We performed all instrumentation according to the previously described protocol ( Figure 1A ) (4, 18, 19). In brief, adult male 12-16-week-old Sprague-Dawley rats (400–500 g, Charles River Laboratories, Wilmington, MA, USA) were anesthetized with 4% isoflurane (Isosthesia, Butler-Schein AHS, Dublin, OH, USA) and intubated with a 14-gauge plastic catheter (Surflo, Terumo Medical Corporation, Tokyo, Japan). We used only male rats to reduce potential confounding among animals that may cause outcome variabilities from hormonal and/or genetic differences. Rats were mechanically ventilated (Ventilator Model 683, Harvard Apparatus, Holliston, MA, USA) at a minute ventilation (MV) volume of 180 mL per minute at a respiratory rate of 45 breaths per minute. In this study, we used one ventilation setting for all animals at all times and did not change the MV or respiratory rate during the experiments. Positive end-expiratory pressure (PEEP) was set at 2 cm H₂O. Anesthesia was maintained with isoflurane at 2% and at a fraction of inspired O₂ (F_IO₂) of 0.3. Core temperature was maintained at 36.5 ± 1.0 °C during the surgical procedure. After instrumentation, neuromuscular blockade was achieved by slow intravenous administration of 2 mg/kg of vecuronium bromide (Hospira, Lake Forest, IL, USA) and asphyxia was induced by turning off the ventilator. CA normally occurred three to four minutes after asphyxia started. The CA group animals received cardiopulmonary resuscitation (CPR) after they were subjected to 12 minutes asphyxia. We defined CA as a mean arterial pressure (MAP) of < 20 mmHg; CA was completely untreated during the initial 10 minutes. After 12 minutes of asphyxia, mechanical ventilation was restarted at an F_IO₂ of 1.0 and manual chest compression CPR was delivered simultaneously. Chest compressions were performed with two fingers over the sternum at a rate of 260 to 300 per minute. At 30 seconds after beginning of CPR, a 20 µg/kg bolus of epinephrine was given to rats through a venous catheter. Following ROSC, defined as MAP > 60 mmHg, CPR was discontinued. At 2 hours after initiating CPR, mechanical ventilation was discontinued, and rats were euthanized for sample collections of whole blood. Whole blood were drawn through the arterial catheter.

We performed all instrumentation according to the previously described protocol ( Figure 1B ) (20). In brief, adult male 14-18-week-old C57BL/6 mice (weight 25 to 30 g; Charles River Production, Wilmington, MA, USA) were freely accessed to food and water before experiments. Mice were anesthetized with 4% isoflurane (Isosthesia, Butler-Schein AHS, Dublin, OH, USA) and the trachea was orally intubated with a 20-gauge catheter (Angiocath, Becton Dickinson). Mechanical ventilation (Minivent, Harvard Apparatus, Holliston, MA, USA) was initiated at rate of 110/min, I:E = 1:1, F_IO₂ = 1.0, and tidal volume was maintained at 8–12 mL/kg per minute. We used only male mice to reduce potential confounding among animals that may cause outcome variabilities from hormonal and/or genetic differences. In this study, we used one ventilation setting for all animals at all times and did not change the MV or respiratory rate during the experiments. PEEP was set at 1 cm H₂O. Anesthesia was maintained with isoflurane at 2%. During surgical preparation, body temperature was maintained between 37.0 ± 0.5 °C by an incandescent heating lamp and monitored by a transesophageal thermocouple probe. A microcatheter (EZ-1101, BioTime Inc, Carlsbad, CA, USA) was inserted into the left femoral vein for drug and fluid administration. The other microcatheter was inserted into left femoral artery to monitor the blood pressure. Electrocardiograph (ECG) was recorded by needle probe. The mouse CA was quickly induced by 100 μL KCl (80 μg/kg) followed by 200 μL room temperature saline flushing and weaned off the ventilator at the same time. The CA was confirmed by loss of ECG activity and blood pressure, which usually sharply decreased, below 10 mmHg. The esophageal temperature of the mice was maintained at 37.0 ± 0.5 °C during the whole procedures by heating lamp. Eight minutes after the induction of CA, resuscitation via manual chest compression with one finger over the sternum at a rate of 300 to 400 per minute was initiated together with 1 μg/100 μL epinephrine followed by flushing with 200 μL room temperature saline via left femoral vein, and ventilation was resumed at the rate of 110/min, I:E = 1:1, F_IO₂ = 1.0 with tidal volume maintained at 8–12 mL/kg per minute. Chest compression was performed to provide a uniform rate and to maintain maximal MAP. ROSC was confirmed by the return of ECG rhythm and significant increase in MAP from that during chest compression. The mouse was maintained on mechanical ventilation after ROSC. At 2 hours after initiating CPR, mechanical ventilation was discontinued, and mice were euthanized for sample collections of the spleen and whole blood. For the assessment of alterations in predominant immune cell populations and the expression of ectonucleotidases at a later time point, 3 mice were monitored until 24 hours after initiating CPR ( Figure 1B ). Subsequent to extubation, a subcutaneous injection of 0.05 mL extended-release buprenorphine (Ethiqa XR, Fidelis Pharmaceuticals, North Brunswick Township, NJ, USA) was administered to the animals for analgesia. At 24 hours after initiating CPR, the mice were euthanized for sample collections of the spleen and whole blood. Whole blood samples were obtained from inferior vena cava under inhaled isoflurane anesthesia prior to perfusion and subsequent spleen extraction. Postsurgical care, including animal housing and observation, were provided by the animal facility.

Whole blood samples were collected into heparinized tubes. In the rat CA experiment, 500 μL of whole blood was collected after catheterization as baseline and again at 2 hours after initiating CPR. In the mouse CA experiment, animals were euthanized 2 hours or 24 hours after initiating CPR and 500 μL of whole blood was collected, and 500 μL of whole blood was collected from a naïve mouse for reference purposes. The collected blood samples were diluted into 5 mL of 1× RBC Lysis Buffer (prepared from 10× RBC Lysis Buffer, BioLegend, San Diego, CA, USA). After an incubation period of 4-5 minutes at room temperature with occasional shaking, the reaction was halted by dilution of the Lysis Buffer with 20-30 mL of 1× PBS. Subsequently, cells were pelleted by centrifugation at 500×g at room temperature for 5 minutes and then resuspended in 2 mL of 1× RBC Lysis Buffer. Following a similar incubation duration with occasional shaking, the reaction was halted by diluting the Lysis Buffer with 10-15 mL of 1× PBS. Cells were pelleted again by centrifugation at 500×g at room temperature for 5 minutes and finally resuspended in 500 μL of Flow Cytometry Staining Buffer (Invitrogen, Waltham, MA, USA).

In the mouse CA experiment, a spleen was harvested 2 hours or 24 hours after initiating CPR, meanwhile a spleen from a naïve mouse was harvested for comparative reference. The harvested spleen sample was finely minced using sharp scissors and then placed onto a 70 μm Cell Strainer (Corning, NY, USA), which was situated atop a 50 mL tube. The minced spleen was further homogenized using the plunger end of a 5 mL syringe and rinsed with 5 mL of 1× PBS. Subsequently, the strained cells were pelleted by centrifugation at 500×g at room temperature for 5 minutes. The resulting cell pellets were resuspended in 2 mL of 1× RBC Lysis buffer. After an incubation period of 1-2 minutes at room temperature with occasional shaking, the reaction was halted by dilution of the Lysis Buffer with 10-15 mL of 1× PBS. Subsequently, cells were pelleted by centrifugation at 500×g at room temperature for 5 minutes and finally resuspended in 500 μL of Flow Cytometry Staining Buffer.

A single fluorophore was conjugated to either the anti-rat CD39/ENTPD antibody (Proteintech, cat no. 14211-1-AP, Rosemont, IL, USA) or the anti-rat NT5E/CD73 antibody (Proteintech, cat no. 12231-1-AP) using the Alexa Fluor® 594 Microscale Protein Labeling Kit or the Alexa Fluor® 647 Microscale Protein Labeling Kit (Invitrogen), respectively, following the manufacturer’s provided protocols. Briefly, 1 M sodium bicarbonate solution was prepared by adding 1 mL deionized water to the vial of sodium bicarbonate (Component B). Next, 50 µL of antibodies and 5 µL of Component B were transferred to a designated reaction tube (Component C). Separately, 10 µL of deionized water was introduced to each vial containing Alexa Fluor 594 and 647 succinimidyl ester (Component A). Subsequently, 0.616 µL (Alexa Fluor 594) or 0.7 µL (Alexa Fluor 647) of Component A was transferred to Component C, followed by a 15-minute incubation period at room temperature. To purify the Alexa Fluor 594-labeled anti-rat CD39 antibody and the Alexa Fluor 647-labeled anti-rat CD73 antibody, 50 µL of Component C was meticulously dispensed onto the center of the resin bed surface within specialized spin columns. The spin columns were then subjected to centrifugation at 16,000×g for 1 minute, effectively yielding the purified labeled antibodies in the collection tubes.

Single-cell suspensions derived from whole blood samples in both the rat and mouse experiments, as well as from the spleen in the mouse experiment, were subjected to flow cytometry analysis. This analysis involved the use of fluorophore-conjugated antibodies (Thermo Fisher Scientific, Waltham, MA, USA) targeting specific cell surface markers, including anti-rat CD3-PE (eBioScience, clone# eBioG4.18), anti-rat CD4-FITC (eBioScience, clone# OX35), anti-rat CD8a-APC (eBioScience, clone# OX8), anti-mouse CD3-APC-eFluor™ 780 (eBioScience, clone# 17A2), anti-mouse CD4-FITC (eBioScience, clone# GK1.5), anti-mouse CD8a-APC (eBioScience, clone# 53-6.7), anti-rat CD39-Alexa Fluor 594 (manually labeled), anti-rat CD73-Alexa Fluor 647 (manually labeled), anti-mouse CD45-PerCP-Cyanine5.5 (eBioScience, clone# 30-F11), anti-mouse CD39-PE-Cyanine7 (eBioScience, clone# 24DMS1), rat IgG2b isotype control-PE-Cyanine7 (eBioScience, clone# eB149/10H5), anti-mouse CD73-PE (eBioScience, clone# eBioTY/11.8), rat IgG1 isotype control-PE (eBioScience, clone# eBRG1), anti-mouse CD11b-FITC (eBioScience, clone# M1/70), and anti-mouse CD19- eFluor™ 450 (eBioScience, clone# eBio1D3). To reduce Fc receptor-mediated binding by antibodies of interest for analyses of both myeloid cells and B lymphocytes, cell suspension were preincubated with anti-mouse CD16/CD32 (BD Biosciences, clone# 2.4G2) at 4˚C for 5 minutes. The cells were incubated with a mixture of these antibodies at saturating concentrations in Flow Cytometry Staining Buffer at room temperature in darkness for 30 minutes. Following this incubation period, the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI, cat no. D1306) for the exclusion of dead and apoptotic cells from flow cytometric analysis and then carefully washed. Subsequently, they were resuspended in 500 μL of Flow Cytometry Staining Buffer. Data were acquired on BD Symphony (BD Biosciences) and subsequently analyzed using FlowJo software 10.8.1 (Becton, Dickinson & Company, Ashland, OR, USA).

Data are shown as means and standard errors of the means (SEM) for all variables. Two-tailed P-values of Wilcoxon matched-pairs signed rank test and Mann-Whitney test were used for two-group comparisons: between baseline and 2 hours after CA in the rat model; and between naïve and 2 hours after CA in the mouse model, respectively. Dunn’s multiple comparisons test was used for three-group comparisons: between naïve, 2 hours, and 24 hours after CA in the mouse model. P < 0.05 was considered statistically significant. Prism 10.0.3 (GraphPad, San Diego, CA, USA) was used for these statistical analyses.

In a rat model of CA, ROSC was achieved at 58.4 ± 4.4 seconds ( Table 1 ). At 2 hours after resuscitating from 12 minutes of asphyxia CA, whole blood samples were collected for subsequent flow cytometry analyses. The population of whole lymphocytes was gated on forward scatter/side scatter dot plots, and specific populations of immune cells, including CD3⁺CD4⁺ helper T lymphocytes and CD3⁺CD8a⁺ cytotoxic T lymphocytes, were identified ( Figures 2A, B ). In comparison to the baseline, CD3⁺CD4⁺ helper T lymphocytes markedly increased ( Figure 2C , baseline, 29.02 ± 2.08 %; CA 2h, 53.27 ± 2.80 %, P < 0.01), while CD3⁺CD8a⁺ cytotoxic T lymphocytes increased slightly ( Figure 2D , baseline, 15.42 ± 1.45 %; CA 2h, 19.34 ± 1.72 %, P < 0.01), consequently resulting in an increase of CD4/CD8a ratio ( Figure 2E , baseline, 2.00 ± 0.19; CA 2h, 2.93 ± 0.27, P < 0.01). Similarly, in a mouse model of CA, ROSC was achieved at 81.3 ± 6.0 seconds ( Table 1 ). At 2 hours after resuscitating from 8 minutes of KCl-induced CA, whole blood and spleen samples were collected. As depicted in Figures 3A, B , CD3⁺CD4⁺ helper T lymphocytes in whole blood displayed a notable increase ( Figure 3C , Naïve, 10.05 ± 0.68 %; CA 2h, 26.16 ± 0.92 %, P < 0.001), while CD3⁺CD8a⁺ cytotoxic T lymphocytes increased ( Figure 3D , Naïve, 10.82 ± 0.66 %; CA 2h, 19.91 ± 1.33 %, P < 0.001), ultimately leading to an increase in the CD4/CD8a ratio ( Figure 3E , Naïve, 0.94 ± 0.06; CA 2h, 1.36 ± 0.10, P < 0.01). Although CD3⁺CD4⁺ helper T lymphocytes in the spleen also significantly increased ( Figures 4A–C , Naïve, 12.17 ± 0.78 %; CA 2h, 14.83 ± 0.63 %, P < 0.05), CD3⁺CD8a⁺ cytotoxic T lymphocytes ( Figure 4D , Naïve, 13.71 ± 0.32 %; CA 2h, 14.93 ± 0.52 %, P = 0.12) and the CD4/CD8a ratio did not exhibit significant increases ( Figure 4E , Naïve, 0.90 ± 0.06; CA 2h, 0.99 ± 0.03, P = 0.31). These findings collectively suggest that there is an upregulation of circulatory immune activity, predominantly driven by an increased population of CD4⁺ T lymphocytes after CA.

To further investigate immune responses after CA, our study focused on ectonucleotidases, specifically CD39 and CD73, known for their involvement in promoting immunosuppression through purinergic signaling and the metabolism of eATP (21–23). In a rat model of CA, median fluorescent intensity (FI) of both CD39 and CD73 were measured among leukocytes isolated from the whole blood ( Figures 5A–E ). Manually fluorophore-conjugated polyclonal antibodies targeting CD39 and CD73 detected a significant increase in the median FI of both antibodies 2 hours after CA ( Figures 5C, E , CD39: baseline, 301.88 ± 42.94; CA 2h, 564.51 ± 127.80, P < 0.05 and CD73: baseline, 341.32 ± 47.03; CA 2h, 524.35 ± 81.23, P < 0.05). For a more detailed analysis, the expression of both ectonucleotidases were evaluated in a mouse model of CA. As shown in Figures 6A, B , CD45⁺ live leukocytes were gated in the whole blood and spleen, and positive ratios of both CD39 and CD73 were determined. In the whole blood, CD45⁺CD39⁺ leukocytes markedly increased ( Figure 6C , Naïve, 19.18 ± 1.40 %; CA 2h, 44.90 ± 4.32 %, P < 0.05; CA 24h, 59.40 ± 5.48 %, P < 0.01), as did CD45⁺CD73⁺ leukocytes ( Figure 6D , Naïve, 14.40 ± 0.12 %; CA 2h, 36.70 ± 2.50 %, P < 0.05; CA 24h, 46.77 ± 2.56 %, P < 0.01). However, in the spleen, both CD45⁺CD39⁺ ( Figure 6E , Naïve, 18.35 ± 1.32 %; CA 2h, 18.98 ± 0.86 %, P = 0.88; CA 24h, 31.63 ± 3.12 %, P < 0.05) and CD45⁺CD73⁺ leukocytes ( Figure 6F , Naïve, 18.88 ± 0.76 %; CA 2h, 20.75 ± 0.92 %, P = 0.49; CA 24h, 27.07 ± 3.09 %, P < 0.05) had increases at 24 hours but not at 2 hours after CA. These findings collectively indicated prompt ectonucleotidase increases in the circulatory blood and late increases in the spleen after CA.

To elucidate the specific immune cell populations in the whole blood and the spleen that can increase ectonucleotidases on the cell surface after CA, we conducted further analyses focusing on CD11b⁺ myeloid cells, CD3⁺ T lymphocytes, and CD19⁺ B lymphocytes by using the mouse model. Following gating of CD45⁺DAPI^- leukocytes ( Figure 7A ), CD11b⁺ myeloid cells were gated, showing the remarkable increase of myeloid cells in the circulatory blood ( Figure 7B , Naïve, 14.70 ± 1.94 %; CA 2h, 58.95 ± 1.83 %, P < 0.05; CA 24h, 73.03 ± 5.59 %, P < 0.01). The expression of CD39 or CD73 in myeloid cells was graphically represented and analyzed ( Figure 7C ). Notably, myeloid cells essentially had high levels of CD39, which was retained until 24 hours after CA ( Figure 7D , Naïve, 86.22 ± 1.10 %; CA 2h, 87.58 ± 2.06 %, P > 0.99; CA 24h, 88.80 ± 2.05 %, P = 0.43), while, the expression of CD73 markedly increased in the whole blood ( Figure 7E , Naïve, 28.63 ± 1.07 %; CA 2h, 58.17 ± 2.71, P < 0.05; CA 24h, 62.17 ± 1.81, P < 0.05). Similarly, in the spleen, the expression of both CD39 and CD73 in myeloid cells were shown to increase ( Figures 7F, G , CD11b⁺CD39⁺: Naïve, 79.43 ± 1.68 %; CA 2h, 86.90 ± 0.76 %, P < 0.05; CA 24h, 87.53 ± 2.22 %, P < 0.05 and CD11b⁺CD73⁺: Naïve, 19.85 ± 1.21 %; CA 2h, 38.47 ± 1.71, P < 0.05; CA 24h, 51.10 ± 6.30 %, P < 0.01).

Regarding the expression of ectonucleotidases within lymphocytes, following gating of CD3⁺ T lymphocytes or CD19⁺ B lymphocytes in DAPI^- live leukocytes population ( Figures 8A, B and 9A, B ), the expression of these enzymes was evaluated in both CD3⁺ T lymphocytes and CD19⁺ B lymphocytes ( Figures 8C , 9C ). The population of CD19⁺ B lymphocytes remarkably decreased in the whole blood ( Figure 9B , Naïve, 68.37 ± 1.43 %; CA 2h, 23.22 ± 0.99 %, P < 0.05; CA 24h, 14.40 ± 4.33, P < 0.01). Both CD3⁺CD39⁺ and CD3⁺CD73⁺ T lymphocytes had decreases in the whole blood ( Figures 8D, E , CD3⁺CD39⁺: Naïve, 2.97 ± 0.33 %; CA 2h, 1.97 ± 0.22 %, P = 0.07; CA 24h, 1.53 ± 0.07, P < 0.01 and CD3⁺CD73⁺: Naïve, 72.83 ± 1.57 %; CA 2h, 61.88 ± 1.46, P < 0.01; CA 24h, 71.53 ± 1.11, P > 0.99). In contrast, both CD19⁺CD39⁺ and CD19⁺CD73⁺ B lymphocytes were shown to have significant increases ( Figures 9D, E , CD19⁺CD39⁺: Naïve, 5.39 ± 0.34 %; CA 2h, 7.78 ± 0.49 %, P < 0.05; CA 24h, 7.14 ± 0.92, P = 0.18 and CD19⁺CD73⁺: Naïve, 1.96 ± 0.09 %; CA 2h, 2.99 ± 0.31, P < 0.05; CA 24h, 2.86 ± 0.12, P = 0.07) in the whole blood. In the spleen, no marked differences were evident in either CD3⁺ T lymphocytes ( Figures 8F, G , CD3⁺CD39⁺: Naïve, 10.06 ± 0.70 %; CA 2h, 9.39 ± 0.52 %, P > 0.99; CA 24h, 10.21 ± 1.40, P > 0.99 and CD3⁺CD73⁺: Naïve, 63.70 ± 1.33 %; CA 2h, 61.02 ± 0.76, P = 0.27; CA 24h, 66.90 ± 2.25, P = 0.95) or CD19⁺ B lymphocytes ( Figures 9F, G , CD19⁺CD39⁺: Naïve, 15.00 ± 1.45 %; CA 2h, 13.77 ± 0.91 %, P > 0.99; CA 24h, 21.43 ± 2.15, P = 0.11 and CD19⁺CD73⁺: Naïve, 4.14 ± 0.46 %; CA 2h, 3.77 ± 0.21, P > 0.99; CA 24h, 6.25 ± 0.50, P = 0.09). These findings provide insight into the immune cell populations that are predisposed to ectonucleotidase expression, with a distinct upregulation observed in myeloid cells in the blood circulation and the spleen after CA.