disturbances in CaN signaling (Chigurupati et al., 2010;Wang et al., 2016).In our study, Trpc1 knockdown reduced CaN activity in microglia, indicating that TRPC1-mediated [Ca 2+ ] i is sufficient to activate CaN in microglia.This observation is in accordance with an expanding body of reports indicating that TRPCs serves as an important Ca 2+ source for CaN activation (Kuwahara et al., 2006;Greka and Mundel, 2011).It is well established that TRPC-CaN participates in multiple pathological processes by activating transcriptional factors.Previous studies have demonstrated that NFATc1, which is directly dephosphorylated by CaN, is the most frequently involved transcription factor downstream of CaN, leading to its translocation to the nucleus (Hogan et al., 2003;Park et al., 2020).NFATc1 plays a critical role in inflammation and immunity and has previously been reported to regulate the expression of multiple genes involved in chemokine generation (Bécart et al., 2007;Fric et al., 2012).In our research, we found that silencing of Trpc1 reduced CCL5 and CCL2 expression in BV2 cells upon OGD/R, this effect is reversed by overexpression of NFATc1, indicating that TRPC1 regulates CCL5 and CCL2 expression via NFATc1.
When interpreting the results, it is important to acknowledge a limitation in the current study.The use of young adult mice to model stroke presents potential pitfalls, as stroke predominantly affects the elderly population.To better reflect the true pathological situation of the disease, utilizing aging mice would be more appropriate given that aging is the most significant independent risk factor for stroke.The profound effects of age on clinical ischemic stroke are mirrored in the results of experimental studies.In stroke animal models, aging rats exhibit more severe neurological impairment and less successful functional recovery compared to young rats.Furthermore, neurogenesis is also markedly attenuated in aging MCAO/R models (Darsalia et al., 2005).Aging animals have less edema formation after stroke compared to the young (Liu et al., 2009).Additionally, astroglial responses to stroke injuries are also accelerated by ageing (Popa-Wagner et al., 2006).Therefore, it is important to consider that the response to microglia Trpc1 knockdown may vary between young and aging stroke mice.Further investigation is needed to validate the impact of microglia Trpc1 knockdown on stroke outcome and elucidate the underlying mechanisms specifically in aging mice.

Conclusion
In summary, we corroborated that TRPC1 knockdown in microglia effectively impedes neutrophil infiltration in stroke-afflicted mice by disrupting the Ca 2+ /NFATc1 pathway, which inhibits the expression of CCL5 and CCL2.TRPC1 plays an important role in orchestrating [Ca 2+ ] i and neutrophil infiltration after ischemic stroke (Fig. 9).Targeting microglia TRPC1 presents a novel and promising therapeutic strategy for neuroinflammation after stroke.

Fig. 2 .
Fig. 2. Generation and characterization of conditional microglia Canonical transient receptor potential channel 1 knockdown (Trpc1 KD) mice.A Construction and breeding procedure of Trpc1 KD mice.Trpc1 KD mice possess both heterozygous Cx3cr1 CreER allele and homozygous loxP-bordered Trpc1 allele.B Microglia were isolated from the cortex of Trpc1 KD mice 24 h after middle cerebral artery occlusion reperfusion (MCAO/R) via magnetic-activated cell sorting method.Knockdown efficiency of TRPC1 was confirmed by Western blot analysis, with microglia isolated from littermate Trpc1 flox/flox /Cre -mice serving as the Control group.Quantitation and statistical analysis of TRPC1 intensity were shown in the right panel, band intensity was normalized to β-actin expression.Data are presented as percentages in comparison to the Control group (n = 3 mice per group).C Body weight of Control and Trpc1 KD mice during 2-12-week development (n = 8 mice per group).D Representative immunofluorescence staining for microglia marker, Ionized calcium-binding adapter molecule 1 (Iba1, Red), astrocytic marker, glial fibrillary acidic protein (GFAP, Green), and neuron marker, neuron-specific nuclear protein (NeuN, Green) in brain section of Trpc1 KD and Control mice.4',6diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue).Scale bar, 100 µm.Quantification of Iba1, GFAP, and NeuN positive cells in Trpc1 KD and Control mice brain was shown in the right panel (n = 4 mice per group).E Quantification of regional cerebral blood flow (rCBF) of Control and Trpc1 KD mice throughout MCAO/R procedure (n = 4 mice per group).* * P < 0.01.n.s.: no significant; KD: Trpc1 KD; CTR: Control.

Fig. 3 .
Fig. 3. Microglia-specific Canonical transient receptor potential channel 1 (Trpc1) knockdown reduces brain injury after middle cerebral artery occlusion reperfusion (MCAO/R).A Modified neurological severity score (mNSS) of indicated groups (n = 8 mice per group).B Foot fault rate of the left forelimb of indicated groups (n = 8 mice per group).C Latency to falls in the rotated cylinder (40 revolutions/min) of indicated groups.D Infarct volumes were determined by triphenyl tetrazolium chloride staining in indicated groups.The red area indicates the non-ischemic tissue, white area indicates the infarct area.Infarct volume percentages were quantified by using Photoshop CC 2019 software in the right panel (n = 6 mice per group).E Impaired blood brain barrier integrity was detected by Evans blue staining in indicated groups.The left panel shows the representative staining of brain slices, blue area indicates the dye leakage tissue.The right panel indicates the quantitative determination of Evans blue content in the hemispheric.Details method will be found in the material and methods section (n = 6 mice per group).* * P < 0.01.KD: microglia-specific Trpc1 knockdown; CTR: Control.

Fig. 5 .
Fig. 5. Neutrophil infiltration after stroke is attenuated with Canonical transient receptor potential channel 1 (Trpc1) knockdown in microglia.A Counts of leukocyte subtypes assayed by flow cytometry in peri-infarct area of microglia-specific Trpc1 knockdown (Trpc1 KD) and Control mice 24 h after middle cerebral artery occlusion reperfusion (MCAO/R) (n = 4 mice per group).B Counts of leukocyte subtypes assayed by flow cytometry in the spleen of Trpc1 KD and Control mice 24 h after MCAO/R (n = 4 mice per group).C Representative images of immunofluorescence staining of neutrophils marker, lymphocyte antigen 6 complex, locus G (Ly6G, red) from peri-infarct area of Trpc1 KD and Control mice after MCAO/R.Nuclei were stained with 4',6-diamidino-2-phenylindole (blue).Scale bar, 20 µm.Quantification of neutrophils in indicated groups was shown in the right panel (n = 4 mice per group).D Counts of neutrophils assayed by flow cytometry in periinfarct area of Trpc1 KD and Control mice 1, 3, 5, and 7 days after MCAO/R (n = 4 mice per group).E Counts of neutrophils assayed by flow cytometry in the spleen of Trpc1 KD and Control mice 1, 3, 5, and 7 days after MCAO/R (n = 4 mice per group).* P < 0.05, ** P < 0.01.n.s.: no significant; KD: Trpc1 KD; CTR: Control.

Fig. 6 .
Fig. 6.Canonical transient receptor potential channel 1 (Trpc1) knockdown in microglia ameliorating stroke outcome via decreasing neutrophil infiltration.Neutrophil in peripheral blood was depleted by intraperitoneal injection of anti-lymphocyte antigen 6 complex, locus G monoclonal antibody in microglia-specific Trpc1 knockdown (Trpc1 KD) and Control mice, middle cerebral artery occlusion reperfusion (MCAO/R) was performed 24 h later.A Modified neurological severity score (mNSS) of indicated groups (n = 8 mice per group).B Foot fault rate of the left forelimb of indicated groups (n = 8 mice per group).C Latency to falls in the rotated cylinder (40 revolutions/min) of indicated groups (n = 8 mice per group).D Infarct volumes were determined by triphenyl tetrazolium chloride staining in indicated groups.The red area indicates the non-ischemic tissue, white area indicates the infarct area.Infarct volume percentages were quantified by using Photoshop CC 2019 software in the right panel (n = 6 mice per group).n.s.: no significant; KD: Trpc1 KD; CTR: Control.

Fig. 7 .
Fig. 7. Canonical transient receptor potential channel 1 (Trpc1) knockdown in microglia reduces neutrophils infiltration via decreasing CC-Chemokines ligand (CCL) 5and CCL2.A Heat map shows the cytokine/chemokine expression profiles of magnetic-activated cell sorted (MACS) microglia from microglia-specific Trpc1 knockdown (Trpc1 KD) and Control mice 24 h after middle cerebral artery occlusion reperfusion (MCAO/R) (n = 6 mice per group).B CCL5 and CCL2 expression levels were detected by enzyme linked immunosorbent assay (ELISA) in MCAS microglia from Trpc1 KD and Control mice 24 h after MCAO/R.C CCL5 and CCL2 expression levels were detected by ELISA in primary cultured microglia from Trpc1 KD and Control mice upon oxygen-glucose deprivation/regeneration (OGD/R) condition (n = 6 per group).D, E Flow cytometry analysis of CCL5-Cy3 (D) and CCL2-Cy3 (E) signal in MACS microglia from Trpc1 KD Control mice 24 h after MCAO/R.Gates were set by using Fluorescence Minus One (FMO) controls.Gray area: FMO control; White area: positive sample.Quantification of flow cytometry was shown in the right panel (n = 4 per group).Two thousand cells were assayed for each experiment.F Illustration of the co-culture and transmigration system in vitro.The former contains primary microglia from Trpc1 KD and Control mice in the lower chamber and neutrophils in the upper chamber.Co-culture media in the microglia chamber are used as conditioned media to stimulate neutrophils migration.G Quantification of neutrophils migrating towards the lower chamber assayed by flow cytometry (n = 4 per group).H Antibodies against CCL5 or/and CCL2 were added to the microglia medium, and the migration rate of neutrophils was measured in the Transwell system by flow cytometry (n = 4 per group).** P < 0.01, n.s.: no significant.KD: Trpc1 KD; CTR: Control.

Fig. 8 .
Fig. 8. Canonical transient receptor potential channel 1 (TRPC1) regulates CC-Chemokines ligand (CCL) 5 and CCL2 via Ca 2+ /calcineurin (CaN)/ nuclear factor of activated T-cells c1 (NFATc1) signaling pathway.A Thapsigargin (Tg) induced store-operated calcium entry (SOCE) was monitored by Fura2 Ca 2+ sensitive fluorescent dye in microglia from microglia specific Trpc1 knockdown (Trpc1 KD) or Control mice underwent Normoxia or oxygen-glucose deprivation and regeneration (OGD/R).Where indicated, 5 μM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 5 μM Tg, and/or 1.8 mM Ca 2+ were added.B Peak Fluorescence 340/380 ratio of indicated groups.C Trpc1 KD or Control microglia were pretreated with SKF96365 to block TRPCs channel mediated Ca 2+ , CCL5 and CCL2 levels were detected by enzyme-linked immunosorbent assay (ELISA) upon OGD/R condition.D CaN activity detected by ELISA in Trpc1 KD or Control microglia underwent OGD/R.E Protein samples were exacted from nuclear of Trpc1 KD or Control microglia upon OGD/R condition, NFATc1 expression was detected by Western blot assay.Each group consisted of 3 bands from one sample.Quantification of band intensity was shown in the right panel, each point represents the average intensity of 3 bands in the left panel.Data are presented as percentages in comparison to the Control group (n = 3).F BV2 cells were transfected with siTrpc1 and Nfatc1 plasmid then underwent OGD/R, CCL5 and CCL2 levels were detected by ELISA in indicated groups.(n = 4 per group).* * P < 0.01.n.s.: no significant; KD: Trpc1 KD; CTR: Control.