Abstract Tissue perfusion and oxygenation, key factors of renal ischemia‐reperfusion injury (RIR), can be assessed using contrast‐enhanced ultrasound (CEUS) and photoacoustic (PA) imaging. We hypothesized that early changes in renal perfusion and oxygenation caused by IRI, or protection by mild therapeutic hypothermia (mTH), could be detected and correlate with fibrosis. C57BL6 mice underwent 15‐min unilateral renal ischemia, with or without mTH, followed by 1‐month reperfusion. They were assigned to sham (n = 5), normothermic renal ischemia (IR‐37°C, n = 7), mTH renal ischemia (IR‐34°C, n = 7). The IR‐37°C and IR‐34°C groups had body temperature maintained at 37°C or 34°C during surgery. CEUS and PA were conducted at baseline, 20 min, and 1 month after reperfusion. Histological analysis was performed at 1 month. IRI induced renal atrophy and fibrosis at 1 month, prevented by mTH. CEUS revealed altered perfusion in IR‐37°C at 20 min, sustained at 1 month, compared to Sham, while perfusion was preserved with mTH in IR‐34°C. Oxygenation assessed by PA was not modified. However, oxygenation measured 20 min after IRI correlated with fibrosis at 1 month. CEUS and PA are promising tools for non‐invasive assessment of renal IRI. IRI induced early and sustained perfusion alterations, while early assessment of oxygenation correlated with fibrosis. mTH prevented these alterations.

In the context of induced blood-brain barrier (BBB) opening, estimating the parenchymal occurrence and concentration of different-sized molecules is an essential aspect for the optimization of focused and non-focused ultrasound protocols. Separate estimations of colocalization and correlation of signals in images partially address these aspects. However, the simultaneous analysis of localization and intensity of two signals holds promise to better understand the mechanistic players in ultrasound-induced BBB permeability. A new theoretical framework is proposed for intensity correlation and uncorrelation analysis (ICUA) enabling the spatial characterization of two signal distributions into 11 behaviors. ICUA was applied to a mice model of BBB opening induced by non-focused ultrasounds followed by 1kDa and 70kDa fluorophore injections and based on (1) parenchymal signal intensities and (2) injected blood concentration. Results showed that the spatial distribution of correlated intensities was strongly influenced by the pressure field pattern of unfocused US. Notably, when normalized by parenchymal signal intensities, the smaller molecule was found in higher intensities compared to the larger molecule in over 30% of the colocalization volume. Conversely, when normalized by injected concentration, the larger molecule was found to have accumulated in 30% of the brain volume for the group with the highest small molecule signal intensity. This two-step approach addressed how the fluorophores were spatially scattered and how this spatial organization was dependent on the efficiency of BBB opening. Overall, the analysis of the spatial localization and intrinsic parameters defining the ICUA helped estimate two physical parameters of BBB opening: size exclusion and duration of opening. Furthermore, the complementary analysis normalized by the injected concentration offered a new potential to explore complex BBB mechanisms such as clearance and binding. These results could help model the efficiency of US to open the BBB to different sizes of molecules and thus optimize the settings to reach the expected BBB opening for drug candidates.

Microglia exhibit diverse morphologies reflecting environmental conditions, maturity, or functional states. Thus, morphological characterization provides important information to understand microglial roles and functions. Most recent morphological analysis relies on classifying cells based on morphological parameters. However, this classification may lack biological relevance, as microglial morphologies represent a continuum rather than distinct, separate groups, and do not correspond to mathematically defined clusters irrelevant of microglial cells function. Instead, we propose a new open-source tool, MorphoCellSorter, which assesses microglial morphology by automatically computing morphological criteria, using principal component analysis and Andrews plots to score cells. MorphoCellSorter properly ranked cells from various microglia datasets in mice and rats of different ages, from in vivo, in vitro, and ex vivo models, that were acquired using diverse imaging techniques. This approach allowed for the discrimination of cell populations in various pathophysiological conditions. Finally, MorphoCellSorter offers a versatile, easy, and ready-to-use method to evaluate microglial morphological diversity that could easily be generalized to standardize practices across laboratories.

Background & Aims:The gut-liver axis and hepatic ER-mitochondria miscommunication (at contact sites called MAMs) are involved in the development of metabolic dysfunction-associated steatotic liver disease (MASLD). We investigated the role of circulating aromatic amino acids (AAA) derived from phenylalanine and tyrosine in MASLD potentially through MAM alterations. Methods:We analyzed AAA metabolomic profiles in individuals with and without MASLD and validated findings in a biopsy-proven cohort. The pro-steatogenic effect of MASLD-associated AAAs was validated in vitro using lipid labeling, MAM structural/functional assays, and palmitate-induced respiration. In vivo effects were tested in mice fed with candidate AAAs, and MAM involvement was confirmed by expressing a specific organelle linker in vitro and in vivo. Results:N-acetyl-phenylalanine (NAPA) was strongly associated with hepatic steatosis and correlated with specific gut microbes. In vitro, NAPA promoted lipid accumulation by impairing ER-mitochondria calcium exchange via a LAT1-dependent electrogenic mechanism, reducing mitochondrial lipid oxidation. Chronic NAPA administration in mice induced steatosis and MAM disruption. Notably, enhancing ER-mitochondria contacts with an organelle linker prevented NAPA-induced steatosis in vitro and in vivo. Additionally, other phenylalanine- and tyrosine-derived AAAs reproduced NAPA's effects, suggesting a class-dependent mechanism. Conclusion:NAPA emerges as a MASLD-promoting metabolite, contributing to hepatic steatosis by disrupting ER-mitochondria calcium coupling and mitochondrial lipid oxidation.

BACKGROUND:The aim of our study was to test different anesthetic mixtures in order to identify the most suitable one for a surgical cardiac ischemia-reperfusion model in mice. METHODS:1) Sixty four mice were submitted to one of the 6 combinations of ketamine or alfaxalone associated to xylazine, medetomidine or midazolam. Depth and quality of anesthesia were evaluated via 5 reflex scores. 2) Impact of analgesic (buprenorphine or butorphanol), anesthesia reversal (with atipamezole) and surgery (cardiac ischemia-reperfusion surgery) have been tested in the selected protocols. 3) infarction size has been measured with TTC (Triphenyl Tetrazolium Chloride) method in mice anesthetized with best protocols. RESULTS:Protocol involving medetomidine induced the longest surgical anesthesia: (median = 120, {interquartile range = 100-125}) min with ketamine and 53 {25-100} min with alfaxalone. Butorphanol substitution with buprenorphine did not alter time-related anesthesia parameters. Atipamezole reversal considerably reduced both recovery and immobilization time (respectively 22 {18-30} min and 98 {88-99} min vs. 55 {40-70} min and 143 {131-149} min, in groups with no reversal, p = 0.001) with no impact on infarction size measurement. CONCLUSION:In this study, the combination alfaxalone/medetomidine/buprenorphine (80/0,3/0,075 mg.kg-1, s.c.) associated with reversal by atipamezole was a reliable anesthetic protocol for murine surgery, particularly for the study of ischemia-reperfusion.

Abstract Background and Aims Renal 3D photo-acoustic imaging (3D-PAI) and contrast-enhanced ultrasound (CEUS) are promising tools in mice models to assess repeatedly and non-invasively renal ischemia-reperfusion (RIR) consequences and damages, or their improvement by nephroprotective strategies like mild therapeutic hypothermia (mTH), at the early phase of reperfusion as well as in the chronic late phase. Method C57Bl6 mice underwent 15 minutes of unilateral renal vascular clamping, with body temperature at 37°C (RIR-37°C, n=7) or mTH at 34°C (RIR-34°C, n=7), or a sham procedure (Sham, n=5). Renal volume and oxygen saturation (sO2-3D; corresponding to the percentage of oxyhemoglobin over the total hemoglobin content) were measured with 3D-PAI (3D reconstruction of the whole kidney through multi-plane acquisition with an automatized mobile support), and renal perfusion parameters (rBV, mTT, rBF) with CEUS (“destruction-replenishment” model of intravenous microbubbles), performed with VEVO3100 echograph (Fujifilm Visualsonic) 1 week before RIR (basal), 20 minutes after reperfusion, and 1 month after. Renal fibrosis was quantified at 1 month with Masson's trichrome coloration on histological samples. Data were compared with Mann-Whitney test or Wilcoxon test (paired data) as appropriate. Correlation was tested by Spearman test. All animal procedures were approved by the Ethics Committee. Results Sham showed no significant changes during follow-up. RIR-37°C led to renal hypotrophy (volume on 3D-PAI) and fibrosis at 1 month, compared to RIR-34°C (median volume 76 IQR [71-119] vs 143 IQR[108-180] mm3; p=0.03), and those parameters were correlated (R=−0.49; p=0.03). In RIR-37°C, sO2-3D tended to lower at 20 minutes compared to basal values (median sO2-3D 37 IQR [37-44] vs 49 IQR [36-57]%; p=0.08), but not in RIR-34°C (p > 0.99), and these early variations of sO2-3D were correlated with fibrosis at 1 month (R=−0.48; p=0.04). Renal perfusion was altered in RIR-37°C at 20 minutes compared to basal values (median rBV 73 IQR [46-105] vs 100 IQR [89-126] a.u.; p=0.047), and remained altered at 1 month (median rBV 71 IQR [57-83] a.u.; p=0.03), but not in RIR-34°C (p=0.47 at both 20 minutes and 1 month). Renal perfusion early alterations at 20 minutes were correlated with late alterations at 1 month (R=0.63; p=0.005), and tended to correlate with fibrosis (R=−0.41; p=0.08). Conclusion Renal 3D-PAI (on the whole 3D-reconstructed kidney) and CEUS can detect early alterations of renal perfusion and oxygen saturation after RIR, and predict chronic disturbances of perfusion and the onset of fibrosis, as well as the protection conferred by mTH. 3D-PAI can also be used to evaluate non-invasively renal volume as a surrogate of renal fibrosis after RIR.

Using a translational approach with an ST-segment myocardial infarction (STEMI) cohort and mouse model of myocardial infarction, we highlighted the role of the secreted IL-6 and MCP-1 cytokines and the STAT3 pathway in heart macrophage recruitment and activation. Cardiac myocytes secrete IL-6 and MCP-1 in response to hypoxic stress, leading to a recruitment and/or polarization of anti-inflammatory macrophages via the STAT3 pathway. In our preclinical model of myocardial infarction, neutralization of IL-6 and MCP-1 or STAT3 pathway reduced infarct size. Together, our data demonstrate that anti-inflammatory macrophages can be deleterious in the acute phase of STEMI.

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic–reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation–reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation–reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia–reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation–reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.

Heart failure is major public health problem with ischemic cardiomyopathy as the main etiology. Despite therapeutic advances, the high mortality of patients highlights that development of new therapeutic strategies is urgently needed. In this context, the establishment of a pre-clinical model mimicking the clinical pathology would be an asset to characterize the functional mechanisms responsible for the development of heart failure following ischemia-reperfusion injury. In this study, male and female C57Bl/6J mice (8–12 weeks old) underwent left anterior coronary artery ligation for 90 minutes followed by 12 weeks reperfusion (n = 18) or no surgical intervention (CTRL; n = 8). Ejection fraction (EF), left ventricle internal diameter (LVID), E-wave deceleration time (EDT) and the isovolumic relaxation time (IVRT) were assessed by echocardiography during the 12 weeks of post-ischemic reperfusion and cellular Ca2 + (dys)regulation mechanisms in failing isolated cardiomyocytes were investigated at 8 weeks with the imaging Ionoptix system. Our results showed that, despite a significant alteration of EF at day 2 post-infarction (due to ischemic stress by itself), ischemic mice developed a chronical heart dysfunction characterized, on one hand, by a systolic dysfunction with a significant decrease of EF averaging 26% and a LV dilatation around 36% from the day 2 to the week 8 of reperfusion (P < 0.05), and on the other hand, associated with a significant diastolic dysfunction at 12 weeks of reperfusion. Mechanistically, Ca2+ phenotyping showed a significant alteration in excitation/contraction coupling and a contractility defect in failing cardiomyocytes isolated at 8 weeks reperfusion (P < 0.05 vs. CTRL). Our experimental conditions show that 90 minutes ischemia followed by 8 weeks reperfusion seems to be a good algorithm to mimic development of heart failure after an ischemia-reperfusion stress, which is characterized by both systolic and diastolic cardiac dysfunctions.

Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca2+ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca2+ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue.

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Uncertainty exists regarding whether cyclophilin D (CypD), a mitochondrial matrix protein that plays a key role in ischemia-reperfusion injury, can be a pharmacological target for improving outcomes after cardiac arrest (CA), especially when therapeutic hypothermia is used. Using CypD knockout mice (CypD-/-), we investigated the effects of loss of CypD on short-term and medium-term outcomes after CA. CypD-/- mice or their wild-type (WT) littermates underwent either 5 minute CA followed by resuscitation with and/or without hypothermia at 33°C-34°C (targeted temperature reached within minutes after resuscitation), or a sham procedure. Brain and cardiac injury were assessed using echocardiography, neurological scores, MRI and biomarkers. Seven day survival was compared using Kaplan-Meier estimates. The rate of restoration of spontaneous circulation was significantly higher in CypD-/- mice (with shorter cardiac massage duration) than in WT mice (P < 0.05). Loss of CypD significantly attenuated CA-induced release of troponin and S100ß protein, and limited myocardial dysfunction at 150 minutes after CA. Loss of CypD combined with hypothermia led to the best neurological and MRI scores at 24 hours and highest survival rates at 7 days compared to other groups (P < 0.05). In animals successfully resuscitated, loss of CypD had no benefits on day 7 survival while hypothermia was highly protective. Pharmacological inhibition of CypD with cyclosporine A combined with hypothermia provided similar day 7 survival than loss of CypD combined with hypothermia. CypD is a viable target to improve success of cardiopulmonary resuscitation but its inhibition is unlikely to improve long-term outcomes, unless therapeutic hypothermia is associated.

Ischemia-reperfusion (IR) leads to systemic inflammation. Mild therapeutic hypothermia (mTH) has been suggested to bring protection against IR lesions. However, mTH remains limited to intensive care units (ICU) after resuscitated cardiac arrest (rCA) and is not currently used in nephrology. Extracellular cold-inducible RNA-binding protein (eCIRP) is a pro-inflammatory cytokine that can be secreted during stress situations such as hypoxia. To test if eCIRP plasma level is correlated to renal IR injuries in mice with or without mTH. A 20-minutes bilateral renal ischemia by clamping (or a sham procedure) was conducted on C57BL6 mice with core body temperature maintained at 37 °C (normothermia) or 34 °C (mTH). Plasma eCIRP, IL-6, IL-10 and urea were dosed 2 h and 24 h after reperfusion. Acute tubular necrosis (ATN) was scored (from 0 to 4) on histological exam of kidneys at 24 h. Bilateral renal ischemia in mice was associated with AKI compared to sham as soon as 2 h after reperfusion (median urea 18.7 vs. 11.7 mmol/L, P = 0.02) and more severe 24 h after reperfusion (median urea 60.8 vs. 6.2 mmol/L, P = 0.004), and with ATN 24 h after reperfusion (median histological score 2.23 vs. 0.80, P < 0.001). Plasma eCIRP levels were significantly increased by renal IR 24 h after reperfusion compared to sham (median 123.1 vs. 21.5 pg/mL, P = 0.03). Renal IR also led to a peak of IL-6 secretion 2 h after reperfusion, and of IL-10 secretion 24 h after reperfusion. We showed a correlation between eCIRP and urea levels 2 h after reperfusion (Spearman r = 0.60, P = 0.007) and 24 h after reperfusion (Spearman r = 0.84, P < 0.001), and with ATN score (Spearman r = 0.65, P < 0.001). The use of mTH during renal ischemia was followed by a significantly reduced plasma urea (median 22.7 mmol/L, P = 0.001), ATN score (median 1.45, P = 0.03) and eCIRP elevation (median 60.8 pg/mL, P = 0.03) 24 h after reperfusion, and an inhibition of IL-6 and IL-10 secretion (Fig. 1). Plasma eCIRP increases 24 h after renal IR in mice and is correlated to AKI and ATN. The use of mTH during ischemia alleviates eCIRP elevation in parallel of renal IR injury and inflammation. We aim to measure eCIRP level in ICU after rCA (i.e. global IR) at admission, and at day 1 and 3 to confirm our results in a cohort of 33 patients in order to test if eCIRP is correlated to relevant clinical and biological outcomes, such as AKI and its severity.

Background & Aims: Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic re-ticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment.Methods: We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations.Results: Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mech-anistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER -mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glyce-mia, HbA1c and HOMA-IR index.Conclusions: ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis.Lay summary: The literature suggests that interactions between the endoplasmic reticulum and mitochondria could play a role in hepatic insulin resistance and steatosis during chronic obesity. In the present study, we reappraised the time-dependent regulation of hepatic endoplasmic reticulum-mitochondria interactions and calcium exchange, investigating reversibility and causality, in mice with diet-induced obesity. We also assessed the relevance of our findings to humans. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis that can be improved by nutritional strategies.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).