Determinants of the dynamic cerebral critical closing pressure response to changes in mean arterial pressure.

Objective. Cerebral critical closing pressure (CrCP) represents the value of arterial blood pressure (BP) where cerebral blood flow (CBF) becomes zero. Its dynamic response to a step change in mean BP (MAP) has been shown to reflect CBF autoregulation, but robust methods for its estimation are lacking. We aim to improve the quality of estimates of the CrCP dynamic response.Approach. Retrospective analysis of 437 healthy subjects (aged 18-87 years, 218 males) baseline recordings with measurements of cerebral blood velocity in the middle cerebral artery (MCAv, transcranial Doppler), non-invasive arterial BP (Finometer) and end-tidal CO2(EtCO2, capnography). For each cardiac cycle CrCP was estimated from the instantaneous MCAv-BP relationship. Transfer function analysis of the MAP and MCAv (MAP-MCAv) and CrCP (MAP-CrCP) allowed estimation of the corresponding step responses (SR) to changes in MAP, with the output in MCAv (SRVMCAv) representing the autoregulation index (ARI), ranging from 0 to 9. Four main parameters were considered as potential determinants of the SRVCrCPtemporal pattern, including the coherence function, MAP spectral power and the reconstruction error for SRVMAP, from the other three separate SRs.Main results. The reconstruction error for SRVMAPwas the main determinant of SRVCrCPsignal quality, by removing the largest number of outliers (Grubbs test) compared to the other three parameters. SRVCrCPshowed highly significant (p< 0.001) changes with time, but its amplitude or temporal pattern was not influenced by sex or age. The main physiological determinants of SRVCrCPwere the ARI and the mean CrCP for the entire 5 min baseline period. The early phase (2-3 s) of SRVCrCPresponse was influenced by heart rate whereas the late phase (10-14 s) was influenced by diastolic BP.Significance. These results should allow better planning and quality of future research and clinical trials of novel metrics of CBF regulation.

Cerebrovascular Effects of Sildenafil in Small Vessel Disease: The OxHARP Trial.

BACKGROUND: Vascular cognitive impairment due to cerebral small vessel disease is associated with cerebral pulsatility, white matter hypoperfusion, and reduced cerebrovascular reactivity (CVR), and is potentially improved by endothelium-targeted drugs such as cilostazol. Whether sildenafil, a phosphodiesterase-5 inhibitor, improves cerebrovascular dysfunction is unknown. METHODS: OxHARP trial (Oxford Haemodynamic Adaptation to Reduce Pulsatility) was a double-blind, randomized, placebo-controlled, 3-way crossover trial after nonembolic cerebrovascular events with mild-moderate white matter hyperintensities (WMH), the most prevalent manifestation of cerebral small vessel disease. The primary outcome assessed the superiority of 3 weeks of sildenafil 50 mg thrice daily versus placebo (mixed-effect linear models) on middle cerebral artery pulsatility, derived from peak systolic and end-diastolic velocities (transcranial ultrasound), with noninferiority to cilostazol 100 mg twice daily. Secondary end points included the following: cerebrovascular reactivity during inhalation of air, 4% and 6% CO2 on transcranial ultrasound (transcranial ultrasound-CVR); blood oxygen-level dependent-magnetic resonance imaging within WMH (CVR-WMH) and normal-appearing white matter (CVR-normal-appearing white matter); cerebral perfusion by arterial spin labeling (magnetic resonance imaging pseudocontinuous arterial spin labeling); and resistance by cerebrovascular conductance. Adverse effects were compared by Cochran Q. RESULTS: In 65/75 (87%) patients (median, 70 years;79% male) with valid primary outcome data, cerebral pulsatility was unchanged on sildenafil versus placebo (0.02, -0.01 to 0.05; P=0.18), or versus cilostazol (-0.01, -0.04 to 0.02; P=0.36), despite increased blood flow (∆ peak systolic velocity, 6.3 cm/s, 3.5-9.07; P<0.001; ∆ end-diastolic velocity, 1.98, 0.66-3.29; P=0.004). Secondary outcomes improved on sildenafil versus placebo for CVR-transcranial ultrasound (0.83 cm/s per mm Hg, 0.23-1.42; P=0.007), CVR-WMH (0.07, 0-0.14; P=0.043), CVR-normal-appearing white matter (0.06, 0.00-0.12; P=0.048), perfusion (WMH: 1.82 mL/100 g per minutes, 0.5-3.15; P=0.008; and normal-appearing white matter, 2.12, 0.66-3.6; P=0.006) and cerebrovascular resistance (sildenafil-placebo: 0.08, 0.05-0.10; P=4.9×10-8; cilostazol-placebo, 0.06, 0.03-0.09; P=5.1×10-5). Both drugs increased headaches (P=1.1×10-4), while cilostazol increased moderate-severe diarrhea (P=0.013). CONCLUSIONS: Sildenafil did not reduce pulsatility but increased cerebrovascular reactivity and perfusion. Sildenafil merits further study to determine whether it prevents the clinical sequelae of small vessel disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03855332.

A Multi-Parametric Approach for Characterising Cerebral Haemodynamics in Acute Ischaemic and Haemorrhagic Stroke.

BACKGROUND AND PURPOSE: Early differentiation between acute ischaemic (AIS) and haemorrhagic stroke (ICH), based on cerebral and peripheral hemodynamic parameters, would be advantageous to allow for pre-hospital interventions. In this preliminary study, we explored the potential of multiple parameters, including dynamic cerebral autoregulation, for phenotyping and differentiating each stroke sub-type. METHODS: Eighty patients were included with clinical stroke syndromes confirmed by computed tomography within 48 h of symptom onset. Continuous recordings of bilateral cerebral blood velocity (transcranial Doppler ultrasound), end-tidal CO2 (capnography), electrocardiogram (ECG), and arterial blood pressure (ABP, Finometer) were used to derive 67 cerebral and peripheral parameters. RESULTS: A total of 68 patients with AIS (mean age 66.8 ± SD 12.4 years) and 12 patients with ICH (67.8 ± 16.2 years) were included. The median ± SD NIHSS of the cohort was 5 ± 4.6. Statistically significant differences between AIS and ICH were observed for (i) an autoregulation index (ARI) that was higher in the unaffected hemisphere (UH) for ICH compared to AIS (5.9 ± 1.7 vs. 4.9 ± 1.8 p = 0.07); (ii) coherence function for both hemispheres in different frequency bands (AH, p < 0.01; UH p < 0.02); (iii) a baroreceptor sensitivity (BRS) for the low-frequency (LF) bands that was higher for AIS (6.7 ± 4.2 vs. 4.10 ± 2.13 ms/mmHg, p = 0.04) compared to ICH, and that the mean gain of the BRS in the LF range was higher in the AIS than in the ICH (5.8 ± 5.3 vs. 2.7 ± 1.8 ms/mmHg, p = 0.0005); (iv) Systolic and diastolic velocities of the affected hemisphere (AH) that were significantly higher in ICH than in AIS (82.5 ± 28.09 vs. 61.9 ± 18.9 cm/s), systolic velocity (p = 0.002), and diastolic velocity (p = 0.05). CONCLUSION: Further multivariate modelling might improve the ability of multiple parameters to discriminate between AIS and ICH and warrants future prospective studies of ultra-early classification (<4 h post symptom onset) of stroke sub-types.

Effect of drug interventions on cerebral hemodynamics in ischemic stroke patients.

The ischemic penumbra is sensitive to alterations in cerebral perfusion. A myriad of drugs are used in acute ischemic stroke (AIS) management, yet their impact on cerebral hemodynamics is poorly understood. As part of the Cerebral Autoregulation Network led INFOMATAS project (Identifying New Targets for Management and Therapy in Acute Stroke), this paper reviews some of the most common drugs a patient with AIS will come across and their potential influence on cerebral hemodynamics with a particular focus being on cerebral autoregulation (CA). We first discuss how compounds that promote clot lysis and prevent clot formation could potentially impact cerebral hemodynamics, before focusing on how the different classes of antihypertensive drugs can influence cerebral hemodynamics. We discuss the different properties of each drug and their potential impact on cerebral perfusion and CA. With emerging interest in CA status of AIS patients, either during or soon after treatment when timely reperfusion and salvageable tissue is at its most critical, the properties of these pharmacological agents may be relevant for modelling cerebral perfusion accuracy and for setting individualised treatment strategies.

Extremes of cerebral blood flow during hypercapnic squat-stand maneuvers.

Squat-stand maneuvers (SSMs) are a popular method of inducing blood pressure (BP) oscillations to reliably assess dynamic cerebral autoregulation (dCA), but their effects on the cerebral circulation remain controversial. We designed a protocol whereby participants would perform SSMs under hypercapnic conditions. Alarmingly high values of cerebral blood flow velocity (CBFV) were recorded, leading to early study termination after the recruitment of a single participant. One healthy subject underwent recordings at rest (5 min sitting, 5 min standing) and during two SSMs (fixed and random frequency). Two sets of recordings were collected; one while breathing room air, one while breathing 5% CO2 . Continuous recordings of bilateral CBFV (transcranial Doppler), heart rate (ECG), BP (Finometer), and end-tidal CO2 (capnography) were collected. Peak values of systolic CBFV were significantly higher during hypercapnia (p < 0.01), and maximal values exceeded 200 cm.s-1 . Estimates of dCA (ARI) during hypercapnia were impaired relative to poikilocapnia (p = 0.03). The phase was significantly reduced under hypercapnic conditions (p = 0.03). Here we report extremely high values of CBFV in response to repeated SSMs during induced hypercapnia, in an otherwise healthy subject. Our findings suggest that protocols performing hypercapnic SSMs are potentially dangerous. We, therefore, urge caution if other research groups plan to undertake similar protocols.

Cerebral critical closing pressure and resistance-area product: the influence of dynamic cerebral autoregulation, age and sex.

Instantaneous arterial pressure-flow (or velocity) relationships indicate the existence of a cerebral critical closing pressure (CrCP), with the slope of the relationship expressed by the resistance-area product (RAP). In 194 healthy subjects (20-82 years, 90 female), cerebral blood flow velocity (CBFV, transcranial Doppler), arterial blood pressure (BP, Finapres) and end-tidal CO2 (EtCO2, capnography) were measured continuously for five minutes during spontaneous fluctuations of BP at rest. The dynamic cerebral autoregulation (CA) index (ARI) was extracted with transfer function analysis from the CBFV step response to the BP input and step responses were also obtained for the BP-CrCP and BP-RAP relationships. ARI was shown to decrease with age at a rate of -0.025 units/year in men (p = 0.022), but not in women (p = 0.40). The temporal patterns of the BP-CBFV, BP-CrCP and BP-RAP step responses were strongly influenced by the ARI (p < 0.0001), but not by sex. Age was also a significant determinant of the peak of the CBFV step response and the tail of the RAP response. Whilst the RAP step response pattern is consistent with a myogenic mechanism controlling dynamic CA, further work is needed to explore the potential association of the CrCP step response with the flow-mediated component of autoregulation.

The critical closing pressure contribution to dynamic cerebral autoregulation in humans: influence of arterial partial pressure of CO2.

KEY POINTS: Dynamic cerebral autoregulation (CA) is often expressed by the mean arterial blood pressure (MAP)-cerebral blood flow (CBF) relationship, with little attention given to the dynamic relationship between MAP and cerebrovascular resistance (CVR). In CBF velocity (CBFV) recordings with transcranial Doppler, evidence demonstrates that CVR should be replaced by a combination of a resistance-area product (RAP) with a critical closing pressure (CrCP) parameter, the blood pressure value where CBFV reaches zero due to vessels collapsing. Transfer function analysis of the MAP-CBFV relationship can be extended to the MAP-RAP and MAP-CrCP relationships, to assess their contribution to the dynamic CA response. During normocapnia, both RAP and CrCP make a significant contribution to explaining the MAP-CBFV relationship. Hypercapnia, a surrogate state of depressed CA, leads to marked changes in dynamic CA, that are entirely explained by the CrCP response, without further contribution from RAP in comparison with normocapnia. ABSTRACT: Dynamic cerebral autoregulation (CA) is manifested by changes in the diameter of intra-cerebral vessels, which control cerebrovascular resistance (CVR). We investigated the contribution of critical closing pressure (CrCP), an important determinant of CVR, to explain the cerebral blood flow (CBF) response to a sudden change in mean arterial blood pressure (MAP). In 76 healthy subjects (age range 21-70 years, 36 women), recordings of MAP (Finometer), CBF velocity (CBFV; transcranial Doppler ultrasound), end-tidal CO2 (capnography) and heart rate (ECG) were performed for 5 min at rest (normocapnia) and during hypercapnia induced by breathing 5% CO2 in air. CrCP and the resistance-area product (RAP) were obtained for each cardiac cycle and their dynamic response to a step change in MAP was calculated by means of transfer function analysis. The recovery of the CBFV response, following a step change in MAP, was mainly due to the contribution of RAP during both breathing conditions. However, CrCP made a highly significant contribution during normocapnia (P < 0.0001) and was the sole determinant of changes in the CBFV response, resulting from hypercapnia, which led to a reduction in the autoregulation index from 5.70 ± 1.58 (normocapnia) to 4.14 ± 2.05 (hypercapnia; P < 0.0001). In conclusion, CrCP makes a very significant contribution to the dynamic CBFV response to changes in MAP and plays a major role in explaining the deterioration of dynamic CA induced by hypercapnia. Further studies are needed to assess the relevance of CrCP contribution in physiological and clinical studies.

Cerebral autoregulation and response to intravenous thrombolysis for acute ischemic stroke.

We hypothesized that knowledge of cerebral autoregulation (CA) status during recanalization therapies could guide further studies aimed at neuroprotection targeting penumbral tissue, especially in patients that do not respond to therapy. Thus, we assessed CA status of patients with acute ischemic stroke (AIS) during intravenous r-tPA therapy and associated CA with response to therapy. AIS patients eligible for intravenous r-tPA therapy were recruited. Cerebral blood flow velocities (transcranial Doppler) from middle cerebral artery and blood pressure (Finometer) were recorded to calculate the autoregulation index (ARI, as surrogate for CA). National Institute of Health Stroke Score was assessed and used to define responders to therapy (improvement of ≥ 4 points on NIHSS measured 24-48 h after therapy). CA was considered impaired if ARI < 4. In 38 patients studied, compared to responders, non-responders had significantly lower ARI values (affected hemisphere: 5.0 vs. 3.6; unaffected hemisphere: 5.4 vs. 4.4, p = 0.03) and more likely to have impaired CA (32% vs. 62%, p = 0.02) during thrombolysis. In conclusion, CA during thrombolysis was impaired in patients who did not respond to therapy. This variable should be investigated as a predictor of the response to therapy and to subsequent neurological outcome.

COHmax: an algorithm to maximise coherence in estimates of dynamic cerebral autoregulation.

OBJECTIVE: The reliability of dynamic cerebral autoregulation (dCA) parameters, obtained with transfer function analysis (TFA) of spontaneous fluctuations in arterial blood pressure (BP), require statistically significant values of the coherence function. A new algorithm (COHmax) is proposed to increase values of coherence by means of the automated, selective removal of sub-segments of data. APPROACH: Healthy subjects were studied at baseline (normocapnia) and during 5% breathing of CO2 (hypercapnia). BP (Finapres), cerebral blood flow velocity (CBFV, transcranial Doppler), end-tidal CO2 (EtCO2, capnography) and heart rate (ECG) were recorded continuously during 5 min in each condition. TFA was performed with sub-segments of data of duration (SEGD) 100 s, 50 s or 25 s and the autoregulation index (ARI) was obtained from the CBFV response to a step change in BP. The area-under-the curve (AUC) was obtained from the receiver-operating characteristic (ROC) curve for the detection of changes in dCA resulting from hypercapnia. MAIN RESULTS: In 120 healthy subjects (69 male, age range 20-77 years), CO2 breathing was effective in changing mean EtCO2 and CBFV (p < 0.001). For SEGD = 100 s, ARI changed from 5.8 ± 1.4 (normocapnia) to 4.0 ± 1.7 (hypercapnia, p < 0.0001), with similar differences for SEGD = 50 s or 25 s. Depending on the value of SEGD, in normocapnia, 15.8% to 18.3% of ARI estimates were rejected due to poor coherence, with corresponding rates of 8.3% to 13.3% in hypercapnia. With increasing coherence, 36.4% to 63.2% of these could be recovered in normocapnia (p < 0.001) and 50.0% to 83.0% in hypercapnia (p < 0.005). For SEGD = 100 s, ROC AUC was not influenced by the algorithm, but it was superior to corresponding values for SEGD = 50 s or 25 s. SIGNIFICANCE: COHmax has the potential to improve the yield of TFA estimates of dCA parameters, without introducing a bias or deterioration of their ability to detect impairment of autoregulation. Further studies are needed to assess the behaviour of the algorithm in patients with different cerebrovascular conditions.

Chasing the evidence: the influence of data segmentation on estimates of dynamic cerebral autoregulation.

OBJECTIVE: Transfer function analysis (TFA) of dynamic cerebral autoregulation (dCA) requires smoothing of spectral estimates using segmentation of the data (SD). Systematic studies are required to elucidate the potential influence of SD on dCA parameters. APPROACH: Healthy subjects (HS, n = 237) and acute ischaemic stroke patients (AIS, n = 98) were included. Cerebral blood flow velocity (CBFV, transcranial Doppler ultrasound) was recorded supine at rest with continuous arterial blood pressure (BP, Finometer) for a minimum of 5 min. TFA was performed with durations SD = 100, 50 or 25 s and 50% superposition to derive estimates of coherence, gain and phase for the BP-CBFV relationship. The autoregulation index (ARI) was estimated from the CBFV step response. Intrasubject reproducibility was expressed by the intraclass correlation coefficient (ICC). MAIN RESULTS: In HS, the ARI, coherence, gain, and phase (low frequency) were influenced by SD, but in AIS, phase (very low frequency) and ARI were not affected. ICC was excellent (>0.75) for all parameters, for both HS and AIS. For SD = 100 s, ARI was different between HS and AIS (mean ± sdev: 5.70 ± 1.61 vs 5.1 ± 2.0; p < 0.01) and the significance of this difference was maintained for SD = 50 s and 25 s. Using SD = 100 s as reference, the rate of misclassification, based on a threshold of ARI ⩽ 4, was 6.3% for SD = 50 s and 8.1% for SD = 25 s in HS, with corresponding values of 11.7% and 8.2% in AIS patients, respectively. SIGNIFICANCE: Further studies are warranted with SD values lower than the recommended standard of SD = 100 s, to explore possibilities of improving the reproducibility, sensitivity and prognostic value of TFA parameters used as metrics of dCA.

Can we assess dynamic cerebral autoregulation in stroke patients with high rates of cardiac ectopicity?

It is unclear whether physiological recordings containing high numbers of ectopic heartbeats can be used to measure the cerebral autoregulation (CA) of blood flow. This study evaluated the utility of such data for assessing dynamic CA capacity. Physiological recordings of cerebral blood flow velocity, heart rate, end-tidal CO2 and beat-to-beat blood pressure from acute ischaemic stroke (AIS) patients (n = 46) containing ectopic heartbeats of varying number (0.2 to 25 occurrences per minute) were analysed. Dynamic CA was determined using the autoregulation index (ARI) and the normalised mean square error (NMSE) was used to evaluate the fitting of the step response between BP and CBFV to Tiecks' model. We fitted linear mixed models on the CA variables incorporating ectopic burden, age, sex and hemisphere as predictor variables. Ectopic activity demonstrated an association with mean coherence (p = 0.006) but not with ARI (p = 0.162), impaired CA based on dichotomised ARI (p = 0.859) or NMSE (p = 0.671). Dynamic CA could be reliably assessed in AIS patients using physiological recordings with high rates of cardiac ectopic activity. This provides supportive data for future studies evaluating CA capability in AIS patients, with the potential to develop more individualised treatment strategies. Graphical Abstract.

Can we use short recordings for assessment of dynamic cerebral autoregulation? A sensitivity analysis study in acute ischaemic stroke and healthy subjects.

OBJECTIVE: It is unclear whether the duration of recordings influences estimates of dynamic cerebral autoregulation (dCA). Therefore, we performed a retrospective study of the effects of reducing recording durations on dCA estimates; with the potential to inform recording duration for reliable estimates in challenging clinical populations. APPROACH: Seventy-eight healthy control subjects and 79 acute ischaemic stroke (AIS) patients were included. Cerebral blood flow (CBF) velocity was recorded with transcranial Doppler (TCD) and continuous blood pressure (BP) with a Finapres device. The autoregulation index (ARI), derived with transfer function analysis (TFA), was calculated for recording durations at one-minute intervals between 1 and 5 min using the same starting point of each recording. MAIN RESULTS: Though recording duration did not affect the overall ARI value, when compared to control subjects, AIS patients had significantly lower ARI values for durations between 3 and 5 min (p  < 0.0001), but not 1 and 2 min. The intraclass correlation coefficient of all participants, for reproducibility of the five recording durations, was 0.69. AIS patients classified as having impaired cerebral autoregulation (CA; ARI ⩽ 4) at 5 min, had a 7.1% rate of false negatives for both 4 and 3 min recordings, reaching 42.9% for 1 min recording. The percentage of false-positives also increased with reduced recording durations (from 0% at 5 to 16.2% at 1 min). SIGNIFICANCE: Reducing recording durations from 5 to 3 min can still provide reliable estimates of ARI, and may facilitate CA studies in potentially medically unstable AIS patients, as well as in other patient groups.

Cerebral Haemodynamics following Acute Ischaemic Stroke: Effects of Stroke Severity and Stroke Subtype.

BACKGROUND: Acute ischaemic stroke (AIS) patients often show impaired cerebral autoregulation (CA). We tested the hypothesis that CA impairment and other alterations in cerebral haemodynamics are associated with stroke subtype and severity. METHODS: AIS patients (n = 143) were amalgamated from similar studies. Data from baseline (< 48 h stroke onset) physiological recordings (beat-to-beat blood pressure [BP], cerebral blood flow velocity (CBFV) from bilateral insonation of the middle cerebral arteries) were calculated for mean values and autoregulation index (ARI). Differences were assessed between stroke subtype (Oxfordshire Community Stroke Project [OCSP] classification) and severity (National Institutes of Health Stroke Scale [NIHSS] score < 5 and 5-25). Correlation coefficients assessed associations between NIHSS and physiological measurements. RESULTS: Thirty-two percent of AIS patients had impaired CA (ARI < 4) in affected hemisphere (AH) that was similar between stroke subtypes and severity. CBFV in AH was comparable between stroke subtype and severity. In unaffected hemisphere (UH), differences existed in mean CBFV between lacunar and total anterior circulation OCSP subtypes (42 vs. 56 cm•s-1, p < 0.01), and mild and moderate-to-severe stroke severity (45 vs. 51 cm•s-1, p = 0.04). NIHSS was associated with peripheral (diastolic and mean arterial BP) and cerebral haemodynamic parameters (CBFV and ARI) in the UH. CONCLUSIONS: AIS patients with different OCSP subtypes and severity have homogeneity in CA capability. Cerebral haemodynamic measurements in the UH were distinguishable between stroke subtype and severity, including the association between deteriorating ARI in UH with stroke severity. More studies are needed to determine their clinical significance and to understand the determinants of CA impairment in AIS patients.

Effects of dominant and non-dominant passive arm manoeuvres on the neurovascular coupling response.

PURPOSE: Models designed to study neurovascular coupling (NVC) describe a possible cerebral hemisphere dominance dependent on task completed and preference in handedness. We investigated whether passive arm manoeuvre performed with dominant (Dom-Arm) or non-dominant arm (ND-Arm) stimulated haemodynamic differences in either contralateral (Cont-H) or ipsilateral (Ipsil-H) cerebral hemisphere. METHODS: Healthy individuals lying in supine position, had measurements of beat-to-beat blood pressure (BP, mmHg), electrocardiogram (HR, bpm), end-tidal CO2 (etCO2, mmHg), and bilateral insonation of the middle cerebral arteries (MCA, cm s-1). Arm movement was performed for 60 s with passive flexion and extension of the elbow (1 Hz), before manoeuvre was repeated on other arm. Data were normalised and effect of treatment was analysed for differences between manoeuvres and within each time period. RESULTS: Seventeen (eight males) healthy volunteers, aged 56 ± 7 years, were studied. Dom-Arm and ND-Arm manoeuvres stimulated a comparable temporal response in peripheral and cerebral haemodynamic parameters between Cont-H and Ipsil-H. CONCLUSIONS: Both manoeuvres can be used to evoke similar bilateral MCA responses in assessing NVC. This finding should lead to more efficient protocols when using passive arm movement for NVC studies in healthy subjects.

Differences in the profile of protection afforded by TRO40303 and mild hypothermia in models of cardiac ischemia/reperfusion injury.

The mode of protection against cardiac reperfusion injury by mild hypothermia and TRO40303 was investigated in various experimental models and compared to MitoQ in vitro. In isolated cardiomyocytes subjected to hypoxia/reoxygenation, TRO40303, MitoQ and mild hypothermia delayed mPTP opening, inhibited generation of mitochondrial superoxide anions at reoxygenation and improved cell survival. Mild hypothermia, but not MitoQ and TRO40303, provided protection in a metabolic starvation model in H9c2 cells and preserved respiratory function in isolated rat heart mitochondria submitted to anoxia/reoxygenation. In the Langendorff-perfused rat heart, only mild hypothermia provided protection of hemodynamic function and reduced infarct size following ischemia/reperfusion. In biopsies from the left ventricle of pigs subjected to in vivo occlusion/reperfusion, TRO40303 specifically preserved respiratory functions in the peri-infarct zone whereas mild hypothermia preserved both the ischemic core area and the peri-infarct zones. Additionally in this pig model, only hypothermia reduced infarct size. We conclude that mild hypothermia provided protection in all models by reducing the detrimental effects of ischemia, and when initiated before occlusion, reduced subsequent reperfusion damage leading to a smaller infarct. By contrast, although TRO40303 provided similar protection to MitoQ in vitro and offered specific protection against some aspects of reperfusion injury in vivo, this was insufficient to reduce infarct size.