Abstract
Obesity is a recognized risk factor for cardiovascular morbidity and mortality, however its impact on survival following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) remains controversial. Therefore, the aim of this study was to evaluate the feasibility of a preclinical model of CA/CPR in obese rats as well as 72-hour (h) survival, and investigate the effect of obesity on myocardial dysfunction post-CA/CPR. Rats were subjected to 6 min of untreated ventricular fibrillation and to 6 min of CPR. Hemodynamic parameters were continuously recorded up to 4 h, serial blood samples were collected and echocardiography was performed at 2 and 4 h after resuscitation. Rats were sacrificed 72 and 4 h after resuscitation. After CA, no obese rats survived up to 72 h compared to controls whereas each rat developed marked post- return of spontaneous circulation hemodynamic impairment during the 4 h observation; obese rats showed an impairment in diastolic function as well as a significant increase in troponin T plasma concentration compared to controls. This could support that obesity negatively impacts survival, hemodynamic stability and left ventricular function after CA.
Introduction
In Europe, approximately 275,000 out-of-hospital cardiac arrests (OHCA) occur each year1. While immediate, high-quality cardiopulmonary resuscitation (CPR) can double or triple the chances of survival, the overall survival rate to hospital discharge remains strikingly low, i.e. approximately 10%2. Cardiac arrest (CA) frequently occurs in the presence of comorbidities, which can significantly worsen patient outcomes. Among these, obesity poses significant challenges3. Although registry studies indicate the presence of obesity in 6% of patients experiencing OHCA4, the true prevalence may be higher as obesity has increased dramatically in recent decades, reaching global epidemic proportions5. Obesity is linked to various metabolic alterations, such as hypertension, abnormal glucose metabolism and dyslipidemia, which directly contribute to the development of cardiovascular disease and sudden cardiac death (SCD)6. The specific mechanisms leading to CA in obese patients are not fully elucidated but include both electrical and structural cardiac remodeling7. Although the link between obesity and an increased risk of cardiovascular disease and mortality is well-established, the effect of obesity on survival and functional outcome following CPR remains a subject of debate8. Indeed, obesity can influence both the efficacy of resuscitation and post-resuscitation efforts4. The increased body mass can make chest compressions less effective due to the difficulty in achieving adequate compression depth and chest recoil. Airway management can also be complicated by anatomical changes associated with obesity, such as a larger neck circumference and increased soft tissue, which can hinder intubation and ventilation9. Furthermore, vascular access may be more difficult to obtain, delaying the administration of medications10. These factors suggest that obese patients may experience similar or worse outcomes compared to non-obese patients following OHCA4. Conversely, other studies suggest that obese patients may have better survival rates post-CA, creating the concept known as the “obesity paradox"11,12. Several hypotheses have been proposed to explain this paradox, suggesting that obese patients may have a greater metabolic reserve, which can provide a survival advantage during critical illness13,14,15. Additionally, adipose tissue may have protective effects, such as buffering against the harmful effects of circulating toxins and inflammatory mediators. Furthermore, obese patients often receive more aggressive medical management due to their higher perceived risk, which may contribute to improved outcomes16.
In this context, preclinical research clarifying the impact of obesity on CA/CPR outcomes is limited. Further, most experimental studies in CA in vivo models are conducted in healthy animals, limiting the understanding of the effect of comorbidities on CPR outcome17. Thus, the present study aims to characterize the impact of obesity on survival and functional outcome in a rat model of CA/CPR. Specifically, the Zucker Fatty (ZF) rats were used in this study, as animal model of obesity. The overall hypothesis was that obese rats would exhibit worse post-resuscitation outcome compared to lean (ZL), non-obese rats.
Results
Study 1
Population characteristics
The table below summarizes the key resuscitation parameters across the three experimental groups (Table 1). CA was induced in 9 male SD, 6 male ZL rats and 4 male ZF rats (22–26 weeks). Of those, 6 SD, 4 ZL and 4 ZF rats obtained ROSC after CA. As expected, ZF rats had a significantly higher body weight compared to both control groups (p < 0.01). Moreover, ZF rats required a greater number of shocks to achieve ROSC (median 3, IQR 3–5), although this difference did not reach statistical significance when compared with SD and ZL rats, which each required a median of only 1 shock (IQR 1–1). In line with this, the total energy delivered to restore circulation was significantly higher in obese rats (p < 0.05) than in ZL and SD animals. Resuscitation efforts also lasted considerably longer in ZF rats (median 8 min, IQR 5–13) compared with the much shorter duration observed in SD and ZL groups (both median 2 min, IQR 2–2). Despite these prolonged and more demanding resuscitation efforts, all animals were successfully resuscitated. However, none of the ZF rats survived to 72 h post-ROSC, whereas survival at this time point was achieved by 3/6 (50%) of SD rats and 3/4 (75%) of ZL rats. Neurological deficit score (NDS) was assessed daily after ROSC to evaluate the overall neurological performance of the animals. ZF rats exhibited markedly worse outcomes compared to both control groups.
Zucker fatty rats exhibited the poorest survival, with a steep decline within the first 24 h and no animals surviving beyond 48 h. In contrast, ZL and SD rats showed a more favorable outcome, surviving until 72 h post ROSC. In particular, ZL group showed no mortality during the first 24 h and a stable survival rate of approximately 75% maintained up to 72 h. SD rats displayed an initial decline within the first 12–24 h, resulting in around 50% survival at 72 h. Overall, these findings highlight that obese Zucker rats experienced significantly worse post-CA/CPR outcomes compared to both Lean and SD rats (Fig. 1).
Kaplan-Maier survival curve of SD, ZL and Zucker fatty rats. Log rank test, p = 0.05.
Hemodynamic
At baseline, no significant differences were observed between obese, ZL and SD rats for all hemodynamic parameters. After ROSC, all rats showed a significant reduction in heart rate (HR), mean arterial pressure (MAP), systolic arterial pressure (SAP), diastolic arterial pressure (DAP) compared to baseline, and coronary perfusion pressure (CPP) (ANOVA p < 0.001 for both) (see Figure S1 A for MAP values during CPR). At 2 and 4 h post-ROSC, HR was significantly reduced in ZF rats compared to both SD and ZL rats. MAP, SAP and DAP were also significantly lower at 2 and 4 h post-ROSC in ZF rats, compared to controls. After resuscitation, CPP was significantly decreased at 1, 2 and 4 h post-ROSC in ZF rats compared to both ZL and SD groups (Fig. 2). To note, no significant differences in hemodynamic parameters and in left ventricular ejection fraction (Baseline: SD 80.5% vs. ZL 81.2%; 72 h: SD 73.1% vs. ZL 81.8%) were observed between ZL and SD rats, indicating that they were comparable and therefore can be both used as controls in this experimental setting.
Hemodynamic parameters. HR heart rate, MAP mean arterial pressure, SAP systolic arterial pressure, DAP diastolic arterial pressure, CPP coronary perfusion pressure. SD (n = 6), ZL (n = 4) and ZF (n = 4). Data are shown as mean ± SD. Mixed-effects model followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 ZF vs. SD; ° p < 0.05, °° p < 0.01 ZF vs. Zucker Lean rats.
Study 2
Population characteristics
CA was induced in 6 SD rats and 5 ZF rats. Also in this case, ZF rats had a significantly higher body weight compared to the control groups (p < 0.001). we did not observed any difference in terms of number of shocks to achieve ROSC (SD median 3, IQR 2–4; ZF median 2, IQR 1–2 ), and for total energy required to obtain ROSC (SD median 5, IQR 4–8; ZF median 5, IQR 3–6). In line with this, time to ROSC was equal between the two experimental groups. All animals were successfully resuscitated and all of them survived up to 4 h after CA (100%) (Table 2).
Hemodynamic
At baseline, no significant differences were observed between obese and SD rats for all hemodynamic parameters. After ROSC, all rats showed a significant reduction in HR, MAP, SAP and DAP compared to baseline, and CPP (ANOVA p < 0.001 for both) (see Figure S1 B for MAP values during CPR). At 2 and 3 h post-ROSC, HR was significantly reduced in ZF rats compared to SD rats. MAP, SAP and DAP were also significantly lower starting from 1, 2 until 4 h post-ROSC in ZF rats, compared to control group. After resuscitation, CPP was decreased at 1, 2, 3 and 4 h post-ROSC in ZF rats compared SD groups, although not significantly (Fig. 3).
Hemodynamic parameters. HR heart rate, MAP mean arterial pressure, SAP systolic arterial pressure, DAP diastolic arterial pressure, CPP coronary perfusion pressure. SD (n = 6) and ZF (n = 5). Data are shown as mean ± SD. Mixed-effects model followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 SD vs. Zucker Fatty.
Echocardiography
All echocardiographic parameters of SD and ZF rats were shown supplementary material (Table S1). ZF animals showed no significant differences in ejection fraction (EF) and fractional shortening (FS) at baseline as well as left ventricular internal dimension systolic (LVIDs) and left ventricular internal dimension diastolic (LVIDd) (Fig. 4). Moreover, no significant differences were observed in diastolic function parameters (Fig. 5) as well as cardiac output (CO) and stroke volume (SV, Fig. 6).
At 2 and 4 h post-ROSC, rats showed a significant decrease in EF and FS compared to baseline (ANOVA p value < 0.001 for both), while LVIDs and LVIDd were significantly increased after ROSC (ANOVA p < 0.001) (Fig. 4). Post-ROSC early diastolic mitral inflow velocity (E vel) and late diastolic mitral inflow velocity (A vel) were also significantly lower compared to baseline after resuscitation (ANOVA p < 0.05), and mean of septal and lateral TDI e’ velocity (e’ mean) displayed the same trend. No differences in these echocardiographic parameters were observed between SD and ZF rats. By contrast, mitral E waves velocity ratio on tissue Doppler (E/e’) did not vary shortly after resuscitation in both strain of rats, although at 4 h after ROSC, e/e’ was significantly higher in ZF rats compared to SD controls (E/e’. ZF vs. SD: +35%; p < 0.05). Deceleration time (DT) showed the same trend of E/e’ (p < 0.05, + 68%, Fig. 5).
At 2 and 4 h post-ROSC, CO and SV were significantly decreased compared to baseline in both ZF and SD rats (ANOVA p < 0.0001 for both). Interestingly, ZF rats showed a significant 50% reduction in both variables compared to SD rats (CO: p < 0.01 at both times point; SV: p < 0.05 at both times point, Fig. 6).
HR heart rate, EF ejection fraction, FS fractional shortening, LVIDs left ventricular internal dimension systolic, LVIDd left ventricular internal dimension diastolic. SD (n = 6) and ZF (n = 5). Data are shown as mean ± SD. Mixed-effects model followed by Sidak’s multiple comparison test.
E vel, early diastolic mitral inflow velocity; A vel, late diastolic mitral inflow velocity; DT, deceleration time; E/e’, E waves velocity ratio; e’ mean, mean of septal and lateral e velocities. SD (n = 6) and ZF (n = 5). Data are shown as mean ± SD. Mixed-effects model followed by Sidak’s multiple comparison test. * p < 0.05, ** p < 0.01 vs. SD.
CO, cardiac output and SV, stroke volume. SD (n = 6) and ZF (n = 5). Data are shown as mean ± SD. Mixed-effects model followed by Sidak’s multiple comparison test. * p < 0.05, ** p < 0.01 vs. SD.
Cardiac circulating biomarker
Post-resuscitation high sensitivity cardiac troponin-T (hs-cTnT) significantly increased in both strain of rats (ANOVA p value < 0.001). At 2 h post-ROSC, hs-cTnT plasma concentration was significantly higher in ZF rats to SD ones (Fig. 7).
Plasma levels of hs-cTnT after CA/CPR. SD (n = 6) and ZF (n = 5). Two-way Anova followed by Tukey’s multiple comparison test on log transformed concentration. Data are shown as median and interquartile range, ** p < 0.01 vs. SD.
Discussion
In this study, we sought to investigate the specific impact of obesity on post-resuscitation myocardial dysfunction. To this end, Study 1 served as an initial investigation to evaluate the feasibility of CA and CPR model in ZF rats. The findings highlighted a markedly reduced survival in obese animals, with no rats surviving beyond 48 h after ROSC. Additionally, no significant differences in hemodynamic parameters and in LVEF were observed between ZL rats and SD rats, supporting the use of SD rats as a suitable control group. Building on these observations, Study 2 was specifically designed to investigate the role of obesity in left ventricular dysfunction in the early post-resuscitation phase. Given the comparable hemodynamic and systolic function profiles between ZL and SD rats observed in Study 1, and considering the greater availability and lower cost of SD rats, we selected SD animals as controls for this second study.
In literature, contrasting findings suggest a complex interplay between obesity, resuscitation effectiveness and CA outcomes. Berrington de Gonzalez et al. demonstrated that overweight individuals exhibited increased overall mortality, potentially due to the anatomical effects of obesity on the efficacy of chest compressions and resuscitation maneuvers. Specifically, they suggested that excess adipose tissue may hinder effective chest compression depth, thereby compromising resuscitation quality18. Conversely, several studies have reported a paradoxical association between overweight status and improved short-term survival after CA. Ma and collaborators and Geri and collaborators both indicated that overweight patients experiencing CA had more favorable clinical outcomes19,20. Similarly, Testori and colleagues found that overweight subjects displayed better survival and neurological outcomes compared to normal-weight patients21. This was clearly not the case in our model, in which obese animals presented an impaired post-resuscitation myocardial function, higher release of hs-cTnT and poor survival, compared to normal-weighted rats, suggesting that other variables beyond subject susceptibility—such as differences in treatment intensity or resuscitation strategies—may also influence survival outcomes in obese patients.
The results from the proposed preclinical animal model might elucidate the discordance in previous studies. In this controlled experimental setting, characterized by homogeneous population and standardized resuscitation protocol, CPR was effective in both groups, yet no protective effect of obesity was observed. A slightly decrease in CPP and increased defibrillation energy to achieve ROSC were observed in obese animals compared to controls. Moreover, none of the obese rats survived beyond 72 h, whereas more than 50% of the control group did, further indicating the absence of a survival advantage associated with obesity. The previously reported potential for improved survival in overweight individuals may instead be attributed to differences in patient populations, variations in resuscitation protocols, or the specific criteria used to define overweight and obesity3.
A marked hemodynamic impairment in terms of reduced MAP and right atrial pressure (RAP) was observed in ZF rats compared to controls both during CPR and following ROSC. This is consistent with existing evidence, suggesting that the obese heart is prone to a series of hemodynamic disorders, ultimately leading to ventricular remodeling and an increased risk of arrhythmogenesis22. Indeed, Yao and colleagues highlighted that chronic obesity induces structural and functional alterations in the myocardium, including increased left ventricular mass, fibrosis, and impaired diastolic function, which can cause hemodynamic instability following CA22. These obesity-related cardiac changes could have explained the sustained reduction in arterial pressure and CPP observed in our ZF rats, finally contributing to the poor outcome.
Transthoracic echocardiography is a non-invasive, reliable tool for monitoring LV in rat models of CA/CPR23. We performed a complete assessment of the LV function and structure in obese rats after CA/CPR. At baseline, no significant differences between obese animals and SD rats were observed, suggesting that obesity does not impact LV function. Vammen and collaborators recently investigated cardiac function in an asphyxia-induced CA model in Zucker diabetic fatty rats24. In line with our results, the authors did not demonstrate any impairment in FS in diabetic obese rats compared to SD controls. However, they reported smaller LV diameters and increased wall thickness in diabetic obese rats compared to SD controls 3 h after ROSC. These structural changes might be partly attributed to the long-term effects of diabetes on LV morphology and function, as previously described25. Likely, we did not observe the above effects, since our rats underwent CA prior to diabetes onset.
Although we did not observe significant differences in systolic function between obese and control animals, hs-cTnT levels were significantly elevated shortly after ROSC in the ZF group compared to SD rats. Our findings suggest that obese rats experienced a greater degree of myocardial injury following CA/CPR, likely reflecting their increased vulnerability to ischemia-reperfusion injury24, and explaining the high mortality observed in the long-term study.
Following resuscitation, diastolic function was however impaired in obese rats compared to controls. Previous research showed that diastolic dysfunction is a prominent feature both in the early and late phases after resuscitation in rats23. Also, in a clinical study, Jentzer and collaborators emphasized the prognostic value of early LV diastolic dysfunction as a predictor of long-term mortality following OHCA26. To our knowledge, this is the first comprehensive assessment of post-resuscitation LV diastolic function in obese animals. Our findings underscore the critical role of LV diastolic function in shaping outcomes after CPR, particularly in the context of obesity.
In addition, LV CO and SV were significantly reduced in obese rats compared to controls following resuscitation. Traditionally, obesity is associated with increased total blood volume and elevated resting CO, leading to left ventricular dilation and hypertrophy. This reflects a general correlation between body size and higher baseline CO and SV27. In our study, CO and SV declined significantly shortly after resuscitation23. Notably, obesity appeared to exacerbate this decline, likely due to an increased vulnerability to myocardial ischemia-reperfusion injury22.
Strengths and limitations
Obese rats exhibited reduced survival after CA, which was associated with hemodynamic impairment and myocardial injury. While these factors likely contributed, we did not investigate other potential mechanisms that may also play a role. Indeed, we performed a comprehensive assessment of LV function and structure combining hemodynamic and echocardiographic variables together with measurement of circulation hs-cTnT, allowing for a detailed characterization of myocardial injury in the presence of a comorbidity such as obesity in a clinically relevant rat model of CA/CPR. The inclusion of multiple control groups further strengthens the validity of our findings, ensuring that the observed effects are specifically related to obesity rather than other confounding factors. Moreover, this is the first study to use a VF induced rat model of CA/CPR to evaluate the effects of obesity on survival and myocardial injury after CA/CPR. Indeed, this experimental model leads to highest degree of myocardial injury compared to the asphyxia induced one28,29, making our findings more translatable to patients.
The following limitations must be considered. The small number of animals per time point from each experimental group limits the ability to detect a more pronounced difference in myocardial dysfunction shortly after resuscitation. Future studies should consider our observations on low survival rates of obese rats in this CA/CPR rat model to confirm our results. Furthermore, the high mortality rate of obese rats prevented the evaluation of neurological outcome, which is central to the assessment of post-CA outcome. Additionally, underpinning mechanisms such as altered metabolic state (Figure S2) and dysregulated inflammation that may contribute to worse outcomes still need to be addressed in future research. In preclinical studies where obesity is studied as a comorbid factor of a disease such as heart failure, diabetes and CA/CPR, ZL rats are commonly employed as a controls, due to their genetic similarity to obese ZF rats. However, due to cost effectiveness evaluation and observed similar post resuscitation hemodynamic effects to SD rats, we chose to use SD as control animals.
Conclusions
Obese rats demonstrated reduced survival rates, impaired hemodynamic, and diastolic dysfunction, while maintaining normal systolic function. Contrary to the debated “obesity paradox,” our findings indicate that obesity is associated with worse outcomes following cardiac arrest and resuscitation. This study underscores the importance of considering obesity as a critical factor in post-resuscitation care and outcome prediction, with potential implications for clinical management strategies in obese patients. Furthermore, establishing a preclinical CA/CPR model that incorporates obesity—a major comorbidity—lays the groundwork for future research utilizing more relevant animal models better reflecting the human pathology.
Materials and methods
Ethics declaration
Ethical approval to animal procedures was obtained from the Italian Ministry of Health (approval no. 210/2010-B, issued on November 11th, 2010). All procedures regarding the study design, animal experiments, statistical analysis, and data reporting fulfil the criteria of the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://www.nc3rs.org.uk/arrive-guidelines) check list provided in supplemental materials. All methods were performed in accordance with the relevant guidelines and regulation.
Animals
SD, ZF and ZL rats (Harlan Laboratory, Italy) were used in this study. ZF rats are the most widely used animal model for studying obesity-related pathologies. Their genotype is characterized by a homozygous mutation in the leptin receptor gene, resulting in dysfunctional leptin signaling, which leads to hyperphagia, obesity, hyperinsulinemia, hyperlipidemia, and insulin resistance, typically evident by 4–5 weeks of age30,31. ZL rats carry at least one normal allele, display normal body weight, appetite, and metabolic parameters, and serve as healthy controls.
Experimental design
Study 1
The aim of this study was to evaluate feasibility and survival after CA/CPR in the obese rat. The experimental design is illustrated in Fig. 8. Nine male SD rats, 6 male ZL rats, and 4 male ZF rats were subjected to CA/CPR and monitored for up to 4 h. Animals that survived beyond this period were subsequently followed until 72 h and then sacrificed.
Experimental design study 1. VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; ECG, electrocardiogram.
Study 2
The aim of this study was to investigate left ventricular dysfunction after CA/CPR in obese rats. The experimental design is described in Fig. 9. Si × SD rats and 5 ZF rats were subjected to CA/CPR and sacrificed 4 h after resuscitation.
Experimental design study 2. VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; ECG, electrocardiogram.
Animal preparation
An established model of electrically induced CA/CPR32 was used. In brief, animals were anesthetized by intraperitoneal (IP) injection of thiopental (50 mg/kg). Additional doses of thiopental (10 mg/kg) were given at intervals of approximately 40 min or when required to maintain anaesthesia. Ampicillin (50 mg/kg) was injected intramuscularly (IM) as prophylaxis after induction of anaesthesia, and buprenorphine (0.16 mg/kg) was given to prevent infection and pain. The trachea was orally intubated with a 14-gauge cannula. A PE-50 catheter (Becton Dickinson, Franklin Lakes, NJ) was advanced into the descending aorta from the left femoral artery for measurement of arterial pressure and sampling arterial blood. Through the left external jugular vein, another PE-50 catheter was advanced into the right atrium for measurement of right atrial pressures. Aortic and right atrial pressures were measured with reference to the mid chest with high-sensitivity transducers. A 3-F PE catheter (model C-PMS-301 J, Cook Critical Care, Bloomington, IN) was advanced through the right external jugular vein into the right atrium. A pre-curved guide wire supplied with the catheter was then advanced through the catheter into the right ventricle and confirmed by endocardial electrocardiogram for inducing VF. All the catheters were flushed intermittently with saline containing 2.5 IU/ml of bovine heparin. A conventional lead II ECG was continuously monitored. Temperature was continuously monitored with the aid of a rectal probe and maintained at 37 ± 0.5 °C throughout the experiment.
Experimental procedures
Fifteen mins prior to inducing VF, baseline measurements were obtained, and mechanical ventilation was initiated with an inspired FiO2 of 0.21. VF was electrically induced with progressive increases in 60-Hz current to a maximum of 4 mA delivered to the right ventricular endocardium. The current flow was continued for 3 min to prevent spontaneous defibrillation. Chest compression was begun after 6 min of untreated VF with a pneumatically driven mechanical chest compressor as previously described32. After 6 min of CPR, rhythm analysis was performed. If a shockable rhythm was detected, defibrillation was attempted, with up to three consecutive shocks delivered (CodeMaster XL, Philips Heartstream, Seattle, WA). ROSC was defined as the presence of sinus rhythm accompanied by a MAP greater than 25 mmHg. If ROSC was not achieved, CPR was resumed for 1 min before the next defibrillation attempt. This protocol was repeated until ROSC was achieved or a maximum of three resuscitation cycles had been completed. In cases where VF recurred after ROSC, immediate defibrillation was performed.
The compression depth was initially set to reduce the anteroposterior chest diameter by 30%, minimizing the risk of visceral injury. Compression depth was then fine-tuned to achieve a target CPP of ≥ 20 mmHg, a threshold previously shown to correlate with successful resuscitation in rats.
Coincident with the start of chest compression, animals were mechanically ventilated at a frequency of 55/mins with a tidal volume 0.6 ml/100 g and a FiO2 of 1.0. Chest compression was maintained at a rate of 200/mins with equal compression-relaxation duration (i.e., 50% duty cycle). A single intra-atrial dose of epinephrine (0.02 mg/kg) was administered 2 min after the start of CPR. No additional doses were given during the resuscitation phase. Following resuscitation, animals were monitored for 4 h. All the catheters and the endotracheal tube were then removed. In the study 1, animals were returned to their cages and were observed for up to 72 h after resuscitation and then sacrificed with an intraperitoneal injection of thiopental (150 mg/kg). In study 2, blood samples were serially collected from the femoral artery cannula in 3 K-EDTA tubes 15 min before CA (pre-CA), 2 and 4 h post-ROSC, and plasma stored at -70 °C prior to perform biochemical analyses. Four h after ROSC, echocardiography was performed, and animals were then killed painlessly with an IP injection of tiopenthal (150 mg/kg).
Measurements
Hemodynamics
Hemodynamics were recorded as previously described32; briefly, ECG, AP and RAP were continuously monitored for up to 4 h after ROSC on a personal computer-based data acquisition system supported by CODAS hardware and software (DataQ, Akron, Oh). CPP was calculated in the same time range as the difference between time-coincident diastolic aortic and right atrial pressures.
Echocardiography
Transthoracic echocardiography (ALOKA SSD-5500, Tokyo, Japan) was done on anesthetized rats 2 and 4 h after resuscitation using a 13 MHz linear transducer at high frame rate imaging (102 Hz) and a 7.5 MHz phased array probe for pulsed-wave and tissue Doppler measurements. Parasternal long-axis and apical four- and five-chamber views were used as well as apical four and five chamber view with color flow and pulsed or tissue Doppler. End-diastolic and end-systolic wall thicknesses, systolic wall thickening, LV internal dimensions and LV volumes were measured and calculated from 2D parasternal short- and long-axis views. LV EF was calculated from parasternal long-axis views by the modified simple plane Simpson’s rule. Mitral E and A velocities together with tissue Doppler velocities were measured from apical four-chamber views. Aortic outflow velocities were measured from an apical five-chamber view by pulsed-wave Doppler and LV CO and SV were calculated.
Echocardiographic recordings were saved on a USB storage device for off-line analysis by a sonographer blind to study groups. All measurements and calculations were taken in three or five consecutive cardiac cycles according to the recommendations of the latest American and European Societies Guidelines for echocardiographic assessment33,34,35 as previously reported23.
High sensitivity cardiac troponin T
Plasma high sensitivity cardiac troponin T (hs c-TnT) levels were assessed at baseline, 2 and 4 h after resuscitation with an electrochemiluminescence assay (ECLIA, Elecsys 2010 analyzer, Roche Diagnostics, Germany).
Neurological deficit score
Neurological Deficit Score (NDS) rated level of consciousness, respiration, motor and sensory functions, and overall behaviour (normal = 0, brain dead = 500)36.
Statistical analysis
The study 1 was a pilot study aimed at implementing an experimental model of CA/CPR in ZF Rats. This preliminary investigation served to refine the methodology, establish the feasibility of the model, and identify optimal temporal time points for analysing myocardial dysfunction and survival outcomes. For this reason, a formal sample size calculation was not applied, as the primary goal of this pilot study was exploratory in nature, focusing on model development and feasibility rather than hypothesis testing.
For Study 2, the sample size was calculated to assess the impact of obesity on myocardial dysfunction following CA/CPR. Plasma levels of hs-cTnT after cardiac arrest were used as the primary endpoint. In this experimental model, early post-CA/CPR plasma hs-cTnT levels in SD rats are approximately 5102 ± 1585 ng/L23. We anticipated a 50% increase in hs-cTnT levels ZF rats. Based on this, a sample size of 6 rats per group was required to achieve a significance level (α) of 0.05 and a power (1–β) of 0.8 (two-sided test).
The GraphPad Prism® program (GraphPad Software, Inc. La Jolla, CA, USA) was used for data processing and statistical analysis. Timebased variables not normally distributed analyzed with Mixed-effects model followed by Tukey’s multiple comparison test. For comparisons of variables with only a time point, Kruskall-wallis with Dunn’s multiple comparison test was used for non parametric variables. P < 0.05 was considered significant.
Data availability
The datasets generated and analyzed during the current study can be found at: https://zenodo.org/communities/irfmn-irccs.
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GR conceived the study. GR and FF performed the experiments and coordinate the study.MC, DDG, AM and FF analysed and interpreted the data. MC and FF drafted the manuscript.GR, ERZ, revised and critically edited the manuscript.All authors read and approved the final manuscript.
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Cerrato, M., De Giorgio, D., Magliocca, A. et al. Impact of obesity on myocardial function and survival after cardiac arrest and cardiopulmonary resuscitation in a rat model. Sci Rep 15, 37316 (2025). https://doi.org/10.1038/s41598-025-21209-w
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DOI: https://doi.org/10.1038/s41598-025-21209-w
