Ki20227 aggravates apoptosis, inflammatory response, and oxidative stress after focal cerebral ischemia injury.

The survival of microglia depends on the colony-stimulating factor-1 receptor (CSF1R) signaling pathway under physiological conditions. Ki20227 is a highly selective CSF1R inhibitor that has been shown to change the morphology of microglia. However, the effects of Ki20227 on the progression of ischemic stroke are unclear. In this study, male C57BL/6 mouse models of focal cerebral ischemic injury were established through the occlusion of the middle cerebral artery and then administered 3 mg/g Ki20227 for 3 successive days. The results revealed that the number of ionized calcium-binding adaptor molecule 1/bromodeoxyuridine double positive cells in the infarct tissue was reduced, the degree of edema was increased, neurological deficits were aggravated, infarct volume was increased, and the number of peri-infarct Nissl bodies was reduced. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the peri-infarct tissue was increased. The expression levels of Bax and Cleaved caspase-3 were up-regulated. Bcl-2 expression was downregulated. The expression levels of inflammatory factors and oxidative stress-associated factors were increased. These findings suggested that Ki20227 blocked microglial proliferation and aggravated the pathological progression of ischemia/reperfusion injury in a transient middle cerebral artery occlusion model. This study was approved by the Animal Ethics Committee of Lanzhou University Second Hospital (approval No. D2020-68) on March 6, 2020.

Chinese Library Classification No. R453; R741; R364.5

Introduction

Ischemic stroke is a disease characterized by a high incidence of disability and mortality (Datta et al., 2020). Neurons, glial cells, and peripheral blood vessels are damaged after ischemic stroke due to the disruption of the blood supply, resulting in corresponding neurological impairment, such as hemiplegia, aphasia, and impaired cognitive abilities (Hillis et al., 2002; Jiang et al., 2018). The current treatment for stroke is mainly focused on improving the plasticity of the neural loop (Wang et al., 2019; Al-Kuraishy et al., 2020; Huang et al., 2020). Therapeutic research has focused on the complex relationship between the various cell types involved in tissue repair (Hayakawa et al., 2016; Xiong et al., 2016; Upadhya et al., 2020). Microglia are immunoreactive cells in the brain that react rapidly after stroke (Kanazawa et al., 2017). During ischemic injury, microglia undergo large changes in morphology, density, and function, including cell body enlargement, debranching, cell wall thickening, inflammatory protein production, and various states of proliferation, migration, and phagocytosis (Hou et al., 2016; Qin et al., 2019; Mo et al., 2020). The chronic inflammatory activation of microglia can last for years after ischemia/reperfusion (I/R) injury (Sekeljic et al., 2012; Cui et al., 2020; Radenovic et al., 2020). However, the function of microglia in stroke remains controversial (Ma et al., 2017; Qin et al., 2019). Activated microglia release pro-inflammatory factors that cause neuronal death and contribute to secondary tissue damage (Kanazawa et al., 2017). However, activated microglia also remove damaged neural cells, cellular debris, and dysfunctional synapses and produce anti-inflammatory factors to promote recovery after stroke (Wang et al., 2018). Microglia may, therefore, serve a dual role during the pathological process of ischemic stroke (Xiong et al., 2016; Ma et al., 2017), and the specific functions of microglia remain debatable.

Colony-stimulating factor 1 receptor (CSF1R)—the specific receptor for colony-stimulating factor 1 (CSF1) and interleukin (IL)-34—regulates the proliferation, differentiation, and function of myeloid lineage cells, including microglia, macrophages, and osteoclasts (Hume et al., 2020). Microglia are the predominant CSF1R-expressing cell type in the central nervous system under physiological conditions (Hu et al., 2020). The selective inhibition of CSF1R was shown to change the physiological state of microglia in vivo (Hou et al., 2016). Although CSF1R has been detected in other cell types, such as neurons and astrocytes, the expression levels were minimal, and no significant changes were observed in these cells after CSF1R inhibition (Hou et al., 2016; Fu et al., 2020; Neal et al., 2020). The specific effect of CSF1R inhibition on microglia represents a new strategy for use in microglial research (Huang et al., 2018; Neal et al., 2020). The highly selective CSF1R tyrosine kinase inhibitor Ki20227 has been administered orally to manipulate myeloid cells in different animal models (Ohno et al., 2006; Hou et al., 2016; Xie et al., 2020). Although Ki20227 administration has been reported to result in morphological changes in microglia (Hou et al., 2016), the effect of Ki20227 on I/R injury remains unclear. In this study, we investigated the impacts of Ki20227 in a mouse model of transient middle cerebral artery occlusion (tMCAO).

Materials and Methods

Animals

Male C57BL/6 mice (specific-pathogen-free level, n = 108, aged 10–12 weeks, weighing 23–25 g) were used for all experiments. All animals were purchased from the Animal Experimental Center of Lanzhou University Second Hospital [license No. SYXK (Gan) 2018-0003]. Mice had free access to water and food at all times and were maintained under the conditions of a 12-hour light/dark cycle at 22 ± 2°C. All animal procedures were performed strictly following the guidelines set by Lanzhou University Second Hospital, and the study was approved by the Animal Ethics Committee of Lanzhou University Second Hospital (approval No. D2020-68) on March 6, 2020. The mice were randomly divided into four groups: sham + vehicle (n = 16), sham + Ki20227 (n = 16), tMCAO + vehicle (n = 38), and tMCAO + Ki20227 (n = 38).

tMCAO model

The method used to generate the tMCAO model, in which a focal I/R injury was induced, was described in a previous study (Chiang et al., 2011). The mice were anesthetized with isoflurane (RWD, Shenzhen, China) inhalation and fixed in a supine position on a mouse plate. After iodophor disinfection, the median skin of the neck was cut open to expose the left common carotid artery, internal carotid artery, and external carotid artery. The external carotid artery was cut after coagulation, and the proximal end was placed in a straight line with the internal carotid artery. A thread bolted with a silicone head (Doccol, Sharon, MA, USA) was inserted from the external carotid artery, through the internal carotid artery, to the entrance of the middle cerebral artery. The embolization process lasted for 60 minutes, and then the threaded bolt was removed. To ensure that ischemia was effectively induced in mice, reduced blood flow was confirmed by laser Doppler flowmetry (RWD). The mice were placed on a constant temperature test pad to ensure that the anal temperature was maintained at approximately 37°C. In the sham groups, the left common carotid artery, internal carotid artery, and external carotid artery were exposed for 60 minutes without thread insertion.

Ki20227 treatment

Mice were treated intragastrically with Ki20227 (Med Chem Express, Deer Park, NJ, USA) at a dose of 2 mg/g daily for 3 days starting immediately after tMCAO to inhibit CSF1R in vivo (). The sham + vehicle and tMCAO + vehicle groups received the same volume (0.04 mL/g) of vehicle [0.5% methylcellulose (Sigma, St. Louis, MO, USA), which promotes the dispersion and dissolution of Ki20227, in distilled water]. Bromodeoxyuridine (BrdU; 50 mg/kg, Sigma) was also administered intragastrically to mice twice a day, starting immediately following the surgery. All mice were sacrificed on the 3rd day after I/R, except for those used for neurological functional assessment (n = 8 in each of the tMCAO + vehicle and tMCAO + Ki20227 groups).

Treatment steps before sample harvest.

MCAO: Middle cerebral artery occlusion.

Peri-infarct microglial proliferation assessment

The proliferation of peri-infarct microglia was analyzed by immunofluorescence staining. Mice were perfused via the left heart ventricle with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. After brain tissues were fixed overnight in 4% paraformaldehyde at 4°C, the brain tissues were dehydrated in 30% sucrose/0.01 M PBS for several days, and then the brain tissues were sectioned (30 µm) on a microtome (Leica, Wetzlar, Hessen, Germany). To label microglia, three or four brain slices were selected from each mouse in each treatment group and stained in a 24-well plate. First, the sections were rinsed three times with 0.1 mL PBS for 5 minutes and treated with 0.5% PBS-Triton X-100 for 10 minutes to increase cell membrane permeability. The sections were blocked in 1% bovine serum albumin solution for 60 minutes at 37°C and incubated overnight at 4°C with rabbit anti-ionized calcium-binding adapter protein 1 (Iba1) antibody (1:100, Abcam, Cambridge, MA, USA, Cat# EPR16589). The brain sections were incubated with the corresponding secondary antibody (fluorescein isothiocyanate, goat anti-rabbit, 1:200, ZSGB-BIO, Beijing, China, Cat# ZF-0312) for 1 hour at 37°C. For BrdU immunofluorescence, the brain sections were incubated with 2 M HCl for 30 minutes at 37°C, neutralized by three incubations with 0.1 M borate buffer (pH 8.5) for 15 minutes each, and incubated overnight with rat anti-BrdU monoclonal antibody (1:500; Abcam, Cat# ab6326). The brain sections were incubated with the corresponding secondary antibody (1:200, tetramethylrhodamine, goat anti-rat, ZSGB-BIO, Cat# ZF-0318) for 1 hour at 37°C. The sections were incubated with Hoechst 33258 (1:10,000; Sigma) for 15 minutes to label the nucleus. The brain slices stained with immunofluorescent labels were scanned sequentially using a laser-scanning confocal microscope (Olympus, Tokyo, Japan). Cells co-expressing Iba1 and BrdU were considered newly proliferated microglia.

Infarct volume assessment

The infarct volume was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Briefly, the mouse brain was cut into seven 2-mm-thick coronal slices, incubated with 2% TTC (Sigma) at 37°C for 30 minutes, and fixed in 10% formalin to stop the staining. The infarct area was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The percentage of infarct volume = total infarct volume/volume of the contralateral hemisphere × 100%. The analysis was conducted by researchers blinded to the treatment strategy.

Nissl staining

Sectioned brain tissues (30 µm) were obtained using a vibrating microtome (Leica) after cardiac perfusion, as described above. The brain slices were incubated in 1% methyl violet for 15 minutes, treated with Nissl differentiation solution for 4–8 seconds, and then immersed in an ethanol gradient and xylene. Finally, the sections were observed with an optical microscope (Olympus).

Neurological functional assessment

The modified neurological severity score (Bieber et al., 2019) was determined based on the performance on a series of behavioral tests, including sensory, motor, reflex, and balance disturbance tests, to assess the neurological function of mice 1, 3, 7, and 14 days after tMCAO. The total score was the sum of the test scores, ranging from 0 to 18. A higher score indicates more serious neurological impairment. The assessment was conducted by researchers blinded to the treatment strategy.

Cerebral edema assessment

On the third day after the operation, seven mice in each group were killed to determine brain edema. The cerebral hemispheres on the infarct side were separated and weighed to obtain the wet weight and measured again after 24 hours of air drying to obtain the dry weight. The formula for calculating edema is (wet weight – dry weight)/wet weight × 100%.

TdT-mediated dUTP nick-end labeling apoptosis assessment

TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to determine peri-infarct apoptosis 72 hours after tMCAO. The process was conducted in accordance with the manufacturer’s instructions. The 30-μm-thick coronal brain slices were washed in PBS, incubated in 0.3% PBS-Tween 20, at room temperature for 5 minutes, and then incubated with the TUNEL reaction mixture (Roche, Basel, Switzerland) in the dark at 37°C for 1 hour. To label the nucleus, the sections were incubated with Hoechst 33258 (1:10,000; Sigma) for 15 minutes. The brain slices were scanned sequentially by a laser-scanning confocal microscope (Olympus). Quantification of TUNEL assay was described as the positive cell density in the peri-infarct region, as measured by ImageJ software.

Western blot analysis

The total proteins of ischemic tissues were extracted with radioimmunoprecipitation assay buffer, evenly separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane. After transfer, the membrane was incubated in 5% skim milk at 37°C for 1 hour and then incubated with gentle shaking with primary antibodies at 4°C overnight. Primary antibodies against Cleaved caspase-3 (rabbit, 1:1000, Cat# 9664, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (rabbit, 1:1000, Cat# 12789-1-AP, Proteintech Group, Wuhan, China), Bax (rabbit, 1:1000, Cat# 50599-2-lg, Proteintech Group) and β-actin (mouse, 1:3000, Cat# TA-09, ZSGB-BIO) were used in our study. After incubation with the primary antibody, the membrane was exposed to goat anti-rabbit secondary antibody (1:5000, ZSGB-BIO, Cat# ZB-2301) or goat anti-mouse secondary antibody (1:5000, Cat# ZB-2305, ZSGB-BIO) at 37°C for 1 hour. Enhanced chemiluminescence was used for visualization, and ImageJ software was used to measure the gray value of each protein. Target proteins were normalized against β-actin expression.

Quantitative real-time-polymerase chain reaction

Total RNA from ischemic tissues was extracted by RNA prep pure Tissue Kit (TIANGEN, Beijing, China) and reverse transcribed to complementary DNA using a reverse-transcription kit with gDNA Eraser (TIANGEN). Quantitative polymerase chain reaction was performed using a SYBR Green kit (TIANGEN) in accordance with the manufacturer’s instructions. All mRNA expression levels were normalized against the expression of glyceraldehyde 3-phosphate dehydrogenase as the endogenous control. The fold-change rate was calculated using the comparative threshold cycle (Ct) method. The primer sequences are shown in .

Enzyme-linked immunosorbent assay

Supernatants of homogenates extracted from ischemic tissues were assessed using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Mlbio, Shanghai, China). The expression levels of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) were quantified following the ELISA kit’s protocols.

Statistical analysis

GraphPad Prism 8.3 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Two-tailed unpaired t-test were used to analyze changes in the quantification of Iba1+/BrdU+ cells, infarct volume, brain water content, neurological severity score, TUNEL-positive cells, and apoptotic protein expression. One-way analysis of variance followed by Tukey’s post hoc test was used to analyze changes in Nissl bodies, inflammatory factors, and oxidative stress-associated factors. Data are expressed as the mean ± standard deviation (SD). P < 0.05 indicated a significant difference.

Results

Ki20227 inhibits the proliferation of microglia in the peri-infarct region after tMCAO

Iba1 and BrdU immunofluorescence was performed to evaluate the effects of Ki20227 on the proliferation of microglia. We quantified the Iba1+/BrdU+ cells in the peri-infarct region through immunofluorescence staining and found that a large number of microglia were activated after tMCAO. Furthermore, stroke-induced microglial proliferation was significantly inhibited by Ki20227 treatment. Fewer Iba1+/BrdU+ cells were observed in mice treated with Ki20227 than in mice treated with vehicle after tMCAO (P < 0.05, Figure and ).

Ki20227 blocks microglial proliferation in the peri-infarct region after tMCAO.

(A) Representative confocal images of Iba1-positive (green, stained by fluorescein isothiocyanate) microglia and BrdU-positive (red, stained by tetramethylrhodamine) cells in the peri-infarct region. No difference was observed between the sham + vehicle and sham + Ki20227 groups. Fewer Iba1+/BrdU+ cells were observed in the tMCAO + Ki20227 group than in the tMCAO + vehicle group. Scale bar: 50 µm. (B) Quantification of microglial proliferation (Iba1+/BrdU+ cells) in the peri-infarct region. Data are expressed as the mean ± SD (n = 6). **P < 0.01 (two-tailed unpaired t-test). BrdU: Bromodeoxyuridine; Iba1: ionized calcium-binding adaptor molecule 1; tMCAO: transient middle cerebral artery occlusion.

Ki20227 aggravates tMCAO-induced I/R injury

Assessments of infarct volume, Nissl body density, brain water content, and neurologic deficits tests were performed to evaluate the pathological progression of I/R injury. We applied TTC staining to measure the infarct volume 3 days after tMCAO and found that Ki20227 treatment significantly increased the infarct volume compared with that in the untreated tMCAO group (P < 0.05; Figure and ). No infarct area was observed in the sham groups. Fewer peri-infarct Nissl bodies were observed in Ki20227-treated mice after tMCAO compared with those in the tMCAO + vehicle group (P < 0.05; Figure and ). No influence of Ki20227 on Nissl bodies was observed in the sham group. Worse neurological behavioral performance was detected in the Ki20227-treated stroke model mice than in the vehicle-treated tMCAO mice on days 3, 5, 7, and 14 after reperfusion (). Brain edema increased in mice treated with Ki20227 compared with that in mice in the tMCAO + vehicle group (P < 0.05; ).

Ki20227 aggravates tMCAO-induced infarct, apoptosis, and edema in mice with ischemia/reperfusion injury.

(A) Representative infarct volume images following TTC staining. No infarct was observed in the sham groups. Larger infarct volumes were observed in the tMCAO + Ki20227 group than tMCAO + vehicle group. (B) Quantification of infarct volumes. (C) Representative Nissl staining images in the peri-infarct region. Fewer peri-infarct Nissl bodies (arrows) were observed in the tMCAO + Ki20227 group than in the tMCAO + vehicle group. Scale bar: 100 µm. (D) Quantification of Nissl bodies. (E) The results of neurological behavior tests. (F) Water content. Data are expressed as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. #P < 0.05, ##P < 0.01, vs. tMCAO + Ki20227 group (two-tailed unpaired t-test in B, E, and F; one-way analysis of variance followed by Tukey’s multiple comparisons test in D). tMCAO: Transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyl tetrazolium chloride.

Ki20227 promotes peri-infarct apoptosis after tMCAO

TUNEL staining was performed to evaluate the effects of Ki20227 on peri-infarct apoptosis after tMCAO. We found that the density of TUNEL-positive cells in the tMCAO + Ki20227 group was significantly higher than that in the tMCAO + vehicle group (P < 0.05, Figure and ). Furthermore, the results of western blot analysis showed that the expression levels of pro-apoptotic proteins Bax and Cleaved caspase-3 increased in the tMCAO + vehicle group compared with those in the sham + vehicle group (Figure ). The expression level of the anti-apoptotic factor Bcl-2 was downregulated in the tMCAO groups compared with that in the sham groups. Comparing between the tMCAO + vehicle and tMCAO + Ki20227 groups, the mice treated with Ki20227 showed a lower expression level of Bcl-2 (P < 0.05, ) and higher expression levels of Bax (P < 0.05, ) and Cleaved caspase-3 (P < 0.05, ). No significant change was observed after Ki20227 administration in the sham group.

Ki20227 promotes peri-infarct apoptosis after tMCAO.

(A) TUNEL (red) and Hochest33258 (blue) staining in the peri-infarct region. The density of TUNEL-positive cells (arrows) was higher in the tMCAO + Ki20227 group than in the tMCAO + vehicle group. Scale bar: 50 µm. (B) Quantification of TUNEL-positive cells. (C) Western blot analysis of Bcl-2, Bax, and Cleaved caspase-3. (D–F) Quantitation of Bcl-2, Bax, and Cleaved caspase-3 expression, which was normalized against β-actin expression. Data are expressed as the mean ± SD (n = 4). **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test). tMCAO: Transient middle cerebral artery occlusion; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Ki20227 exacerbates the inflammatory response and oxidative stress in the peri-infarct region after tMCAO

We detected the mRNA expression of CSF1R using quantitative real-time polymerase chain reaction. CSF1R mRNA was expressed at a higher level after tMCAO and decreased significantly after Ki20227 treatment (P < 0.05, ). To explore the role played by the CSF1R signaling pathway in the tMCAO-induced inflammatory response and oxidative stress, we used quantitative real-time polymerase chain reaction to measure the levels of inflammatory factors and ELISA to quantify the expression of oxidative stress-associated proteins. A significant change in inflammatory gene expression levels was observed after tMCAO, showing the activation of the inflammatory response cause by tMCAO. The mRNA expression levels of pro-inflammatory factors [interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS)] were higher in Ki20227-treated mice than in the vehicle-treated group after tMCAO (all P < 0.05, Figure ). In contrast, transforming growth factor (TGF)-β, which is an anti-inflammatory factor (Ma et al., 2017), showed lower expression in the tMCAO + Ki20227 group than in the tMCAO + vehicle group (P < 0.05, ). The increased expression level of MDA [a pro-oxidant factor (Del Rio et al., 2005)] and the reduced expression of antioxidant factors (including SOD and GSH) were detected in the Ki20227-treated group after tMCAO compared with the vehicle-treated group (all P < 0.05, Figure ).

Ki20227 exacerbates the inflammatory response and oxidative stress in the peri-infarct region after tMCAO.

(A–F) Quantitative polymerase chain reaction analysis of the mRNA expression levels of CSF1R, IL-6, IL-1β, TNF-α, iNOS, and TGF-β. (G–I) Enzyme-linked immunosorbent assay of MDA, GSH, and SOD. Data are expressed as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). CSF1R: Colony-stimulating factor-1 receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSH: glutathione; IL-1β: interleukin-1β; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; MDA: malondialdehyde; SOD: superoxide dismutase; TGF-β: transforming growth factor β; tMCAO: transient middle cerebral artery occlusion; TNF-α: tumor necrosis factor-α.

Discussion

Estrogen has been shown to exert a protective effect against stroke (Koellhoffer and McCullough, 2013; Ritzel et al., 2013; Nematipour et al., 2020). Following the same ischemic intervention treatment, the infarct degree in female mice was shown to be smaller than that of male mice (Manwani et al., 2015). To avoid the potentially confounding effects of estrogen on the experimental results, female mice were not used in the course of this experiment. Age is another factor that affects the severity of stroke (Dinapoli et al., 2010; Zhao et al., 2017); therefore, we used mice at the same age. Young adult mice (10–12 weeks old) display strong vitality, which can reduce the death rate associated with the modeling process. Furthermore, mice at this age range had blood vessels that were the optimal size for the 0.21-mm diameter threat plugs used to generate the model. Some studies have shown the beneficial effects of CSF1R signaling on a variety of nervous system diseases (Fu et al., 2020; Hu et al., 2020), whereas other studies have reported the opposite results (Du et al., 2020; Kerkhofs et al., 2020; Neal et al., 2020). To investigate the role played by CSF1R in I/R injury, we used Ki20227 to inhibit the signaling pathway and observed its effects in tMCAO model mice.

The infarct volume is the most direct reflection of injury degree after cerebral I/R injury (Kalogeris et al., 2016). Nissl bodies are large basophilic masses and granules found in neuronal cell bodies or dendrites. When the neuron is damaged, the number of Nissl bodies decrease, whereas Nissl bodies increase during the recovery process; Nissl body can, therefore, be used as a sign of the functional recovery state of neurons (Niu et al., 2015). After I/R, large numbers of necrotic brain cells can result in an increase in vascular wall permeability, resulting in brain edema (Shadman et al., 2019). Neurobehavioral outcomes are macroscopic reflections of the neurological state of mice (Bieber et al., 2019). In our study, we found that the intragastric injection of Ki20227 into I/R mice could increase the infarct volume, reduce the number of Nissl bodies, increase the degree of hemispheric edema, and worsen behavioral outcomes, indicating that the use of Ki20227 increased the degree of I/R injury in mice.

Iba1 is a specific cytoskeleton protein expressed in microglia (Gheorghe et al., 2020). BrdU is a thymidine analog that binds to the DNA of mitotic cells during the S phase of the cell cycle. Therefore, Iba1 has become a widely accepted microglial marker, and BrdU has been used to monitor cell proliferation (Taupin, 2007). In this study, we use the double-immunofluorescence analysis of Iba1 and BrdU to evaluate microglial proliferation in different treatment groups. This analysis was performed 3 days after reperfusion when microglial proliferation and activation peaked (Inamasu et al., 2000). We found that mice treated with Ki20227 had fewer Iba1+/BrdU+ cells in the peri-infarct zone after tMCAO than mice in the vehicle group, suggesting that the inhibition of CSF1R with Ki20227 blocked the proliferation of microglia.

Unlike the ischemic core, where cells die primarily due to necrosis, apoptosis plays a dominant role in the penumbra. The penumbra represents the potentially salvageable transition zone in the peri-infarct region that expands after stroke (Uzdensky, 2019). Bcl-2 is an anti-apoptotic protein that inhibits the release of cytochrome c from the mitochondria by preventing mitochondrial membrane permeabilization in cells. Bax is a protein in the Bcl-2 family that facilitates apoptosis (Cui and Placzek, 2018). Cytochrome c further activates various proteases and nucleases, stimulating the caspase cascade (Siddiqui et al., 2015). More TUNEL-positive apoptotic cells were observed in the peri-infarcted regions in mice treated with Ki20227 than in mice treated with vehicle after I/R injury. Additionally, a reduced level of Bcl-2 and higher levels of pro-apoptotic proteins (Bax and Cleaved caspase-3) were detected in Ki20227-treated ischemic mice compared with those in the vehicle-treated group. Our results indicated that treatment with the CSF1R kinase inhibitor Ki20227 further aggravated apoptosis in the peri-infarcted region.

In this study, we observed the increased release of inflammatory cytokines in response to ischemic injury, suggesting that these molecules play crucial roles in the pathological progression of ischemia. Pro-inflammatory cytokines, such as IL-6 and TNF-α, were further released in mice treated with Ki20227, whereas anti-inflammatory cytokines, such as TGF-β, were suppressed, suggesting the inflammatory effects of CSF1R signaling. Microglia play a controversial role in the inflammatory process after stroke and have been divided into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes according to their effects (Kanazawa et al., 2017; Qin et al., 2019). Our study found changes in inflammatory cytokine expression after Ki20227 treatment; however, whether Ki20227 functions to affect the microglial polarization phenotypes requires additional studies. In addition to changes in inflammatory cytokine expression, our results showed that CSF1R was expressed at a significantly higher level after stroke, which was concurrent with changes in microglial proliferation. The expression of CSF1R decreased significantly after Ki20227 treatment. The mechanism of this phenomenon may be associated with the inhibitory effect of Ki20227, which reduces the further expression of CSF1R, inhibiting the proliferation of microglia (Neal et al., 2020). Additionally, the decrease in the microglial population may also contribute to reduced CSF1R expression.

Oxidative stress damages the integrity of the genome and leads to cell death (Li et al., 2018). In this study, the expression levels of MDA, SOD, and GSH were determined to analyze the degree of oxidative stress. Free radicals act on lipid peroxidation, and the final product of oxidation is MDA, which can cause the cross-linking polymerization of macromolecules. Therefore, the MDA concentration has become an important indicator that reflects the degree of tissue peroxidation (Del Rio et al., 2005). SOD is an important antioxidant enzyme within the biological system. GSH can protect sulfhydryl groups in proteins from oxidation and has been widely accepted as a physiological antioxidant against free radicals (Dinapoli et al., 2010). The production and metabolism of reactive oxygen species are maintained in a dynamic balance under physiological conditions. Our study found that MDA increased sharply, whereas the antioxidant factors (SOD and GSH) decreased significantly, indicating that this balance was disrupted after tMCAO. More importantly, we found that mice treated with Ki20227 expressed higher levels of MDA and lower levels of antioxidant factors, such as SOD and GSH, after tMCAO, indicating that CSF1R inhibition exacerbated oxidative stress.

Our study had several limitations. First, although CSF1R is present primarily in microglia in the central nervous system, CSF1R is also expressed at low levels on neurons and astrocytes. The impacts of Ki20227 on other cell types cannot be excluded, and the experimental results cannot be attributed solely to microglial CSF1R signaling. Second, we analyzed peri-infarct apoptosis in a general manner without identifying the types of cells that are affected, such as neurons, astrocytes, and microglia. All cell types will undergo apoptosis under I/R conditions, but our study did not address the specific outcomes of any specific cell types.

In conclusion, this study found that the inhibition of CSF1R with Ki20227 blocked microglial proliferation and aggravated the pathological progression of ischemia. Enhanced apoptosis, an aggravated inflammatory response, and intense oxidative stress may be associated with the damage and deterioration observed after I/R. Therefore, the CSF1R signaling pathway may play a beneficial role in the pathological process and can potentially be considered as a therapeutic target for ischemic stroke.

Additional files:

: Open peer review reports .

: Original data of the experiment.

Additional Table 1

Primer sequences for quantitative polymerase chain reaction

Gene	Forward (5’-3’)	Reverse (5’-3’)	Product size (bp)
GAPDH	CGT GCC GCC TGG AGA AAC	AGA GTG GGA GTT GCT GTT GAA	205
	CTG	GTC G
CSF1R	GCA GTA CCA CCA TCC ACT	GTG AGA CAC TGT CCT TCA GTG C	178
	TGT A
IL-6	TAG TCC TTC CTA CCC CAA	TTG GTC CTT AGC CAC TCC TTC	158
	TTT CC
IL-1β	GCA ACT GTT CCT GAA CTC	ATC TTT TGG GGC GTC AAC T	173
	AAC T
TNF-α	GAC GTG GAA CTG GCA GAA	ACT GAT GAG AGG GAG GCC AT	234
	GA
iNOS	GCA TCC CTG TGG AGG ACA	GCA TCC CTG TGG AGG ACA ACC	203
	ACC
TGF-β	GGC GAT ACC TCA GCA ACC G	CTA AGG CGA AAG CCC TCA AT	185

CSF1R: Colony-stimulating factor-1 receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL-1β: interleukin-1β; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; TGF-β: transforming growth factor β; TNF-α: tumor necrosis factor-α.