Hypercapnia intensies cerebral hypoxia via increasing cerebral oxygen extraction ratio: implication in neuroinammation in hypoxemic adult rats

Background: Hypoxemia is a typical symptom of acute respiratory distress syndrome (ARDS). Ventilations are always needed for correcting hypoxemia. To avoid pulmonary morbidity, the low tidal volume ventilation is often applied. The ventilation strategy will certainly result in hypercapnia. Our previous study found that hypercapnia increase neuronal death and aggravate the cognitive function of hypoxic adult rats. However, the underlying mechanism has remained unclear. This study aimed to explore whether hypercapnia would induce neuroinammation through increasing cerebral oxygen extraction ratio in adult rats with hypoxemia. Methods: Cerebral oxygen extraction ratio (CERO2), partial pressure of brain tissue oxygen (PbtO2), and reactive oxygen species (ROS) production in brain tissue in a rat model of hypercapnia/hypoxemia were evaluated. Along with this, the oxygen consumption rate (OCR) and ROS production of BV-2 microglial cells were evaluated after 15% CO2/0.2% O2 treatment. The protein expression level of caspase-1 and IL-1β in microglia cells were detected before and after application of a ROS scavenger in vivo and in vitro. Results: PbtO2 level was elevated by hypercapnia in the hypoxemic rats in the rst 1.5 h, but it was signicantly decreased 2 h after ventilation. This was further evident by the increased levels of CERO2 at 3 h after ventilation. Besides, a high concentration of CO2 treatment could increase the levels of OCR in hypoxic BV-2 microglial cells in vitro. Hypercapnia markedly increased the production of ROS and the expression of caspase-1 and IL-1β in hypoxia-activated microglia both in vivo and in vitro. Pharmacological scavenging ROS inhibited the NLRP3 inammasome activation and expression of IL-1β. Conclusion: Hypercapnia-intensied cerebral hypoxia via increasing cerebral oxygen extraction ratio may induce ROS overproduction, activate the NLRP3 inammasome and enhance IL-1β release in hypoxia-activated microglia. IL-1β uorescence Hypoxia IL-1β uorescence is reduced with NAC pretreatment (2 mM) in BV-2 microglial cells. Scale bars: 10 μm. IL-1β, interleukin-1 beta; ns, non-signicant; HC group, high concentration of carbon dioxide group.

injury [18,19]. When the levels of oxygen fall, hypoxia would inhibit the respiratory chain and induce ROS production [20][21][22]. It remains to be ascertained whether hypercapnia would activate the NLRP3 in ammasome through increasing ROS overproduction in hypoxia-activated microglia.
Neurons and neuronal functions are highly sensitive to hypoxia. Under hypoxic conditions, the oxidative metabolism rate of the brain would decrease to prevent the partial pressure of brain tissue oxygen (PbtO 2 ) from dropping to a very low level [23,24]. However, it is still unknown whether hypercapnia would exert any effects on oxidative metabolism of the hypoxic brain.
In the present study, we hypothesized that hypercapnia may induce the NLRP3 in ammasome activation in microglia via intensifying cerebral hypoxia in adult rats with hypoxemia. It was surmised that hypercapnia might exert its effect through increasing CERO 2 , decreasing PbtO 2 , and inducing ROS production, which ultimately would activate the NLRP3 in ammasome in microglia and enhance the release of IL-1β.
The rat model of hypercapnia/hypoxemia All rats were fasted with access to water overnight before the experiments. The rat model of hypercapnia/hypoxemia was established as described in our previous study [9]. Brie y, the rats were anesthetized with pentobarbital sodium followed by mechanical ventilation. The tidal volume (9ml/kg body weight), respiratory rate (45 breaths/min), and inspiratory to expiratory ratio (1:1) were xed. Sham group was exposed to the air. Hypercapnia group was exposed to 5% CO 2 to maintain pH of arterial blood at 7.20 -7.25. Hypoxemia group was exposed to 16% O 2 to maintain partial pressure of artery blood oxygen (PaO 2 ) at 55 -60 mmHg. HH group was exposed to 16% O 2 mixing with 5% CO 2 to maintain PaO 2 at 55 -60 mmHg and pH at 7. 20 -7.25. Rats in the HH + NAC group were treated with an intraperitoneal injection of 150 mg/kg NAC (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215) for 30 min before being exposed to 5% CO 2 and 16% O 2 . The rats, which were used for Western blotting analysis and immuno uorescence staining, were not performed invasive manipulation except ventilation.

Measurement of cerebral oxygen extraction ratio (CERO 2 )
The right femoral artery and the right jugular vein was cannulated upstream. The blood samples were collected at 3 h after ventilation. The hemoglobin concentration (Hb), saturation of artery blood oxygen (SaO 2 ), PaO 2 , saturation of jugular vein blood oxygen (SjVO 2 ), and pressure of jugular vein blood oxygen (PjVO 2 ) were measured using a blood gas/electrolyte analyzer (Model 5700, IL, San Diego, CA, USA). The content of artery blood oxygen (CaO 2 ), content of jugular vein blood oxygen (CjVO 2 ), and CERO 2 were calculated using the following formulas: [Due to technical limitations, please see the supplementary les section to access the formulas.] Measurement of the partial pressure of brain tissue oxygen (PbtO 2 ) The levels of PbtO 2 were measured at 0.5, 1, 1.5, 2, 2.5, and 3 h after ventilation. To evaluate the PbtO 2 , a midline incision over vertex was performed after anesthesia. After this, a hole was drilled caudal to the coronal suture, 4 mm from the midline. The dura was punctured and a microsensor for PbtO 2 was inserted into brain tissue [24]. A monitor (Integra CAMO2, Integra LifeSciences Limited, County Offaly, Ireland) was used to measure the PbtO 2 .

ROS evaluation of brain tissue
The ROS of brain tissue was evaluated using a ROS ELISA kit (Dogesce, Beijing, China; cat. no. DG21175D) at 3 h after ventilation following the manufacturers' instructions. Brie y, samples and standards (50 μl/well) were added to the plate wells coated by antibodies labeled with HRP, which were used to capture ROS. The plate was incubated for 1 h at 37 °C . After washing completely, substrate A (50 μl/well) and substrate B (50 μl/well) were added to incubate the plate in a dark place for 15 min at 37 °C .
Then the stop buffer was added, and the optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The concentrations of ROS in the samples were then determined by comparing the optical density of the samples to the standard curve BV-2 microglial cell cultures and treatment BV-2 microglial cells were purchased form CHI Scienti c (cat. no. 7-1502), and were cultured and treated as described in our previous study [9]. Brie y, the cells were cultured with DMEM high glucose (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA; cat. no. 8117121) supplemented with 10% FBS (Invitrogen Life Technologies Corporation; Carlsbad, CA, USA; cat. no. 42F0374K). The microglial cells were randomly divided into ve groups: Control group, high concentration of carbon dioxide group (abbreviated HC group), Hypoxia group, Hypoxia + HC group, and Hypoxia + HC + NAC group. Control group was exposed to 5% CO 2 + 20% O 2 . HC group was exposed to 15% CO 2 + 20% O 2 to maintain pH of the supernatant at 7.20 -7.25. Hypoxia group was exposed to 5% CO 2 + 0.2% O 2 to maintain partial pressure of oxygen (PO 2 ) of the supernatant at 55 -60 mmHg. Hypoxia + HC group was exposed to 15% CO 2 + 0.2% O 2 to maintain PO 2 at 55 -60 mmHg and pH at 7.20 -7.25. The cells in the Hypoxia + HC + NAC group were treated with 2 mM NAC (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215) for 30 min before being exposed to 15% CO 2 + 0.2% O 2 .
Oxygen consumption rate (OCR) evaluation of BV-2 microglial cells The OCR was evaluated using a cellulate OCR Assay Kit (BestBio, Shanghai, China; cat. no. BB-48211) after treatment with 0.2% O 2 and 15% CO 2 for 0 h, 6 h, 12 h, and 24 h. The intervention time and testing time were chosen, when the levels of OCR peaked (In this study, 12 h was chosen as the intervention time, and 18 min was chosen as the testing time). The OCR was measured following the manufacturers' instructions. Brie y, BV-2 microglial cells were seeded in 96-well plates (5000 cells/well) and cultured in DMEM high glucose supplemented with 10% FBS. When the cells spread to 80% of the bottom of the well, they were treated with different concentrations of CO 2 and O 2 . After the treatment, the medium was changed to DMEM high glucose without FBS, and then uorescent probes (10 μl/well) were added sequentially. Finally, oxygen mounting medium was added (2 drops/well). The OCR levels were examined every three minutes until half an hour with a uorescent microplate reader (Model 9260, IL-COR ® inc, LINCOLN, NE, USA). The excited and emitted wavelength were 485/20 nm and 590/35 nm, respectively.

ROS measurement in microglia
The ROS production in BV-2 microglial cells was evaluated using a ROS assay kit (BestBio, Shanghai, China; cat. no. BB-4705-2) following the manufacturers' instructions. Briefly, DCFH-DA was diluted with DMEM high glucose without FBS (1: 1500). The coverslips with adherent BV-2 microglial cells were cultured with DMEM high glucose supplemented with 10% FBS. After the treatment, the medium was changed to diluted DCFH-DA (2 ml/well). Then the plates were incubated for 20 min at 37 ℃, 5% CO 2 .
The coverslips were washed with DMEM high glucose without FBS. Finally, the coverslips were mounted by a uorescent mounting medium and detected using a uorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan).

Western blotting analysis.
Total proteins from the hippocampus tissue and BV-2 microglial cells (n = 4 for each group) were extracted using a Total Protein Extraction Kit (BestBio, Shanghai, China; cat. no. BB-3101-100T). Protein concentrations were determined using a BCA Protein Assay Kit (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA; cat. no. 23227). Equal amounts of protein from each sample were separated in a 15% SDS-PAGE gel and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature. After this, the following primary antibodies were added to incubate the membranes overnight at 4 °C : caspase-1 (1: 1000, Abcam, Cambridge, MA, USA; cat. no. ab1872) and IL-1β (1: 1000, Abcam, Cambridge, MA, USA; Cat. No. ab9722). The membranes were washed on the following day, and HRP-labeled goat anti-rabbit antibody (1: 3000; Cell Signaling Technology; cat. no. 7074S) was added to incubate the membranes for 2 h at 4 °C . The immunoblots were visualized using a chemiluminescence kit (Bioworld Technology, St. Louis Park, MN, USA; cat. no. AC36131), and detected by an imaging densitometer (ImageQuant LAS 500, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The relative density was quanti ed using FluorChem 8900 software (version 4.0.1, Alpha Innotech Corporation, San Leandro CA, USA). β-actin was used as the control.

Double immunofluorescence labeling
In vivo, the rats were anesthetized with pentobarbital sodium and transcardially perfused with normal saline and 4% paraformaldehyde at 3 h after ventilation. The brains were harvested and post-xed in 4% paraformaldehyde. This tissue was then dehydrated in graded sucrose and cut into sections of 10 μm thickness. The sections were blocked in 5% normal donkey serum for 0.5 h at room temperature. In vitro, the coverslips with adherent BV-2 microglial cells were xed with 4% paraformaldehyde at 24 h after treatment. The coverslips were blocked in 5% normal donkey serum for 0.5 h at room temperature sequentially.
After that, the following primary antibodies were added to incubate the sections/coverslips overnight at 4°C no. ATRMR2301) were added to incubate the sections/coverslips for 1 h at room temperature. Finally, the sections/coverslips were mounted by the uorescent mounting medium with DAPI (Sigma, St. Louis, MO, USA; cat. no. SLBW4468) and detected using a uorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan).

Statistical analysis
The statistical analysis was performed by the SPSS19.0 statistical (IBM, New York, USA). All values are expressed as mean ± standard deviation. Repeated measures one-way analysis of variance (ANOVA) was used to analyze the repeated measurement data. Factorial ANOVA was for the interaction effects. When an interaction was examined, simple effects analyses were evaluated. Differences were considered statistically signi cant if the P value < 0.05.

Hypercapnia increased cerebral oxygen extraction rate (CERO 2 ) in hypoxemic rats
Signi cant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < 0.01) (Fig. 1a). Simple effects analyses found an increased CERO 2 in Hypoxemia group (P < 0.05), but not in Hypercapnia group (P > 0.05) compared with Sham group. HH group had the highest CERO 2 levels as compared with Hypoxemia group (P < 0.01) and Hypercapnia group (P < 0.01) (Fig. 1b).

Hypercapnia induced overproduction of ROS in hypoxemic hippocampus
Signi cant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < 0.05) (Fig. 3a). Simple effects analyses found increased levels of ROS in Hypoxemia group (P < 0.01), but not in Hypercapnia group (P > 0.05) compared with Sham group. HH group had the highest ROS levels as compared with Hypoxemia group (P < 0.01) and Hypercapnia group (P < 0.01) (Fig. 3b).
Hypercapnia enhanced NLRP3 in ammasome activation via inducing ROS overproduction in microglia in the hypoxic hippocampus Signi cant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < 0.01) (Fig. 4b). Simple effects analyses found increased protein expression levels of caspase-1 in Hypoxemia group (P < 0.01), but not in Hypercapnia group (P > 0.05) compared with Sham group. HH group had the highest expression levels of caspase-1 as compared with Hypoxemia group (P < 0.01) and Hypercapnia group (P < 0.01) (Fig. 4c). Additionally, the protein expression of caspase-1 was signi cantly suppressed with NAC pretreatment (150 mg/kg) in rats (P < 0.01) (Fig. 4c). Double immuno uorescence was used to examine caspase-1 expression in microglia of hippocampus (Fig. 4d).
Enhanced caspase-1 immuno uorescence was observed in Hypoxemia group, but not in Hypercapnia group compared with Sham group. HH group had the most intense caspase-1 uorescence when compared with Hypoxemia group and Hypercapnia group. In rats given NAC treatment, caspase-1 uorescence was noticeably attenuated (Fig. 4d).

Hypercapnia increased IL-1β expression via inducing ROS overproduction in microglia in the hypoxic hippocampus
Signi cant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < 0.01) (Fig. 5b). Simple effects analyses found increased protein expression levels of IL-1β in Hypoxemia group (P < 0.01), but not in Hypercapnia group (P > 0.05) compared with Sham group. HH group showed the highest expression levels of IL-1β in comparison with Hypoxemia group (P < 0.01) and Hypercapnia group (P < 0.01) (Fig. 5c). Additionally, the protein expression of IL-1β was signi cantly suppressed with NAC pretreatment (150 mg/kg) in rats (P < 0.01) (Fig. 5c). Double immuno uorescence was used to examine IL-1β expression in microglia of hippocampus (Fig. 5d). Enhanced IL-1β immuno uorescence was observed in Hypoxemia group, but not in Hypercapnia group compared with Sham group. HH group had the strongest IL-1β uorescence as compared with Hypoxemia group and Hypercapnia group. IL-1β uorescence was evidently reduced in rats given NAC pretreatment (Fig. 5d).
15% CO 2 increased the oxygen consumption rate (OCR) in hypoxic BV-2 microglial cells The OCR levels of BV-2 microglial cells were examined after the treatment with 0.2% O 2 and 15% CO 2 for 0 h, 6 h, 12 h, and 24 h. The OCR levels were the highest in 12 h group as compared with other groups (12 h vs 0 h: P < 0.01; 12 h vs 6 h: P > 0.05; 12 h vs 24 h: P < 0.01). Besides, the OCR levels were increased time-dependently and achieved a stationary phase at 18 min (Fig. 6a). In view of this, microglia was treated for 12 hours, and the OCR was examined at 18 min after the treatment (Fig. 6b, c). Signi cant interaction effects were observed between 0.2% O 2 treatment and 15% CO 2 treatment (P < 0.01) (Fig. 6b).
Simple effects analyses found increased levels of OCR in Hypoxia group (P < 0.01), but not in HC group (P > 0.05) compared with Control group. Hypoxia + HC group had the highest levels of OCR as compared with Hypoxia group (P < 0.01) and HC group (P < 0.01) (Fig. 6c).
15% CO 2 induced overproduction of ROS in hypoxic BV-2 microglial cells Enhanced ROS immuno uorescence was observed in Hypoxia group, but not in HC group compared with Control group. Hypoxia + HC group had the strongest ROS uorescence as compared with Hypoxia group and HC group. ROS uorescence was obviously reduced with NAC pretreatment (2 mM) in BV-2 microglial cells (Fig. 7).
Enhanced caspase-1 immuno uorescence was observed in Hypoxia group, but not in HC group compared with Control group. Hypoxia + HC group had the strongest caspase-1 uorescence as compared with Hypoxia group and HC group. Of note, caspase-1 uorescence was evidently reduced with NAC pretreatment (2 mM) in BV-2 microglial cells (Fig. 8d).

15% CO 2 increased IL-1β expression via inducing ROS overproduction in hypoxic BV-2 microglial cells
Signi cant interaction effects were observed between 0.2% O 2 treatment and 15% CO 2 treatment (P < 0.01) (Fig. 9b). Simple effects analyses found increased protein expression levels of IL-1β in Hypoxia group (P < 0.01), but not in HC group (P > 0.05) compared with Control group. Hypoxia + HC group had the highest expression levels of IL-1β as compared with Hypoxia group (P < 0.01) and HC group (P < 0.01) (Fig. 9c). Additionally, the protein expression of IL-1β was signi cantly suppressed with NAC pretreatment (2 mM) in BV-2 microglial cells (P < 0.01) (Fig. 9c). Double immuno uorescence con rmed IL-1β expression in BV-2 microglial cells (Fig. 9d). Enhanced IL-1β immuno uorescence was observed in Hypoxia group, but not in HC group compared with Control group. Hypoxia + HC group had the strongest IL-1β uorescence as compared with Hypoxia group and HC group. IL-1β uorescence was markedly suppressed with NAC pretreatment (2 mM) in BV-2 microglial cells (Fig. 9d).

Discussion
The present results have shown that hypercapnia enhanced the NLRP3 in ammasome activation via inducing ROS overproduction in hypoxia-activated microglia in vitro and in vivo. This was evident by the increased CERO 2 , decreased PbtO 2 , and increased expression levels of caspase-1 and IL-1β in hypoxiaactivated microglia cells.
The present rat and cell models of hypercapnia/hypoxemia were established as described in our previous study. In the models, PO 2 levels of the arterial blood and culture supernatant were maintained at 55-60 mmHg and pH levels of the arterial blood and culture supernatant at 7.20-7.25 [9]. These are consistent with the change of hypoxemia and permissive hypercapnia in ARDS [25][26][27][28].
We reporeted in our previous study that hypercapnia-induced NLRP3 in ammasome activation in hypoxia-activated microglia could upregulate the expression of IL-1β. It is well documented that IL-1β promotes a cascade of in ammatory response in the central nervous system that can lead to the aggravation of neuronal injury [9]. Under hypoxic conditions, ROS is known to be critical for NLRP3 in ammasome activation [29][30][31]. Thus, the expression levels of caspase-1 and IL-1β in the microglia in this study were determined, and the production of ROS in the hippocampus was also evaluated. Signi cant interaction effects were observed on ROS production, caspase-1 and IL-1β expression between hypercapnia treatment and hypoxia treatment. More importantly, we have shown that hypercapnia upregulated the expression of caspase-1 and IL-1β in hypoxic hippocampus microglia via inducing ROS overproduction. Remarkably, caspase-1 and IL-1β expression in hypoxic hippocampus microglia was downregulated when ROS was scavenged by NAC. These results indicate that hypercapnia enhanced the NLRP3 in ammasome activation via inducing ROS overproduction.
To determine the effect of hypercapnia on oxidative metabolism of the rat brain, cerebral oxygen extraction ratio (CERO 2 ) and partial pressure of brain tissue oxygen (PbtO 2 ) were examined. Hypoxemia, as a typical symptom of ARDS, decreases oxidative metabolism rate of the brain to avoid or reduce cerebral damage via preventing the PbtO 2 from dropping to a very low level [23]. In this study, we showed hypercapnia alone was not enough to increase or decrease the levels of PbtO 2 and CERO 2 . In the rst 1.5 hours, the levels of PbtO 2 were elevated by hypercapnia in the hypoxemic rats. This may be the reason why hypercapnia was neuroprotective in rats with transient global cerebral ischemia-reperfusion injury [32] and lateral uid percussion injury [33]. However, hypercapnia signi cantly decreased the levels of PbtO 2 in the hypoxemic rats after 2 hours' ventilation. It was further evidenced by the increased levels of CERO 2 at 3 h after ventilation. These results suggest that hypercapnia is neuroprotective within a very short period of time (1.5 h), but hypercapnia could aggravate neuronal functions injury over a protracted period via increasing CERO 2 and decreasing PbtO 2 .
In vitro, to determine the effect of a high concentration of CO 2 on oxidative metabolism in hypoxic BV-2 microglial cells, the oxygen consumption rate (OCR) was evaluated. There was an interaction effect on the levels of OCR between 0.2% O 2 treatment and 15% CO 2 treatment. The high concentration of CO 2 treatment could increase the levels of OCR in hypoxic BV-2 microglial cells, which were consistent with that hypercapnia increased levels of CERO 2 in hypoxic rats. The results suggest that hypercapnia could intensify microglial hypoxia both in vivo and in vitro.
To ascertain if a high concentration of CO 2 would activate NLRP3 in ammasome via inducing ROS overproduction in hypoxia-activated BV-2 microglial cells, the production of ROS and expression of caspase-1 and IL-1β was determined. The results were consistent with in vivo experiments. Signi cant interaction effects were observed on ROS production, caspase-1 and IL-1β expression between 0.2% O 2 treatment and 15% CO 2 treatment. The high concentration of CO 2 upregulated the expression of caspase-1 and IL-1β in hypoxia-activated microglia via inducing ROS overproduction, as evidenced by the downregulated expression of caspase-1 and IL-1β when ROS was scavenged. All in all, the results support that a high concentration of CO 2 enhanced the NLRP3 in ammasome activation in hypoxiaactivated BV-2 microglial cells via inducing and augmenting ROS overproduction.
In summary, the present results have demonstrated the underlying mechanism whereby hypercapnia can enhance the activation of NLRP3 in ammasome in hypoxic microglia. In this connection, hypercapnia was found to intensify the cerebral hypoxia via increasing CERO 2 and decreasing PbtO 2 . As a consequence, ROS was overproduced in hypoxic microglial cells coupled with activation of NLRP3 in ammasome. Remarkedly, caspase-1 and IL-1β expression was doenregulated when ROS was scavenged. Thus, hypercapnia-induced ROS overproduction and NLRP3 in ammasome activation in microglia may be a potential target to mitigate neuronal damage in the central nervous system such as that in the hippocampus.

Conclusions
Hypercapnia-induced ROS overproduction via intensifying cerebral hypoxia may activate the NLRP3 in ammasome and enhance IL-1β release in hypoxia-activated microglia.

Consent for publication
Not applicable Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Authors' contributions DHG participated in making the animal model, assessed IL-1β and caspase-1 expression in the hypoxic hippocampus microglia, collected data, and drafted the manuscript. LXS carried out the measurement of cerebral oxygen extraction ratio and partial pressure of brain tissue oxygen. LXQ conducted BV-2 microglial cells culture and treatment. WKR participated in evaluating ROS production of brain tissue and BV-2 microglial cells. LY participated in the evaluation of oxygen consumption rate evaluation in vitro. WMY performed statistical analysis. ZHK carried out the design of the study. All authors read and approved the nal manuscript. Figure 1 Hypercapnia increased CERO2 in hypoxemic rats (n = 4). a There is an interaction effect between hypoxia treatment and hypercapnia treatment (P < 0.01). b Simple effects analyses show an increased CERO2 in Hypoxemia group (* P < 0.05), but not in Hypercapnia group (ns P > 0.05) compared with Sham group.

Figures
HH group has the highest CERO2 levels as compared with Hypoxemia group (** P < 0.01) and Hypercapnia group (** P < 0.01). ns, non-signi cant; Sham group, sham-operated group; HH group, hypercapnia + hypoxemia group; CERO2, cerebral oxygen extraction rate. The concentrations of O2 and CO2 in the air are 21% and 0.03%.  Hypercapnia induced overproduction of ROS in hypoxemic hippocampus (n = 4). a There is an interaction effect between hypoxia treatment and hypercapnia treatment (P < 0.05). b Simple effects analyses show increased levels of ROS in Hypoxemia group (** P < 0.01), but not in Hypercapnia group (ns P > 0.05) compared with Sham group. HH group has the highest ROS levels as compared with Hypoxemia group (** P < 0.01) and Hypercapnia group (** P < 0.01). ns, non-signi cant; Sham group, sham-operated group; HH group, hypercapnia + hypoxemia group; ROS, reactive oxygen species. The concentrations of O2 and CO2 in the air are 21% and 0.03%.  Hypercapnia increased IL-1β expression via inducing ROS overproduction in hypoxic microglia (n = 4). a Immunoreactive bands of IL-1β (17 kDa) and β-actin (42 kDa). b There is an interaction effect between hypoxia treatment and hypercapnia treatment (P < 0.01). c Simple effects analyses show increased protein expression levels of IL-1β in Hypoxemia group (** P < 0.01), but not in Hypercapnia group (ns P > 0.05) compared with Sham group. HH group has the highest expression levels of IL-1β as compared with Hypoxemia group (** P < 0.01) and Hypercapnia group (** P < 0.01). The protein expression of IL-1β is signi cantly suppressed with NAC pretreatment (150 mg/kg) in rats (** P < 0.01   . Enhanced caspase-1 immuno uorescence is evident in Hypoxia group, but not in HC group compared with Control group. Hypoxia + HC group shows the strongest caspase-1 uorescence as compared with Hypoxia group and HC group. Caspase-1 uorescence is drastically reduced with NAC pretreatment (2 mM) in BV-2 microglial cells. Scale bars: 10 μm. ns, non-signi cant; HC group, high concentration of carbon dioxide group.