Inhaled Carbon Dioxide Improves Neurological Outcomes by Downregulating Hippocampal Autophagy and Apoptosis in an Asphyxia‐Induced Cardiac Arrest and Resuscitation Rat Model
Journal of the American Heart Association
Abstract
Background
Protracted cerebral hypoperfusion following cardiac arrest (CA) may cause poor neurological recovery. We hypothesized that inhaled carbon dioxide (CO2) could augment cerebral blood flow (CBF) and improve post‐CA neurological outcomes.
Methods and Results
After 6‐minute asphyxia‐induced CA and resuscitation, Wistar rats were randomly allocated to 4 groups (n=25/group) and administered with different inhaled CO2 concentrations, including control (0% CO2), 4% CO2, 8% CO2, and 12% CO2. Invasive monitoring was maintained for 120 minutes, and neurological outcomes were evaluated with neurological function score at 24 hours post‐CA. After the 120‐minute experiment, CBF was 242.3% (median; interquartile range, 221.1%–267.4%) of baseline in the 12% CO2 group while CBF fell to 45.8% (interquartile range, 41.2%–58.1%) of baseline in the control group (P<0.001). CBF increased along with increasing inhaled CO2 concentrations with significant linear trends (P<0.001). At 24 hours post‐CA, compared with the control group (neurological function score, 9 [interquartile range, 8–9]), neurological recovery was significantly better in the 12% CO2 group (neurological function score, 10 [interquartile range, 9.8–10]) (P<0.001) while no survival difference was observed. Brain tissue malondialdehyde (P=0.02) and serum neuron‐specific enolase (P=0.002) and S100β levels (P=0.002) were significantly lower in the 12% CO2 group. TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling)‐positive cell densities in hippocampal CA1 (P<0.001) and CA3 (P<0.001) regions were also significantly reduced in the 12% CO2 group. Western blotting showed that beclin‐1 (P=0.02), p62 (P=0.02), and LAMP2 (lysosome‐associated membrane protein 2) (P=0.01) expression levels, and the LC3B‐II:LC3B‐I ratio (P=0.02) were significantly lower in the 12% CO2 group.
Conclusions
Administering inhaled CO2 augmented post‐CA CBF, mitigated oxidative brain injuries, ameliorated neuronal injury, and downregulated apoptosis and autophagy, thereby improving neurological outcomes.
Nonstandard Abbreviations and Acronyms
- CA
- cardiac arrest
- CBF
- cerebral blood flow
- EtCO2
- end‐tidal CO2
- iCO2
- inhaled CO2
- NFS
- neurological function scores
- NSE
- neuron‐specific enolase
- PaCO2
- pressure of arterial CO2
- PbtO2
- partial pressure of brain tissue oxygenation
- ROS
- reactive oxygen species
- ROSC
- return of spontaneous circulation
Clinical Perspective
What Is New?
•
Inhaled CO2 could improve post‐cardiac arrest neurological outcomes by augmenting cerebral blood flow.
•
The mechanisms underlying the improvement of neurological outcomes by inhaled CO2 involve both apoptosis and autophagy pathways.
What Are the Clinical Implications?
•
Cerebral blood flow is a crucial determinant in post‐cardiac arrest neurological recovery.
•
Instead of adjusting ventilator settings to achieve desired pressure of arterial CO2 level, inhaled CO2 might be more applicable for patients with post‐cardiac arrest to facilitate neurological recovery.
Globally, out‐of‐hospital cardiac arrest (CA) affects an estimated 44 individuals per 100 000 on an annual basis.1 The prognosis following this condition is dismal, with only ≈45% of patients2 recovering neurological function after return of spontaneous circulation (ROSC).
Brain injury is a major component of post‐CA syndrome,3 contributing to 65.2% of deaths during post‐CA intensive care.4 Post‐CA brain injury5 is caused by a primary injury (ie, the immediate cessation of cerebral oxygen delivery during CA), and secondary injuries after ROSC, including protracted cerebral hypoperfusion (ie, the so‐called “no‐reflow” phenomenon).6, 7, 8
Cerebral oxygen delivery is determined by cerebral blood flow (CBF) and blood oxygen levels. A major determinant of CBF is partial pressure of arterial CO2 (PaCO2). Mild hypercapnia (end‐tidal CO2 [EtCO2]: 45–50 mm Hg) was reported to be associated with less neuronal degeneration in a porcine post‐CA model.9 The administration of inhaled CO2 (iCO2) showed neuroprotective effects in cerebral ischemia‐reperfusion injury rat models.10, 11
In this study, we hypothesized that iCO2 may facilitate neurological recovery by augmenting CBF in rats resuscitated from asphyxia‐induced CA. We first monitored changes in CBF corresponding to different iCO2 concentrations. Then, we compared clinical outcomes between different iCO2 concentration groups. Finally, we examined the potential mechanisms underlying the neuroprotective effects of iCO2, including mitochondrial biomarkers alterations, apoptosis, and autophagy mechanisms.12
METHODS
This research was approved by the Institutional Animal Care and Use Committee of College of Medicine, National Taiwan University (approval number 20180314), and was conducted according to Animal Research Reporting of In Vivo Experiments guidelines.13
Data Transparency and Openness
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Animal Preparation
Animal studies were performed using an established asphyxia‐induced CA and cardiopulmonary resuscitation (CPR) rat model.8, 14 Briefly, 14‐week‐old male Wistar rats were anesthetized using intraperitoneal pentobarbital (45 mg/kg). Then, tracheal intubation with mechanical ventilation was initiated at a tidal volume of 0.6 mL/100 g body weight, a frequency of 100 breaths/min, and an inspired O2 fraction of 21%. CBF and partial pressure of brain tissue oxygenation (PbtO2) levels were continuously monitored using OxyFlo Pro and OxyLite Pro (Oxford‐Optronix, Oxford, UK) instruments, respectively, via a fiber sensor (NX‐BF/OFT/E, Oxford‐Optronix) inserted through a small cranial window over the left hemisphere. The insertion site was marked on the dura with the stereotaxic coordinate anteroposterior (−3.5 mm) and lateral (2.0 mm) from the bregma. The fiber sensor was inserted 2 mm from the dura into the dorsal hippocampal CA1 region.15 The right femoral artery was cannulated to monitor arterial blood pressure and the left jugular vein cannulated to collect blood samples and administer medication. Rectal temperature was maintained at 36.5 °C to 37.5 °C.
Asphyxia‐Induced CA and CPR
CA was induced 5 minutes after surgical preparation (Figure 1) by clamping the endotracheal tube. CA was defined as mean arterial pressure (MAP) ≤20 mm Hg. CPR was started at 6 minutes after asphyxia, with 1 intravenous epinephrine (0.002 mg/100 g) dose, 1 sodium bicarbonate (1 mEq/kg) dose, and manual chest compressions (200/min). During CPR, ventilatory inspired O2 fraction was increased to 100%. The ROSC was defined as a MAP ≥40 mm Hg for 10 minutes. Rats were excluded if ROSC failed to occur within 10 minutes of CPR.

Figure 1. Study procedures and measurements at baseline, asphyxia, cardiac arrest and cardiopulmonary resuscitation, and reperfusion.
ABG indicates arterial blood gas; CA, cardiac arrest; CBF, cerebral blood flow; EtCO2, partial pressure of end‐tidal carbon dioxide; HR, heart rate; MAP, mean arterial pressure; and ROSC, return of spontaneous circulation.
Rats were randomly allocated to 4 study groups 15 minutes after ROSC. Randomization was performed using a computer‐generated random number list, with a block size of 4. Depending on group assignment, animals were ventilated at different iCO2 concentrations for 105 minutes: 50% O2+50% N2 (control group), 50% O2+4% CO2+46% N2 (4% CO2 group), 50% O2+8% CO2+42% N2 (8% CO2 group), and 50% O2+12% CO2+38% N2 (12% CO2 group). EtCO2 levels were continuously monitored (PhysioSuite, Kent Scientific Corporation, Torrington, CT) after ROSC. Point‐of‐care testing (Epoc Blood Analysis System, Siemens Healthineers, Erlangen, Germany), including arterial blood analyses and concentrations of sodium, potassium, calcium, and creatinine, was conducted 60 minutes post‐ROSC. If necessary, intraperitoneal pentobarbital would be used for sedation after ROSC.
Outcome Measures of Survival and Neurological Recovery
After the 120‐minute experiment, rats were returned to cages. At 24 hours post‐ROSC, rat survival status was recorded; neurological outcomes were assessed using rat neurological function scores (NFS) (Table S1)16 by researchers masked to treatment allocations. Rats were then humanely euthanized using intraperitoneal pentobarbital (45 mg/kg). The brain tissues of 24‐hour survival animals were harvested for Western blotting (WB), thiobarbituric acid reactive substance assay, and TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling) assay. Right atrium blood samples were collected for ELISA and measuring concentrations of aspartate aminotransferase, alanine aminotransferase, and creatinine. Right hemispheres were washed in physiological saline, immediately frozen in liquid nitrogen, and stored at −80 °C. Left hemispheres were immediately fixed in 4% formaldehyde in 0.1 M phosphate buffer.
Thiobarbituric Acid Reactive Substance Assay
The right hemispheres were homogenized in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors (#78441, Thermo Fisher Scientific, Waltham, MA). After centrifugation, the resulting supernatant was used for thiobarbituric acid reactive substance assay kit (#10009055, Cayman Chemical, Ann Arbor, MI) per manufacturer's instructions to measure concentrations of malondialdehyde, a product of lipid peroxidation caused by reactive oxygen species (ROS). Absorbance was measured on a microplate (SpectraMax Plus 384 Microplate reader, Molecular Devices, San Jose, CA).
ELISA Kits
ELISA kits of neuron‐specific enolase (NSE) (#E‐EL‐R0058, Elabscience Biotechnology, Houston, TX), S100β (#E‐EL‐R0868, Elabscience Biotechnology), and cardiac troponin I (#ab246529, Abcam, Cambridge, UK) were used to determine the injury biomarkers of brain and heart according to the manufacturer's instructions. Absorbance was measured on a microplate (SpectraMax Plus 384 Microplate reader, Molecular Devices, San Jose, CA).
TUNEL Assay
Left hemispheres were assayed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) according to manufacturer's instructions. TUNEL assays were performed on 4‐μm paraffin‐embedded sections. We used 4’,6‐diamidino‐2‐phenylindole to stain nuclei. Apoptotic cells were examined by confocal fluorescence microscopy (EVOS FL Auto Imaging System, Thermo Fisher Scientific, Waltham, MA). For each sample, viewing fields were randomly selected in hippocampal CA1 and CA3 regions for quantitative comparisons.17 Fields were photographed under microscopy at 510/542‐nm (green TUNEL) and 447/460‐nm wavelengths (bluish‐violet 4’,6‐diamidino‐2‐phenylindole). PhotoImpact X3 (Corel, Ottawa, Canada) was used to merge images for final counting analyses in ImageJ software (Version 1.52a, National Institutes of Health, Bethesda, MD). TUNEL‐positive cell densities in the aforementioned regions were measured at 50×magnification, with the area of each microscopic field ≈0.48 mm2 (apoptotic cells/mm2).
Western Blotting
Right hemispheres were used for Western blotting. The primary antibodies, including Beclin‐1 (#3738, Cell Signaling Technology, Danvers, MA), LC3B (#83506, Cell Signaling Technology), p62 (#39749, Cell Signaling Technology), LAMP2 (lysosome‐associated membrane protein 2; #PA1‐655, Invitrogen, Waltham, MA), caspase‐3 (#14220, Cell Signaling Technology), PARP (#9542, Cell Signaling Technology), Bcl‐2 (#GTX100064, GeneTex, Irvine, CA), Bax (#GTX109683, GeneTex), and cytochrome c (#GTX108585, GeneTex) were used. Beta‐actin (HRP‐60008, Proteintech Group, Rosemont, IL) was used as a loading control.
Statistical Analysis
We a priori planned to compare the primary outcome, NFS, between the 12% CO2 group and the control group to observe the effects of iCO2. Both 4% CO2 and 8% CO2 concentrations were used for exploratory analyses to investigate if iCO2 effects could be achieved by lower iCO2 concentrations and to also check for trends among study groups. Accordingly, 17 animals/group were required to demonstrate a mean difference of 1.5 in NFS, with a power of 80% at the 5% level, and an SD of 1.5 (MedCalc, version 20.011, MedCalc Software, Ostend, Belgium). From our previous studies,8, 14 the 24‐hour survival rate of post‐ROSC rats in the control group was ≈70%; therefore, the sample size for each group was calculated at 25 to compensate for losses.
CBF was expressed as the proportion of the baseline (%baseline) and delta of PbtO2 was computed by PbtO2 at a certain time point minus the baseline.18 Categorical data were expressed as counts and proportions and compared using a Chi‐squared test. Continuous data were expressed as median and interquartile range and compared using the Kruskal–Wallis test. A post hoc Dunn test was used for pairwise comparisons between each experimental group and the control group. Trend analysis was performed using Jonckheere‐Terpstra test or Chi‐squared test for trend. Time‐based measurements were compared using 2‐way repeated measurement ANOVA. Survival curves were determined with the Kaplan–Meier method and compared with the log‐rank test. Between‐group comparisons were made with all available data. For the NFS, a sensitivity analysis was performed to test whether the missing NFS in rats with early death would influence the results, including (1) best scenario: in each experimental group, the rats with early death would be assigned with the median NFS of the experimental rats surviving 24 hours while in the control group, the rats with early death would be assigned with an NFS of zero point; and (2) worst scenario: in the control group, the rats with early death would be assigned with the median NFS of the control rats surviving 24 hours while in the experimental groups, the rats with early death would be assigned with an NFS of zero point.
A 2‐tailed P<0.05 was considered statistically significant. All statistical tests were performed in GraphPad Prism Version 9.2.0 (GraphPad Software, La Jolla, CA) and IBM SPSS Statistics for Windows, version 28.0.1.1 (IBM Corp., Armonk, NY).
RESULTS
Baseline Characteristics and Resuscitation Variables
In total, 120 rats were used for experiments and 100 rats randomized to study groups (Figure S1). No significant differences were observed in baseline characteristics and resuscitation variables among groups (Table 1).
Variables | Control group (n=25) | 4% CO2 group (n=25) | 8% CO2 group (n=25) | 12% CO2 group (n=25) | P value (Kruskal–Wallis test) | P value (Jonckheere‐Terpstra test) |
---|---|---|---|---|---|---|
Baseline characteristics and resuscitation variables | ||||||
Body weight, g | 450 (420–471) | 460 (440–486) | 445 (440–470) | 456 (424–486) | 0.56 | 0.60 |
Cardiac arrest time, s | 162 (148–170) | 159 (146–175) | 161 (152–174) | 161 (152–174) | 0.90 | 0.83 |
CPR time, s | 65 (55–235) | 61 (56–106) | 74 (44–337) | 77 (58–161) | 0.85 | 0.56 |
Point‐of‐care testing at 60‐min post‐ROSC | ||||||
pH | 7.331 (7.314–7.360) (n=20) | 7.239 (7.226–7.247) (n=21) | 7.140 (7.130–7.151) (n=18) | 7.067 (7.047–7.360) (n=21) | <0.001 | <0.001 |
PaCO2, mm Hg | 51.5 (50.5–54.7) (n=20) | 72.5 (70.3–76.9) (n=21) | 98.7 (93.8–103.1) (n=18) | 121.3 (119.8–126.2) (n=21) | <0.001 | <0.001 |
PaO2, mm Hg | 233.4 (184.9–292.5) (n=20) | 221.9 (208.9–253.5) (n=21) | 231.8 (218.9–247.3) (n=18) | 267.1 (228.7–284.3) (n=21) | 0.09 | 0.05 |
HCO3−, mEq/L | 29.4 (27.0–30.6) (n=20) | 32.2 (29.7–33.5) (n=21) | 33.0 (32.1–34.5) (n=18) | 35.4 (32.8–36.8) (n=21) | <0.001 | <0.001 |
Lactate, mg/dL | 7.3 (5.6–11.3) (n=20) | 4.5 (3.1–6.5) (n=21) | 2.7 (2.7–2.8) (n=18) | 2.7 (2.7–4.8) (n=21) | <0.001 | <0.001 |
Sodium, mEq/L | 142.5 (141.0–143.8) (n=20) | 143.0 (143.0–145.5) (n=21) | 146.0 (142.8–147.0) (n=18) | 146.0 (145.0–147.0) (n=21) | <0.001 | <0.001 |
Potassium, mEq/L | 3.8 (3.7–4.1) (n=20) | 3.8 (3.6–3.9) (n=21) | 3.8 (3.6–3.9) (n=18) | 4.0 (3.6–4.4) (n=21) | 0.60 | 0.98 |
Calcium, mg/dL | 5.1 (5.0–5.3) (n=20) | 5.2 (5.1–5.5) (n=21) | 5.4 (5.3–5.5) (n=18) | 5.4 (5.3–5.8) (n=21) | <0.001 | <0.001 |
Creatinine, mg/dL | 0.64 (0.50–0.67) (n=20) | 0.71 (0.62–0.76) (n=21) | 0.75 (0.62–0.82) (n=18) | 0.74 (0.58–1.03) (n=21) | 0.10 | 0.03 |
Physiological parameters at 120‐min post‐ROSC | ||||||
CBF level, % of baseline | 45.8 (41.2–58.1) (n=20) | 155.5 (134.0–175.1) (n=20) | 192.4 (183.6–215.6) (n=18) | 242.3 (221.1–267.4) (n=21) | <0.001 | <0.001 |
ΔPbtO2, mm Hg | 1.26 (−1.78–11.74) (n=20) | 14.39 (0.20–25.44) (n=20) | 22.22 (8.25–35.09) (n=18) | 29.81 (12.57–55.60) (n=21) | 0.001 | <0.001 |
EtCO2, mm Hg | 51 (47–54) (n=20) | 77 (71–81) (n=20) | 100 (97–104) (n=18) | 127 (124–129) (n=21) | <0.001 | <0.001 |
MAP, mm Hg | 110.4 (103.6–112.0) (n=20) | 116.9 (102.5–129.9) (n=20) | 114.9 (102.0–117.7) (n=18) | 109.2 (100.1–117.2) (n=21) | 0.53 | 0.62 |
Brain injury biomarkers at 24‐h post‐ROSC | ||||||
NSE level at 24 h, ng/mL | 0.552 (0.483–0.732) (n=10) | 0.369 (0.316–0.454) (n=10) | 0.419 (0.394–0.520) (n=9) | 0.374 (0.364–0.443) (n=10) | 0.001 | 0.02 |
S100β level, pg/mL | 1.706 (1.612–1.868) (n=10) | 1.420 (1.384–1.436) (n=10) | 1.432 (1.409–1.500) (n=9) | 1.414 (1.392–1.472) (n=10) | 0.04 | 0.05 |
Malondialdehyde level, μM | 49.15 (47.24–49.84) (n=5) | 43.26 (42.69–46.68) (n=5) | 44.67 (44.04–46.98) (n=5) | 43.35 (42.65–44.05) (n=5) | 0.03 | 0.03 |
Cardiac, renal, and hepatic biomarkers at 24 h post‐ROSC | ||||||
Cardiac troponin I, pg/mL | 0.238 (0.139–0.423) (n=20) | 0.057 (0.031–0.210) (n=20) | 0.214 (0.109–0.420) (n=18) | 0.107 (0.062–0.166) (n=21) | <0.001 | 0.24 |
AST, U/L | 915.5 (248.6–2036.0) (n=20) | 557.7 (157.6–848.0) (n=20) | 764.3 (400.9–1398.0) (n=18) | 576.3 (459.3–838.5) (n=21) | 0.26 | 0.15 |
ALT, U/L | 162.5 (33.8–297.8) (n=20) | 85.5 (43.0–167.5) (n=20) | 128.5 (54.5–222.3) (n=18) | 98.0 (75.5–145.5) (n=21) | 0.45 | 0.33 |
Creatinine, mg/dL | 0.30 (0.20–0.30) (n=20) | 0.25 (0.20–0.30) (n=20) | 0.30 (0.18–0.33) (n=18) | 0.30 (0.20–0.30) (n=21) | 0.92 | 0.86 |
Outcomes at 24 h post‐ROSC | ||||||
Survival, n (%) | 20 (80) | 20 (80) | 18 (72) | 21 (84) | 0.77* | 0.82† |
Neurological function score | 9 (8–9) (n=20) | 9 (8–9) (n=20) | 10 (9–10) (n=18) | 10 (9.8–10) (n=21) | <0.001 | <0.001 |
Data are expressed as the median (interquartile range) or counts (proportions). ALT indicates alanine aminotransferase; AST, aspartate aminotransferase; CBF, cerebral blood flow; CPR, cardiopulmonary resuscitation; ΔPbtO2, delta of partial pressure of brain tissue oxygenation; EtCO2, partial pressure of end‐tidal carbon dioxide; HCO3−, sodium bicarbonate; MAP, mean arterial pressure; NSE, neuron‐specific enolase; PaCO2, arterial partial pressure of carbon dioxide; PaO2, arterial partial pressure of oxygen; ROSC, return of spontaneous circulation; and NA, not available. The results of post hoc pairwise comparisons were annotated in corresponding figures.
*
This comparison was performed using the Chi‐squared test.
†
This comparison was performed using the Cochran–Armitage test for trend.
Physiological Parameters During Invasive Monitoring
As shown (Figure 2 and Table 1), in the control group, after the initial hyperemia state, CBF decreased gradually and fell to ≈45.8% of baseline at 120 minutes post‐ROSC. In contrast, CBF was increased gradually by iCO2. In the 12% CO2 group, CBF at 120 minutes post‐ROSC was 242.3% of baseline, significantly higher than the control group. A significant increasing trend was also observed in CBF levels with increasing iCO2 concentrations. EtCO2 levels plateaued at 30 minutes post‐ROSC after an initial rapid increase. At 60 minutes post‐ROSC, significant differences in PaCO2 were noted among experimental groups. At 120 minutes post‐ROSC, ΔPbtO2 levels were significantly higher in 12% CO2 animals than controls. MAP levels were not significantly different among groups.

Figure 2. Physiological parameters during invasive monitoring.
The administration of inhaled CO2 increased cerebral blood flow, end‐tidal CO2, partial pressure of brain tissue oxygenation without compromising mean arterial pressure. Data are presented as the median and interquartile range. Pairwise comparisons of physiological parameters at 120 minutes post‐return of spontaneous circulation between experimental and control groups were performed by the post hoc Dunn test. The asterisk indicates statistical significance. CA indicates cardiac arrest; CBF, cerebral blood flow; ΔPbtO2, delta of partial pressure of brain tissue oxygenation; EtCO2, partial pressure of end‐tidal carbon dioxide; iCO2, inhaled carbon dioxide; MAP, mean arterial pressure; and ROSC, return of spontaneous circulation.
Survival and Neurological Outcomes
No significant differences in 24‐hour survival rates were observed among groups (Table 1, Figure S2). There was significant difference in cardiac troponin I (Table 1); compared with the control group, the cardiac troponin I level was significantly lower in the 12% CO2 group (P=0.003). For rats surviving to 24 hours, NFS were significantly higher in the 12% CO2 group when compared with control animals (Figure 3A). The sensitivity analysis revealed that the 12% CO2 group still demonstrated higher NFS than the control group after accounting for the rats with early death (Figure S3).

Figure 3. Neurological outcomes and brain injury biomarkers at 24 hours post‐return of spontaneous circulation.
Compared with the control group, the neurological function score was higher in the 12% CO2 group, suggesting better neurological recovery. The oxidative injuries, as indicated by the brain tissue malondialdehyde level, were lower in the 12% CO2 group. Also, the neuronal injuries, as represented by the serum neuron‐specific enolase and S100β concentrations, were lower in the 12% CO2 group. Data are presented as the median and third quartile. Pairwise comparisons of neurological outcomes or biomarker levels between experimental and control groups were performed using the post hoc Dunn test. Samples were randomly selected for measuring malondialdehyde, neuron‐specific enolase, and S100β levels. The asterisk indicates statistical significance. NSE indicates neuron‐specific enolase.
Oxidative and Neuronal Injuries
Apoptosis Downregulation
TUNEL assay data revealed significantly lower apoptotic cell densities in the hippocampal CA1 and CA3 regions of the 12% CO2 group when compared with controls (Figure 4A through 4C). Nonetheless, no significant differences were observed in expression of mitochondrial biomarkers, caspase‐3 activation, or PARP cleavage (Figure 5).

Figure 4. TUNEL staining in hippocampus.
The lower hippocampal TUNEL‐positive cell densities suggest reduced apoptotic cells in the 12% CO2 group than the control group. Data are presented as the median and third quartile. Pairwise comparisons between experimental and control groups were performed by the post hoc Dunn test. Samples were randomly selected for TUNEL stain. A, Brain sections were stained for TUNEL (green) and 4’,6‐diamidino‐2‐phenylindole (bluish‐violet). Confocal microscopy was used to image hippocampal CA1 and CA3 regions at 50× magnification. Red rectangles indicate selected CA1 and CA3 regions for quantification. The scale bar represents 200 μm. B and C, Apoptotic cell densities in CA1 and CA3 regions. The asterisk indicates statistical significance. DAPI indicates 4’,6‐diamidino‐2‐phenylindole; and TUNEL, terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling.

Figure 5. Western blotting of brain tissues.
Reactive oxygen species may activate the mitochondrial pathway leading to execution of apoptosis. Compared with the control group, no difference in the expression of cytochrome c, Bcl‐2, or Bax was observed. Caspase‐3 activation and PARP cleavage served as a proxy of execution of apoptosis pathway; also, no significant between‐group differences are noted. Data are presented as the median and third quartile. A through C, Comparisons of expression level of cytochrome c, Bcl‐2, and Bax in brain tissue by Western blotting. D and E, Comparisons of ratios between full‐length and cleaved caspase 3 or PARP in brain tissue by Western blotting.
Autophagy Downregulation
As shown (Figure 6), beclin‐1 and LAMP2 expression levels, and the LC3B‐II:LC3B‐I ratio were consistently lower in the 12% CO2 group when compared with controls, suggesting autophagy was downregulated in the former group.

Figure 6. Western blot analysis of beclin‐1, LC3B, p62, and LAMP2 expression.
Reduced expression levels of beclin‐1 and LAMP2 and lower LC3B‐II: LC3B:I ratio suggest downregulation of autophagy. Data are presented as the median and third quartile. Pairwise comparisons of protein expression levels or ratios between experimental and control groups were performed using the post hoc Dunn test. The asterisk indicates statistical significance. LAMP2 indicates lysosome‐associated membrane protein 2.
DISCUSSION
Main Findings
Firstly, we observed that increasing iCO2 concentrations would result in increasing CBF. Secondly, when compared with control animals, the 12% CO2 group showed better neurological recovery. Thirdly, the neuroprotective effects induced by iCO2 could be explained by mitigated ROS production, less neuronal injury, and downregulated apoptosis and autophagy.
Augmented Post‐CA CBF Levels and Improved Neurological Outcomes
Previous studies10, 11 demonstrated that iCO2 was neuroprotective for cerebral ischemia‐reperfusion injury; nonetheless, the changes in CBF were not demonstrated. Because vascular reactivity to CO2 may be attenuated in the early post‐CA period,19 the neuroprotective effects of iCO2 may not be directly attributed to augmented CBF if CBF is not monitored. As shown in the control group (Figure 2A), after initial hyperemia peaked at ≈10 minutes post‐ROSC, CBF levels continued decreasing until the end of observations, revealing the so‐called “no‐reflow” phenomenon.20, 21 In contrast, when iCO2 was administered, CBF levels began to rise 20 minutes post‐ROSC. Even when EtCO2 had plateaued at 30 minutes post‐ROSC (Figure 2B), CBF levels were still rising. These data supported our hypothesis that iCO2 exerted neuroprotective effects as it improved post‐CA CBF during the no‐reflow period.
Furthermore, trend analyses indicated a putative linear relationship between iCO2 concentrations and CBF changes (Table 1). However, when compared with the control group, only the 12% CO2 group showed consistently better results across all outcomes. Because sample sizes were estimated by assumed differences in NFS between the control and the 12% CO2 group, our sample size may have been insufficient to show significant differences between the control and the 4% CO2 or 8% CO2 groups. Nevertheless, in terms of all outcomes, significant linear trends were identified for improved neuroprotective effects with increasing iCO2 concentrations, suggesting iCO2 was generally beneficial for post‐CA neurological recovery, rather than being only effective at concentrations of 12%.
Interestingly, 24‐hour survival rates were not significantly different among study groups despite significant between‐group differences in blood pH (Table 1). Acidosis was suggested to exert adverse effects on heart contractility22, 23 and was associated with worse clinical outcomes after ROSC.24 However, MAP was not significantly different among study groups (Figure 2D). Furthermore, lactate levels were significantly lower with increasing iCO2 concentration (Table 1). Previous studies reported that hypercapnic acidosis suppressed systemic lactate production during systemic hypoxemia25 and was protective toward myocardial ischemia.26 Furthermore, lower levels of aspartate transaminase and alanine transaminase were also noted in the groups administered with iCO2. Despite that the between‐group differences in both liver injury markers were not statistically different (Table 1), it was possible that iCO2 facilitated the liver recovery process after ischemia‐reperfusion injuries,27 thereby expediting lactate clearance.
Thus, by improving post‐CA no‐reflow phenomenon, iCO2 facilitated neurological recovery without significantly compromising hemodynamics and survival.
Attenuated Oxidative and Neuronal Injuries
Abrupt cessation of cerebral oxygen delivery caused primary post‐CA brain injury.5 Cerebral oxygen delivery could be adjusted by manipulating CBF or blood oxygen levels. Ventilating patients with high oxygen levels is a frequently used method to increase blood oxygen and increase oxygen delivery to the brain. However, resulting hyperoxia was associated with poor post‐CA neurological recovery,28, 29 which may have been attributed partly to increased ROS production and associated oxidative injuries.30, 31 In our study, iCO2 augmented CBF, thereby increasing cerebral oxygen delivery and PbtO2. This result was consistent with the pilot trial conducted by Jakkula et al,32 which demonstrated higher near‐infrared spectroscopy‐measured regional cerebral oxygen saturation in patients with PaCO2 targeted at 44 to 46 mm Hg than those with PaCO2 targeted at 33 to 35 mm Hg. Cerebral reoxygenation reduced ROS‐associated oxidative injuries, as indicated by lower malondialdehyde levels, and neuronal injuries as indicated by lower NSE and S100β levels (Figure 3).33 In contrast to administering high oxygen levels, administering iCO2 appeared to be a safer method to reoxygenate the brain effectively after asphyxia‐induced CA.
Downregulating Apoptosis and Autophagy
We showed that iCO2 mitigated neuronal apoptosis, as evidenced by reduced TUNEL‐positive cell densities in hippocampal CA1 and CA3 regions (Figure 4). ROS may activate the mitochondrial pathways, leading to the release of cytochrome c and execution of apoptosis.34 Therefore, reduced ROS levels in the 12% CO2 group may have partly accounted for the attenuated apoptosis seen in these animals. Apoptosis is mainly executed by caspase‐3, whereas PARP, a DNA repair enzyme, is selectively cleaved by activated caspase‐3 during apoptosis.35, 36, 37 Therefore, caspase‐3 activation and PARP cleavage have been viewed as hallmark apoptosis indicators. Interestingly, we detected no significant differences in caspase‐3 activation or PARP cleavage among groups (Figure 4), which may be explained by several reasons. Firstly, we used the whole hemisphere rather than specifically the hippocampus to investigate apoptotic pathways. Because TUNEL‐positive cells were most densely distributed in the hippocampus, containing other brain regions in Western blot analysis may dilute the differences. Secondly, Western blot analysis was performed using brain tissue harvested at 24 hours post‐CA. At this time, initial apoptosis phases may have been completed.
It was reported that after ROSC, autophagy was initially activated, followed by apoptosis, and culminated in hippocampal neuronal death.12 Autophagy (or macroautophagy) is an orchestrated cell catabolic process that eliminates damaged cytoplasmic contents38 during various stress conditions (eg, hypoxia, nutrient starvation, and oxidative stress). During the process,39 beclin‐1 initiates autophagosome formation, LC3B elongates the autophagosome, and LAMP2 is required for autophagolysosome formation. During elongation, LC3B is first cleaved to LC3B‐I and then converted to LC3B‐II. Thus, the LC3B‐II:LC3B‐I ratio is used to monitor autophagy flux. We showed that beclin‐1 and LAMP2 expression levels and the LC3B‐II:LC3B‐I ratio were reduced in the 12% CO2 group, suggesting iCO2 putatively downregulated autophagy (Figure 6). In contrast, p62 is a scaffold for protein aggregates destined for autophagolysosome degradation and should have accumulated when autophagy was downregulated. Nonetheless, our study did not identify significant difference in p62 level between the 12% CO2 and control groups (Figure 6). It was possible that p62 may interact with multiple pathways other than autophagy and, therefore, it could be a less specific indicator for autophagy flux.40 It should be emphasized that the changes in these autophagy markers may only be viewed as indirect evidence of the iCO2 effects. Using autophagy inducer, such as rapamycin, or autophagy inhibitor, such as SP600125, to study the autophagic flux may be a better way to delineate the changes in autophagic flux caused by iCO2 and may be considered in future studies, for which our study results may serve as the basis to select the optimal iCO2 concentration for investigation.
Taken together, these results suggest that when post‐CA CBF is augmented by iCO2, neuronal stress is ameliorated, and autophagy might revert to a basal state to function as a potential prosurvival mechanism, concomitant with downregulated apoptosis.41
Future Directions
To date, the clinical evidence on post‐CA PaCO2 effects toward neurological outcomes is inconsistent.42 In 2 small randomized‐controlled pilot trials, Eastwood et al43 reported that PaCO2 at 50 to 55 mm Hg attenuated NSE release when compared with PaCO2 at 35 to 45 mm Hg, while Jakkula et al showed no difference in release of NSE32 and neurofilament light44 between PaCO2 at 44 to 46 mm Hg and PaCO2 at 33 to 35 mm Hg. The latest 2020 American Heart Association45 and 2021 European Resuscitation Council/ European Society of Intensive Care Medicine guidelines46 recommended that post‐CA PaCO2 should be maintained within the normal physiological range, 35 to 45 mm Hg. Our study demonstrated that increasing PaCO2 levels were associated with better neuroprotective effects (Table 1). Nonetheless, in a rat model of transient global cerebral ischemia‐reperfusion injury, Zhou et al10 noted PaCO2 between 100 and 120 mm Hg increased brain injury, which may be caused by increased brain edema. Therefore, further research is warranted to investigate the safety and neuroprotective effects of targeting higher PaCO2 levels for patients with post‐CA. On the other hand, targeting personalized PaCO2 levels by monitoring cerebral tissue oxygenation may also be an alternative to balancing risk and safety of iCO2.
Study Limitations
First, we only assessed short‐term neurological outcomes without performing neurobehavioral tests to evaluate long‐term cognitive injuries. Second, we used inspired O2 fraction of 50% during the initial post‐ROSC period, resulting in elevated PaO2 levels higher than recommended by guidelines.45, 46 Future studies should further investigate whether the neuroprotective effects of iCO2 would be influenced by different PaO2 levels. Third, we used asphyxia‐induced CA model to study the protective effects of iCO2. However, in adult patients, the most prevalent cause of collapse was ventricular fibrillation or pulseless ventricular tachycardia, constituting 24% to 35% of out‐of‐hospital CA events.1 In a rat study, Drabek et al6 noted that both ventricular fibrillation– and asphyxia‐induced CA resulted in delayed hypoperfusion in all regions, including hippocampus. Because the delayed hypoperfusion phenomenon was common in both models, the benefits of iCO2 observed in our asphyxia‐induced CA model may still exist in ventricular fibrillation–induced CA model, which deserved further experiments and clinical studies. Fourth, according to the trend analysis, iCO2 concentration <12% might be effective for post‐ROSC brain injuries in general. Nonetheless, our study results may not be externally generalized to iCO2 concentration >12% because some adverse effects of severe hypercapnia had been observed in different brain injury models.10 Finally, the ability to tolerate hypercapnic acidosis may be different between rats and human subjects. Before its application in clinical trials, more studies are needed to explore the safety of iCO2 in patients with fragile post‐ROSC.
CONCLUSIONS
Administering iCO2 could increase post‐CA CBF and facilitate neurological recovery. The neuroprotective effects of iCO2 could be attributable to mitigated oxidative injury, reduced neuronal injury, and downregulated apoptosis and autophagy. Further research is required to examine the safety and neuroprotective effects of iCO2 in clinical settings.
Sources of Funding
Dr Chih‐Hung Wang received a grant (108‐2628‐B‐002‐016‐, 109‐2628‐B‐002‐045‐, 110‐2628‐B‐002 ‐028‐) from the Ministry of Science and Technology. Ministry of Science and Technology had no involvement in designing the study, collecting, analyzing or interpreting the data, writing the manuscript, or deciding whether to submit the manuscript for publication.
Disclosures
None.
Acknowledgments
We thank the staff of the 3rd Core Lab, Department of Medical Research, National Taiwan University Hospital, for technical support. We thank Dr Chih‐Fan Yeh at Department of Internal Medicine, National Taiwan University Hospital, Taipei, for statistical support.
Footnotes
Supplemental Material is available at Supplemental Material
For Sources of Funding and Disclosures, see page 12.
Supplemental Material
Table S1
Figures S1–S3
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© 2022 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
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Received: 1 August 2022
Accepted: 19 September 2022
Published online: 31 October 2022
Published in print: 1 November 2022
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Ministry of Science and Technology: 110‐2628‐B‐002‐028, 109‐2628‐B‐002‐045, 108‐2628‐B‐002‐016
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- Effects of intrathecal administration of sodium nitroprusside and nicardipine on cerebral pial microcirculation, cortical tissue oxygenation, and electrocortical activity in the early post-resuscitation period in a porcine cardiac arrest model, PLOS ONE, 20, 1, (e0313257), (2025).https://doi.org/10.1371/journal.pone.0313257
- Translational approach to assess brain injury after cardiac arrest in preclinical models: a narrative review, Intensive Care Medicine Experimental, 13, 1, (2025).https://doi.org/10.1186/s40635-024-00710-y
- Neuroprotective Approaches for Brain Injury After Cardiac Arrest: Current Trends and Prospective Avenues, Journal of Stroke, 26, 2, (203-230), (2024).https://doi.org/10.5853/jos.2023.04329
- Pre‐arrest atrial fibrillation and neurological recovery after cardiac arrest among hospitalized patients: A retrospective cohort study, European Journal of Clinical Investigation, 55, 3, (2024).https://doi.org/10.1111/eci.14375
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