Nine- to twelve-week-old male C57BL/6N mice were purchased from Charles River (Sulzfeld, Germany). All mice were housed in the local animal facility. All experiments were conducted strictly according to the German animal protection law and in accordance with good animal practice as defined by the Federation of Laboratory Animal Science Associations (https://www.felasa.eu) and the national animal welfare body GV-SOLAS (https://www.gv-solas.de). The examinations undertaken in this study were approved by the federal authorities in Freiburg and the Institutional Review Board (G-17/44).

All surgeries were performed between 10 a.m. and 2 p.m. to avoid circadian rhythm-associated irregularities according to the guidelines for experimental models of myocardial ischemia and infarction (27). In brief, 9–12 week-old wildtype (WT) mice were anesthetized by intraperitonial (i.p.) injection of 100 mg/kg Ketamine (Ketavet, Pfizer Pharmacia, Berlin, Germany) and 5 mg/kg Xylazine (Rompun, Bayer Vital, Leverkusen, Germany). Analgesia was provided by subcutaneous (s.c.) injection of 0.1 mg/kg buprenorphine. To avoid dehydration 500 µl warm 0.9% NaCl (9 mg/ml) containing 5% glucose (G5%, B. Braun Melsungen, Melsungen, Germany) was injected i.p. Mice were placed on a heating pad to maintain body temperature at 37°C. The operation table was tilted 20°C to allow visualization of the vocal cords and mice were intubated under sight. Oxygen saturation, heart rate, and respiratory rate were monitored throughout the procedure by a MouseOX system (Starr Life Sciences, Oakmont, PA). Anesthesia was maintained by addition of 1.2% isoflurane (Abbott, Wiesbaden, Germany) during surgery. After right lateral positioning of the animal, left lateral thoracotomy was performed and the LAD was identified. To induce ischemia an 8.0 nylon suture (Prolene, Ethicon, Norderstedt, Germany) was placed around the vessel and a loose loop was formed. A fine bore 0.61 mm polythene tube was placed on the LAD and the loop was tied. Ligation was visually confirmed by paling of the left ventricular anterior wall. The tube was removed after 30 min to allow reperfusion of myocardium. After evacuation of the pneumothorax, chest and skin wounds were closed using a 6–0 prolene suture (Prolene, Ethicon, Norderstedt, Germany).

Mice were monitored until spontaneous breathing occurred, extubated and placed under a heating lamp. Analgesia with Buprenorphine was administered every 6 h.

Echocardiography was performed either on day 1, 3 or 7 as previously described (28). In brief, mice were anesthetized by 2% isoflurane i.n. and placed on a heating pad. Echocardiography loops were recorded in B and M modes in longitudinal and short axis views on a Vevo 3100 equipped with a MX550D transducer (both FUJIFILM VisualSonics, Inc., Toronto, ON, Canada). Heart rate was monitored during the procedure. Systole and diastole were defined based on concomitant electrocardiography (ECG) recordings. The end-systolic time point for left ventricular (LV) diameter measurement was defined as the maximum of ventricle contraction just before complete closure of the aortic valve. End-diastole was defined as the maximum of LV dilation and filling just before mitral valve closing (when visible) and aortic valve opening. Left ventricular ejection fraction (LVEF) was determined by LV tracing relating end systolic LV area as the minimal LV cross-sectional area to end-diastolic LV area as the maximum LV cross-sectional area in long axis views. FS was assessed from short axis M-mode using VevoLab 5.5.1 software (FUJIFILM VisualSonics).

Statistical analyses were performed using GraphPad Prism version 8. The Shapiro–Wilk test was employed to assess data for normality.

Normally distributed data are presented as mean ± standard error of the mean (SEM), while non-normally distributed data are reported as median with interquartile range (IQR). Statistical outliers were identified using the ROUT test. Comparisons of normally distributed data were conducted using one-way ANOVA followed by Tukey's multiple comparisons test. Non-normally distributed data were analyzed using the Kruskal–Wallis test.

A significant increase in the percentage of neutrophils of the total leukocytes was observed 1 day after I/R injury in both, blood (9.1% ± 1.5 vs. 42.8% ± 3.6, p ≤ 0.001; Figure 2B) and spleen (11.5 ± 2.3 vs. 21.5% ± 3.1; Figure 2C). Additionally, a significant rise of the proportion of neutrophils was detected in the AAR 3 days post I/R injury (3.7% ± 0.8 vs. 16.2% ± 2.4, p ≤ 0.01; Figure 2A). By 7 days after I/R injury, the percentage of neutrophils in the AAR, blood, and spleen returned to the baseline range (10%–15%) (6). In contrast, neutrophil proportions in the bone marrow showed only minor changes over the observation period. Monocyte proportion increase appeared in parallel to that of neutrophils. Three days post I/R injury, a significant increase in the monocyte percentage was observed in the AAR (4.9% ± 0.9 vs. 20.1% ± 2.4, p ≤ 0.01; Figure 2E), blood (2.3% ± 0.5 vs.7.5% ± 1.3, p ≤ 0.05; Figure 2F) and bone marrow (3.3% ± 0.5 vs. 11.3% ± 1.8, p ≤ 0.05; Figure 2H). In the spleen 1 day after I/R injury a significant increase of monocyte proportion was detected 1 day after I/R injury (12.61 ± 1.6 vs. 6.4% ± 0.8, p ≤ 0.05; Figure 2G). Similarly, by 7 days post I/R injury, monocyte proportions in all examined tissues had nearly returned to baseline levels.

Phenotypically and functionally distinct monocyte subtypes were differentiated using Ly6C expression. One day post I/R injury, the percentage of pro-inflammatory Ly6C^high monocytes peaked significantly in the AAR (43.7% ± 12.6 vs. 85.2% ± 2.1, p ≤ 0.01; Figure 3A). In contrast the percentage of anti-inflammatory Ly6C^low monocytes showed a marked decrease at this time, followed by a significant increase in the AAR 7 days after I/R injury (14.2% ± 2.03 vs. 78.8% ± 4.4, p ≤ 0.001; Figure 3E). Concurrently a percentage decline in pro-inflammatory Ly6C^high monocytes was observed in the blood, followed by a significant rise 7 days post I/R injury (8.11% ± 4.4 vs. 39.5% ± 7.4, p ≤ 0.05; Figure 3B). Conversely, the percentage of anti-inflammatory Ly6C^low monocytes in the blood increased significantly 1 day after I/R injury before returning to baseline values (96.9% ± 1.3 vs. 60.4% ± 7,4; p ≤ 0.05; Figure 3F). Pro-inflammatory Ly6C^high monocytes also showed a significant percentage increase in the spleen (1.3% ± 0.4 vs. 22.6% ± 5.6, p ≤ 0.05; Figure 3D) and bone marrow (21.8% ± 10 vs. 72.8% ± 6.8; Figure 3D) within the first 7 days post I/R injury. Additionally, the bone marrow exhibited consistently lower proportions of anti-inflammatory Ly6C^low monocytes within the first 7 days after I/R injury compared to the baseline (78.1% ± 10.2 vs. 21.2% ± 4,68, p ≤ 0.0001; Figure 3G).

In the blood, there was a marked increase observed in the proportion of platelet-neutrophil-complexes (PNCs) of the total neutrophils following I/R injury (16.8% ± 2.9 vs. 30.6% ± 4.7; Figure 4E). Additionally, changes were observed in the AAR (11.9% ± 2.6 vs. 21.6% ± 2.5; Figure 4A). However, when examining pro- and anti- inflammatory PNC subtypes, identified by neutrophil surface antigen CD206, GMFI, defined CD206⁻ as proinflammatory (N1) and CD206⁺ as anti-inflammatory (N2) neutrophils. At day 7 after I/R injury a significant increase in CD206-GMFI was detected on N2 PNCs in the AAR (GMFI 861 ± 128 vs. 2,395 ± 332, p ≤ 0.05; Figure 4B). Similarly, platelet–monocyte-complexes (PMCs) were identified in both, the AAR and blood (Figures 4C,F). Here also anti -inflammatory Ly6C^low PMCs were distinguished.

In the AAR, a statistically significant reduction in the proportion of anti-inflammatory Ly6C^low. PMCs was observed on day 3 post I/R injury (42.1% ± 2.2 vs. 18.5% ± 3.5, p ≤ 0.05; Figure 4D). In blood, clear changes of PMC proportions were also observed, although not statistically significant (18.8% ± 5.7 vs. 36.1% ± 8.2; Figure 4F).

To confirm the success of the surgery to induce myocardial I/R injury, echocardiographic measurements, including EF and FS, were conducted with the studied animals. A significant reduction in the EF was observed 1 day post I/R injury as compared to baseline, with the decline persisting over the whole observation period of 7 days (62.5% ± 2.7 vs. 37.2% ± 1.2–38.1% ± 2.0, p ≤ 0.0001; Figure 5A). Similarly, FS was significantly reduced in mice following I/R injury compared to baseline levels and remained diminished throughout 7 days post I/R injury (23.9% ± 0.8 vs.15.7% ± 1.5%–15.0% ± 0.5, p ≤ 0.0001; Figure 5B).

Within the first 3 days after I/R injury, there is a continuous increase in neutrophils in the AAR, reaching a significant maximum on the third day, Figure 2A. Neutrophils, stimulated by pro-inflammatory cytokines migrate into the AAR via diapedesis and undergo phagocytosis before apoptosis (7, 29). While neutrophil recruitment typically peaks within 24 h of inflammation, I/R injury demonstrates distinct dynamics with an early increase in neutrophil proportions at day 1 post I/R injury and sustained elevations throughout 3 days (29, 30). The persistence of neutrophils aligns with rising leukocytes in AAR and is likely driven by excessive cytokine release, characteristic for reperfusion injury, establishing a feedback loop that amplifies neutrophil recruitment to the AAR (3, 31, 32). Another possible reason for the persistent increase in neutrophils is their prolonged lifespan. Previous data suggested that neutrophils in mice and humans have a lifespan of just 1.5–8 h (5, 33). However, more modern methods have made it possible to measure neutrophils in vivo. A study measured the lifespan of neutrophil granulocytes as up to 12.5 h in mice and up to 5.4 days in humans (33, 34). It has also been demonstrated that activated and primed neutrophils that reach the site of inflammation have a longer lifespan (35, 36). Furthermore, it has been demonstrated, that fully mature circulating neutrophils can proliferate further. These cells also have the ability to proliferate, prime and persist in inflamed tissue (10, 35, 36). Once in the tissue, activated neutrophils generate high levels of reactive oxygen species, secrete myeloperoxidase (MPO) and proteases, thereby exacerbating local vascular and tissue damage (10). The result is the infiltration of monocytes, which phagocytose tissue debris. Apoptotic neutrophils, thereby activating polarised macrophages (12). These processes form the basis of the necessary scar formation. Neutrophils play a key role in cardiac healing, but an unbalanced and exaggerated immune response after I/R exacerbates tissue damage. Unbalanced and exaggerated immune response after I/R exacerbates tissue damage, leading to maladaptive remodeling (11, 13). Our data also show a significant increase in the proportion of neutrophils in the spleen 1 day after I/R injury, suggesting its involvement in neutrophil recruitment Figure 2C (37, 38). In contrast, the bone marrow displayed a uniform distribution of neutrophils over the duration of our experiment, indicating a balanced turnover of cell degradation, recruitment, and production Figure 2D. These findings suggest that both the bone marrow and spleen act as key reservoirs for neutrophils. The bone marrow continuously produces neutrophils from myeloid progenitor cells, a process regulated by factors such as Granulocyte-Colony Stimulating Factor (G-CSF), Interleukin 17A (IL-17A), and Interleukin 23 (IL-23) (39). G-CSF is the main regulator of granulopoiesis, encouraging the growth, development, and movement of neutrophils from myeloid progenitor cells in the bone marrow (40). IL-17A enhances the production of G-CSF and chemokines by stimulating stromal and myeloid cells, thereby indirectly boosting neutrophil production and recruitment. IL-23 has an indirect effect on granulopoiesis by promoting the expansion of Th17 cells, which activate the IL-17A/G-CSF axis (41). Furthermore, neutrophils themselves contribute to hematopoietic production by releasing Tumor Necrosis Factor-alpha (TNF-α), which stimulates vascular and hematopoietic regeneration in the bone marrow (40). Neutrophils also undergo breakdown in the bone marrow (41). Monocytes are also recruited to the AAR, peaking alongside neutrophils Figures 2E–H. These monocytes, likely originating from the bone marrow and spleen, play a key role in phagocytosing tissue debris and apoptotic neutrophils. These processes also activate polarized macrophages (12). The kinetics of monocyte recruitment follow a similar pattern to the observed neutrophils. Monocytes also show a significant increase in bone marrow, observed as early as 24 h post I/R injury, peaking at day 3, Figure 2H. This suggests that monocytes are newly formed and released from the bone marrow in response to I/R injury.

The spleen contributes to the early monocyte recruitment within 24 h post I/R injury, Figure 2G. It is estimated, that the spleen contributes approximately 50% of the monocytes, that migrate into the infarcted myocardium (42).

Together, these findings underscore the pivotal roles of neutrophils and monocytes in I/R injury, where a dysregulated immune response exacerbates tissue damage and contributes to maladaptive cardiac remodeling. The bone marrow and spleen play essential roles in the mobilization and clearance of activated leukocytes, as evidenced by our observations across the analyzed time points. Notably, neutrophils emerge as integral players in cardiac repair; however, their disproportionate and overactive immune response following I/R injury amplifies tissue damage and drives maladaptive remodeling (11, 13).

Upon closer examination N1 and N2 neutrophils were differentiated based on their surface expression of the antigen CD206. CD206 expression on neutrophils in the AAR tended to increase on day 7 post I/R injury, Supplementary Figure S1, indicating a phenotypic change of the present neutrophils into an N2 neutrophil subtype. This functional and phenotypic plasticity, as observed in our data, aligns with earlier descriptions of neutrophil alterations (43). It suggests, that neutrophils are not just important during inflammation, cytokine release, cell death and phagocytosis, but also play an important role in regeneration of the damaged tissue. We assume that activated neutrophils exhibit a longer lifespan (33, 35), Figure 2A, and therefore are capable of undergoing a phenotypic change, redefining their relevance in inflammation and repairment after I/R injury. Furthermore, depletion of neutrophils does not appear to be a rational approach to prevent excessive inflammation. Recent echocardiographic data from neutrophil—depleted mice show impaired myocardial repair, with reduced contractility and increased collagen deposition, leading to fibrosis (9). These findings highlight the therapeutic potential of targeting the inflammatory phase and modulating N1 neutrophils to mitigate reperfusion injury, supported by neuroprotective effects of N2 neutrophils in neurological studies (44). Like neutrophils, monocyte subtypes were differentiated based on their inflammatory profiles. pro-inflammatory Ly6C^high monocytes showed a significant increase in the AAR within 24 h post I/R injury, Figure 3A. These cells produce cytokines, facilitate proteolytic degradation of necrotic material, and phagocytose cellular debris (14). Over time, pro-inflammatory Ly6C^high monocytes transition into anti-inflammatory Ly6C^low monocytes, promoting the differentiation of regenerative Ly6C^low F4/80⁺ macrophages essential for tissue repair (45). In the AAR, anti-inflammatory Ly6C^low monocyte proportion significantly increased by day 3 after I/R injury, peaking at day 7, Figure 3E. This transition mostly reflects the differentiation of resident pro-inflammatory Ly6C^high monocytes, whereas the recruitment through blood is playing a minor role, as supported by kinetic data, Figure 3F. Unlike findings in the spleen, Figures 2D,H, no significant differentiation was observed in splenic pro-inflammatory Ly6C^high and anti-inflammatory Ly6C^low monocyte subtypes. However, bone marrow analysis revealed distinct kinetics, with pro-inflammatory Ly6C^high and anti-inflammatory Ly6C^low monocytes stabilizing within 7 days post I/R injury, Figures 3C,G. This suggests that monocyte subtypes recruited to the AAR partly originate from the bone marrow (45, 46).

The data indicate that neutrophils remain in the AAR longer than previously assumed. Importantly, anti-inflammatory and regenerative functions are evident not only in monocytes and macrophages but also in neutrophils. This suggests that the extent of reperfusion injury is influenced more by the phenotypic characteristics of recruited cells than by their overall numbers. Our findings further validate the dynamic kinetics and phenotypic transitions of monocytes in the AAR described in the literature (46, 47).

The shift from pro-inflammatory Ly6C^high to anti-inflammatory Ly6C^low monocytes three after post I/R injury, along with normalization by day 7, is consistent with established models. Moreover, our results highlight the role of the bone marrow in supporting these transitions and delivering functionally altered monocytes to the affected tissue.

This underscores the importance of understanding immune cell subtypes in developing targeted therapies for I/R injury.

Our data confirm the presence and kinetic change of PNCs and PMCs in the AAR (Figure 4). While these complexes do not show significant changes at first glance, further analysis reveals important trends in functional phenotypes. Notably, the surface expression of the neutrophil—specific antigen CD206 increases significantly in PNCs 7 days post I/R injury (Figure 4B), suggesting an elevated presence of N2 neutrophils, some bound to platelets. This trend indicates that N2 PNCs might increase further at later time points, such as 10–21 days post I/R injury. Despite this potential, current evidence suggests that PNCs themselves have no direct functional impact on ischemic myocardium after I/R (48). Following I/R injury, platelets are activated alongside leukocytes (49, 50). Activated neutrophils express the P-selectin glycoprotein ligand-1 (PSGL-1), which binds to P-selectin on platelets, triggering intracellular signaling cascades that enhance Reactive Oxygen Species (ROS) production (51, 52). This interaction promotes Neutrophil Extracellular Trap (NET) formation, observed in acute myocardial infarction, which contributes to micro thrombosis and may worsen myocardial no-reflow after I/R injury (53, 54). Additionally, platelet—neutrophil binding activates integrins, such as Lymphocyte Function Associated Antigen 1 (LFA-1) and Macrophage -1- Antigen (Mac-1), which mediate adhesion to endothelial molecules like the Intracellular Adhesion Molecule 2 (ICAM-2), promoting neutrophil extravasation into the AAR (55). Platelets act as “binding bridges,” decelerating neutrophils and facilitating their transmigration into affected tissues via direct and indirect interactions with fibrinogen, GPIbα, and P-selectin (21, 55–57). Activated platelets interact with monocytes via P-selectin and glycoprotein Ib platelet subunit alpha (GPIb), triggering monocyte activation, degranulation, and transmigration into the AAR (18, 58–60). This also activates platelets to release Platelet Factor 4 (PF4) and Regulated And Normal T cell Expressed an Secreted (RANTES), enhancing monocyte adhesion and prolonging their lifespan, while PF4 stimulates macrophage inflammatory protein 1 alpha (MIP-1α), promoting monocyte differentiation into proinflammatory Ly6C^high macrophages (61–64).

In our data, PMCs were observed in both, the AAR and blood, Figures 4C,F. While no statistically significant changes were detected overall, anti-inflammatory Ly6C^low PMCs show a significant percentage decrease in the AAR during the first 7 days after I/R injury, Figure 4D. This reduction may reflect increased adherence of these complexes to the vascular endothelium, facilitating monocyte migration into the AAR rather than accumulation within the tissue or circulation.

Taken together, these findings suggest that the impact of PNCs and PMCs on myocardial regeneration and scarring may arise not from the complexes themselves but from their role in facilitating leukocyte infiltration and differentiation. It is conceivable that these complexes represent a transient intermediate state, reflecting a dynamic step in the immune cascade. Given the highly time-dependent nature of post-I/R immune responses, the detection of such complexes at specific time points may capture only a narrow but biologically meaningful window. Understanding these interactions may offer promising avenues for modulating the inflammatory response and mitigating reperfusion injury.

To place the cellular data in a clinically meaningful context and to confirm the technical success of the I/R procedure, echocardiography was performed at all time points studied. This allowed parallel assessment of myocardial kinetics, mobility and function. At the time of neutrophil and monocyte invasion into the area at risk (AAR) (Figures 2A,E) and during the dominance of pro-inflammatory subtypes within the myocardial tissue (Figure 3A), myocardial function, as represented by EF and FS, was significantly impaired compared to baseline from day 1 to day 7 post I/R injury (Figures 5A,B). As expected, this decline is consistent with previous reports (65). While these findings provide functional support for the observed temporal immune cell dynamics, a direct mechanistic link between cellular composition and myocardial function cannot be established at this time. Further studies are warranted to clarify the relationship between immune cell kinetics and functional cardiac recovery.

I/R injury triggers systemic immune activation, marked by neutrophil and monocyte infiltration into the AAR. Neutrophil proportions peak by day 3, exacerbating tissue damage via reactive oxygen species and protease release, while monocyte proportions, recruited through neutrophils, clear debris and transition to anti-inflammatory phenotypes to support repair. We could show that anti-inflammatory and regenerative functions are evident, not only in monocytes and macrophages, but also in neutrophils. Echocardiographic analysis shows impaired cardiac function, with reduced EF and FS within the first 24 h after I/R injury. During this periode we observed high levels of proinflammatory leukocytes and peak inflammation. PNCs and PMCs facilitate leukocyte migration and differentiation, indirectly influencing myocardial repair.