Min Of Perfusion Research Articles

Cardioprotection by Preconditioning with Intralipid Is Sustained in a Model of Endothelial Dysfunction for Isolated-Perfused Hearts.

Endothelial dysfunction (ED) is closely associated with most cardiovascular diseases. Experimental models are needed to analyze the potential impact of ED on cardioprotection in constant pressure Langendorff systems (CPLS). One cardioprotective strategy against ischemia/reperfusion injury (I/RI) is conditioning with the lipid emulsion Intralipid (IL). Whether ED modulates the cardioprotective effect of IL remains unknown. The aim of the study was to transfer a protocol using a constant flow Langendorff system for the induction of ED into a CPLS, without the loss of smooth muscle cell functionality, and to analyze the cardioprotective effect of IL against I/RI under ED. In isolated hearts of male Wistar rats, ED was induced by 10 min perfusion of a Krebs-Henseleit buffer containing 60 mM KCl (K+), and the vasodilatory response to the vasodilators histamine (endothelial-dependent) and sodium-nitroprusside (SNP, endothelial-independent) was measured. A CPLS was employed to determine cardioprotection of pre- or postconditioning with 1% IL against I/RI. The constant flow perfusion of K+ reduced endothelial response to histamine but not to SNP, indicating reduced vasodilatory functionality of endothelial cells but not smooth muscle cells. Preconditioning with IL reduced infarct size and improved cardiac function while postconditioning with IL had no effect. The induction of ED neither influenced infarct size nor affected the cardioprotective effect by preconditioning with IL. This protocol allows for studies of cardioprotective strategies under ED in CLPS. The protection by preconditioning with IL seems to be mediated independently of a functional endothelium.

An Isolated Perfused Rat Liver Model: Simultaneous LC-MS Quantification of Pitavastatin, Coproporphyrin I, and Coproporphyrin III Levels in the Rat Liver and Bile.

The isolated perfused rat liver (IPRL) model provides a mechanistic understanding of the organic-anion-transporting polypeptide (OATP/Oatp)-mediated pharmacokinetics in the preclinical evaluation, which often requires the use of control substrates (i.e., pitavastatin) and monitoring endogenous biomarkers (coproporphyrin I and III). This study aimed to develop and validate an LC-MS method allowing the simultaneous quantification of pitavastatin, coproporphyrin I (CPI), and coproporphyrin III (CPIII) in rat liver perfusion matrices (perfusate, liver homogenate, bile). The analysis was performed on a C18 column at 60 °C with 20 μL of sample injection. The mobile phases consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid with a gradient flow of 0.5 mL/min. The assay was validated according to the ICH M10 Bioanalytical Method Validation Guideline (2022) for selectivity, calibration curve and range, matrix effect, carryover, accuracy, precision, and reinjection reproducibility. The method allowing the simultaneous quantification of pitavastatin, CPI, and CPIII was selective without having carryover and matrix effects. The linear calibration curves were obtained within various calibration ranges for three analytes in different matrices. Accuracy and precision values fulfilled the required limits. After 60 min perfusion with pitavastatin (1 μM), the cumulative amounts of pitavastatin in the liver and bile were 5.770 ± 1.504 and 0.852 ± 0.430 nmol/g liver, respectively. CPIII was a more dominant marker than CPI in both liver (0.028 ± 0.017 vs 0.013 ± 0.008 nmol/g liver) and bile (0.016 ± 0.011 vs 0.009 ± 0.007 nmol/g liver). The novel and validated bioanalytical method can be applied in further IPRL preparations investigating Oatp-mediated pharmacokinetics and DDIs.

Perfusion index (PI) measured in the brachial plexus blocks performed by ultrasonography: investigation of correlation with block success

BackgroundBrachial plexus block is one of the peripheral nerve blocks commonly used in regional anesthesia. We aimed to investigate whether the perfusion index (PI) measurement can be used to evaluate the adequacy of the upper limb blocks accompanied by ultrasonography. Thirty-three patients between the ages of 18 and 65 who were in the ASA I–II group who will have a hand, forearm, and arm operation were included in the study. An appropriate type of brachial plexus block was applied to these patients under the guidance of ultrasonography. After the block application was completed, the sensory and motor blocks were evaluated at the 5th, 10th, and 20th min. In addition, perfusion indices measured from both extremities of the patient were recorded at the 5th, 10th, and 20th min.ResultsIn 32 of 33 patients who underwent brachial plexus block, our block was successful, and perfusion index measurements in the applied limb increased continuously from the 5th min over the 20-min observation period. In one patient who failed the block and in the arm group without block, no statistically significant difference was detected in the 5th, 10th, and 20th min perfusion index measurements.ConclusionsPerfusion index measurement is a very valuable and usable method to evaluate the success of peripheral nerve blocks early, easily, and objectively.

Aortic ischemia-reperfusion injury and potency of fluoxetine.

Due to cross-clamping of the aorta during aortic aneurysm surgeries, ischemia-reperfusion (IR) develops, and it may cause damage to the aorta itself or even to remote organs by oxidative stress or inflammation. Fluoxetine (FLX) which might be used in the preoperative period for its tranquilizing effect also has antioxidant effects in short-term use. The purpose of our study is to examine whether FLX protects aorta tissue, against the damage caused by IR. Three groups of Wistar rats were formed randomly. 1) Control group (sham-operated), 2) IR group (60 min ischemia+120 min perfusion), and 3) FLX+IR group (FLX dose was 20 mg/kg for 3 days IP before IR). At the end of each procedure, aorta samples were collected, and oxidant-antioxidant, anti-inflammatory, and anti-apoptotic status of the aorta were evaluated. Histological examinations of the samples were provided. Levels of LOOH, MDA, ROS, TOS, MPO, TNFα, IL-1β, IL-6, NF-kB, MMP-9, caspase-9, 8-OHdG, NO, and HA were found to be significantly increased in the IR group compared with control (P<0.05) and SOD, GSH, TAS, and IL-10 levels were significantly lower (P<0.05). FLX significantly decreased LOOH, MDA, ROS, TOS, MPO, TNFα, IL-1β, IL-6, NF-kB, MMP-9, caspase-9, 8-OHdG, NO, and HA levels in the FLX+IR group compared with IR group (P<0.05) and increased IL-10, SOD, GSH, and TAS (P<0.05). FLX administration prevented the deterioration of aortic tissue damage. Our study is the first study that demonstrates FLX-mediated suppression of IR injury in the infrarenal abdominal aorta by antioxidant, anti-inflammatory, and anti-apoptotic properties.

Zonal pattern of blood flow distribution in the Pliss lymphosarcoma microcirculatory bed caused by single exposure of high-intensity focused ultrasound

Introduction. Cell damage is not the only important mechanism of high-intensity focused ultrasound (HIFU) for cancer therapy; concomitant microcirculation disorders having alterations patterns not studied enough also have high significance. The aim of the study was to analyze perfusion along the tumor perimeter depending on the HIFU exposure. Materials and methods. We used a stand for HIFU therapy (MTL LLC). Mode: 8.2 kW/cm2 intensity at 0.6 mm focal spot at 1.4 MHz, exposure time: 150, 200, 250 and 500 ms. The object of the study is the vascular bed of Pliss lymphosarcoma. Tumor tissue temperature was monitored by a T-8 thermograph (Russia), blood flow was measured by the BLF21 laser Doppler recorder (USA). Results. After HIFU exposure time from 150–250 ms, perfusion was noted to have decreased to 1.06 perfusion units (pf.u) in the central zone (p&lt;0.01) compared to the control, but by the 30 min perfusion has increased to 1.22 pf.un. No signs of perfusion improvement were found in exposure time of 500 ms. In the peripheral zone, against the background of a decrease in blood flow to 3.6 pf. u. (p &lt; 0.05) by 30 min of the experiment, hyperperfusion was restored to 5.86 pf. u. in all exposure time. In the central zone, a sharp decrease in microcirculation was observed, probably caused by damage of blood vessels; in the periphery of the tumor, perfusion changes had a phasic and reversible pattern. Conclusion. An increase of HIFU exposure correlates with an increase of temperature of Pliss lymphosarcoma tissue near the focus of exposure. Specific perfusion changes are recorded in the central and peripheral zones of the tumor when single exposed to HIFU with different time exposure; the severity of changes depends on ultrasound exposure-time and is characterized by a zonal pattern of blood flow distribution due to the formation of zones of irreversible and reversible damage to microcirculation.

Empagliflozin attenuates arrhythmogenesis via inhibition of O-GlcNAcylation in diastolic phase of diabetic cardiomyopathy

Abstract Background Diabetic cardiomyopathy is an important complication of diabetes mellitus (DM) and reported to increase the risk of fatal ventricular arrhythmias. Recent clinical trials showed that empagliflozin (EMPA), a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, improved cardiovascular outcomes regardless of the presence of diabetes and the traditional cardiovascular risk factors. Despite the promising benefit of EMPA on heart failure treatment, its beneficial effect in the context of anti-arrhythmic therapy has not been fully examined. We therefore aimed to examine anti-arrhythmic effect of acute EMPA treatment especially onto calcium (Ca2+) handling in diabetic cardiomyocytes. Methods We assessed echocardiography, hemodynamic study, electrophysiology, Ca2+ handling and protein expression in C57BLKS/J-leprdb/db mice (db/db mice), a leptin receptor-deficient model of obesity and Type 2 diabetes, and their non-diabetic lean heterozygous Leprdb/+ littermates (db/+ mice). Results The 16-week-old db/db mice had preserved systolic function but exhibited diastolic dysfunction. In arrhythmia induction using ex-vivo Langendorff-perfused hearts, db/db mice showed a significantly increased premature ventricular complex (PVC) by 2x Ca2+ and 1μM isoproterenol load than control, which was attenuated by EMPA perfusion (1 μM). Diabetic cardiomyocytes showed an increased frequency of spontaneous Ca2+ sparks and waves, and decreased Ca2+ transient amplitude and sarcoplasmic reticulum (SR) Ca2+ content. Ca2+ transient decay tau and time to 50% decay were significantly prolonged in diabetic cardiomyocytes. These data indicating the impaired Ca2+ handling in diabetic cardiomyocytes were normalized by acute administration of EMPA (1 μM), while the administration of NHE inhibitor (Cariporide 10 μM) did not show significant differences. In the protein expression analysis, CaMKII Thr287 autophosphorylation and CaMKII-dependent RyR2 S2814 phosphorylation were significantly increased in diabetic hearts, which were inhibited by short-term (30 min) perfusion of EMPA (1 μM). The expressions of SERCA2a and phospholamban were not significantly different among three groups. Lastly, whole hearts O-GlcNAcylation, one of the important post translational modifications, was significantly reduced by EMPA treatment. Conclusion EMPA improved intracellular Ca2+ handling and attenuated arrhythmogenesis in mice with diabetic cardiomyopathy at the diastolic dysfunction phase, suggesting that EMPA may exhibit this anti-arrhythmic effect by normalization of intracellular calcium handling via inhibiting O-GlcNAcylation. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Boehringer Ingelheim

Optimizing transcardial perfusion of small molecules and biologics for brain penetration and biodistribution studies in rodents.

Efficiently removing blood from the brain vasculature is critical to evaluate accurately the brain penetration and biodistribution of drug candidates, especially for biologics as their blood concentrations are substantially higher than the brain concentrations. Transcardial perfusion has been used widely to remove residual blood in the brain; however, the perfusion conditions (such as the perfusion rate and time) reported in the literature are quite varied, and the performance of these methods on blood removal has not been investigated thoroughly. In this study, the effectiveness of the perfusion conditions was assessed by measuring brain hemoglobin levels. Sodium nitrite (NaNO2 ) as an additive in the perfusate was evaluated at different concentrations. Blood removal was significantly improved with 2% NaNO2 over a 20 min perfusion in mouse without disrupting the integrity of the blood-brain barrier (BBB). In mice, the optimized perfusion method significantly lowered the measured brain-to-plasma ratio (Kp,brain ) for monoclonal antibodies due to the removal of blood contamination and small molecules with a moderate-to-high BBB permeability and with a high brain-unbound-fraction (fu,brain ) presumably due to flux out of the brain during perfusion. Perfusion with or without NaNO2 clearly removed the residual blood in rat brain but with no difference observed in Kp,brain between the perfusion groups with or without 2% NaNO2 . In conclusion, a perfusion method was successfully developed to evaluate the brain penetration of small molecules and biologics in rodents for the first time. The transcardial perfusion with 2% NaNO2 effectively removed the residual blood in the brain and significantly improved the assessment of brain penetration of biologics. For small molecules, however, transcardial perfusion may not be performed, as small molecule compounds could be washed away from the brain by the perfusion procedure.

Protective Effect of Hydrogen Sulphide in Cardioplegia on Global Myocardium Ischemia‐reperfusion Injury

ObjectiveThe endogenous gaseous transmitter hydrogen sulfide (H2S) is an important regulator of the cardiovascular system and is reported to have cardioprotective activity against ischemia/reperfusion (I/R) injury. We investigated the cardioprotective effect of H2S added in cardioplegia against I/R injury in global cardioplegic arrest.MethodsIsolated Wistar rat hearts were perfused in Langendorff apparatus and underwent 30 min perfusion, 120 min cold ischemia, and 90 min reperfusion. The rats were randomly divided into 5 groups: Control group, ischemic and reperfusion (I/R), St. Thomas cardioplegia (St. Thomas +I/R), St. Thomas plus H2S donor NaHS (NaHS+St.Thomas+I/R), St.Thomas plus NaHS and glibenclamide (an antagonist of KATP channel; NaHS+Glib+St.Thomas+I/R). The following indices were compared among the groups: the change of left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), heart rate, heart rhythm, and coronary blood flow. The difference in left ventricular developed pressure (LVDP), the maximum left ventricular pressure rise rate (+dp/dtmax), and the maximum left ventricular pressure drop rate (‐dp/dtmax) were also compared. Western blot was used to detect the changes of related myocardial enzymes among different groups.ResultsNaHS+St.Thomas+I/R treatment significantly improved cardiac function (LVSP, LVDP, +dp/dtmax and ‐dp/dtmax) compared to other groups (p&lt;0.05), although no significant differences on coronary flow and heart rate were observed. After reperfusion, the frequency of ventricular arrhythmia in I/R group and St.Thomas+I/R group was significantly higher than that in NaHS+St.Thomas+I/R group. Further, the expression of cardiac Troponin T, troponin I, and creatine kinase in the NaHS+St.Thomas+I/R group was significantly lower than that in other groups (p&lt;0.05).ConclusionCardioplegia containing H2S confers significant protection against global I/R injury during cold cardioplegic arrest. This effect is associated with opening of the myocardial KATP channels. Therefore, H2S may be a useful adjunct to cardioplegic solutions in heart surgery.

Transplacental transfer of venlafaxine evaluated by ex vivo perfusion.

Depression is frequent among pregnant women and decision for treatment with antidepressants needs careful consideration of risks for the fetus. Since data regarding fetal antidepressant exposure are rare, we aimed to evaluate transplacental transfer of venlafaxine, a selective norepinephrine reuptake inhibitor. Ex vivo human placental perfusion experiments were conducted in double closed set-up. Venlafaxine (18.1±2.1μg/L) was offered in maternal circuit and maternal-to-fetal transfer was monitored over a period of 3h. Venlafaxin and O-desmethylvenlafaxine concentrations were determined by HPLC-MS in maternal and fetal perfusion medium. We observed maternal-to-fetal transfer of venlafaxine within 5min perfusion. The concentration equilibrium was approximated between maternal (7.5±0.5μg/L) and fetal (6.5±0.6μg/L) compartment at time point 180min, which corresponds to a fetal-maternal (FM) ratio of 0.89. Our results are comparable with in vivo data from an observational study which emphasizes that the ex vivo placental perfusion model is suitable for systematic evaluation of fetal antidepressant exposure.

Bioanalytical Method Validation of an RP-HPLC Method for Determination of Rifampicin in Liver Perfusion Studies

Background: The number of validated quantification methods for rifampicin, a prototypical Oatp inhibitor, in biological rat samples was limited. Objective: This study was conducted to validate a modified reversed-phase liquid chromatographic method for the determination of rifampicin in rat liver tissue according to the current ICH M10 Bioanalytical Method Validation Draft Guideline (2019) for application to samples of in situ rat liver perfusion studies. Methods: Liver tissue samples were obtained from recirculatory in situ rat liver perfusion studies. The analysis was performed on a C18 column with a mobile phase composed of 0.05 M phosphate buffer (pH 4.58): acetonitrile (55:45, v/v). The assay was validated for selectivity, calibration curve and range, matrix effect, carry-over, accuracy and precision, reinjection reproducibility, and stability. Results: he method was considered selective and stable, without having carry-over and matrix effects. The calibration curve was linear (R2: 0.9983) within the calibration range (0.5-60 ppm). Accuracy and precision values fulfilled the required limits. Liver concentrations of rifampicin in liver tissue, obtained after 60 min perfusion with 10 μM and 50 μM of rifampicin, were 45.1 ± 11.2 and 313.4 ± 84.4 μM, respectively. Conclusion: The bioanalytical method validation was completed and the method was successfully applied for the determination of rifampicin in rat liver tissue.

Dexmedetomidine pretreatment alleviates ischemia reperfusion injury-induced inflammatory reaction via inhibition of TLR-4/NF-ΚB signaling pathway

Purpose: To study the effect of dexmedetomidine on ischemia-reperfusion injury (IRI)–induced inflammatory response, as well as its underlying mechanism of action.&#x0D; Methods: Three groups of healthy adult Sprague Dawley (SD) rats (mean weight, 275 ± 10 g): control, IRI and treatment groups were used. With the exception of control group, ligation was performed on left anterior descending coronary arteries for 30 min and blood perfusion was restored within 100 min to establish IRI. Control group rats were without left ligation. Rats in control and IRI groups received normal saline intraperitoneally 30 min prior to surgery, while the treatment group received 100 μg/kg dexmedetomidine via intraperitoneal injection 30 min before operation. Infarct volume was determined using triphenyl tetrazolium chloride (TTC) staining. IL-6) and TNF-α levels of myocardial tissues and serum were determined using enzyme-linked immunosorbent assay (ELISA). Western blotting was used to determine protein expressions of NF-ΚB and TLR-4 in myocardial tissues.&#x0D; Results: Pretreatment with dexmedetomidine markedly decreased infarct volume caused by IRI (p &lt; 0.05). Serum and myocardial TNF-α and IL-6 were significantly upregulated in IRI group, relative to control group, but were downregulated by pretreatment with dexmedetomidine (p &lt; 0.05). There was marked upregulation of NF-ΚB and TLR-4 proteins in IRI rats, relative to untreated rats (p &lt; 0.05). Dexmedetomidine also down-regulated the expressions of these proteins (p &lt; 0.05).&#x0D; Conclusion: Pretreatment with dexmedetomidine alleviates IRI-induced inflammatory reaction via suppression of TLR-4/NF-ΚB signaling pathway. This finding provides a basis for large-scale clinical trials with dexmedetomidine.

Treating Ischemically Damaged Porcine Kidneys with Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stromal Cells During Ex Vivo Normothermic Machine Perfusion.

Pretransplant normothermic machine perfusion (NMP) of donor kidneys offers the unique opportunity to perform active interventions to an isolated renal graft before transplantation. There is increasing evidence that mesenchymal stromal cells (MSCs) could have a paracrine/endocrine regenerative effect on ischemia-reperfusion injury. The purpose of this study was to determine which cytokines are secreted by MSCs during NMP of a porcine kidney. Viable porcine kidneys and autologous whole blood were obtained from a slaughterhouse. Warm ischemia time was standardized at 20 min and subsequent hypothermic machine perfusion was performed during 2-3 h. Thereafter, kidneys were machine perfused at 37°C during 7 h. After 1 h of NMP, 0, 107 cultured human adipose tissue-derived MSCs, or 107 cultured bone marrow-derived MSCs were added (n = 5 per group). In a fourth experimental group, 7-h NMP was performed with 107 adipose tissue-derived MSCs, without a kidney in the circuit. Kidneys perfused with MSCs showed lower lactate dehydrogenase and neutrophil gelatinase-associated lipocalin levels in comparison with the control group. Also, elevated levels of human hepatocyte growth factor, interleukin (IL)-6, and IL-8 were found in the perfusate of the groups perfused with MSCs compared to the control groups. This study suggests that MSCs, in contact with an injured kidney during NMP, could lead to lower levels of injury markers and induce the release of immunomodulatory cytokines.

Micromolar concentrations of Zn2+ depress cellular excitability through a blockade of calcium current in rat adrenal slices

The present work, using chromaffin cells in rat adrenal slices (RCCs), aims to describe what type of ionic current alterations induced by zinc underlies their effects reported on synaptic transmission. Thus, Zn2+ blocked calcium channels of RCCs in a time- and concentration-dependent manner with an IC50 of 391 μM. This blockade was partially reversed upon washout and was greater at more depolarizing holding potentials (i.e. 32 ± 5% at −110 mV, and 43 ± 6% at -50 mV, after 5 min perfusion). In ω-toxins-sensitive calcium channels (N-, P- and Q-types), Zn2+caused a lower blockade of ICa, 33.3%, than in ω-toxins-resistant ones (L-type, 55.3%; and R-type, 90%). This compound inhibited calcium current at all test potentials and shows a shift of the I-V curve to more depolarized values of about 10 mV. The sodium current was not blocked by acute application of high Zn2+concentrations. Voltage-dependent potassium current was marginally affected by high Zn2+ concentrations showing no concentration-dependence. Nevertheless, calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 453 μM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, RCCs exhibit a resting potential of -50.7 mV, firing spontaneous APs (1–2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of voltage and Ca2+-activated K+-channels (BK). We found that the blockade of these ionic currents by Zn2+ led to a drastic alteration of cellular excitability with a depolarization of the membrane potential, the slowdown and broadening of the APs and the severe reduction of the after hyperpolarization (AHP) which led to a decrease in the APs firing frequency. Taken together, these results point to a neurotoxic action evoked by zinc that is associated with changes to cellular excitability by blocking the ionic currents responsible for both the neurotransmitter release and the action potentials firing.

SUN-717 SG-2 a Novel Multi-Target Directed Ligand (MTDL) for the Treatment of Neurodegenerative Diseases (NDDS)

NDDs are progressive multifactorial disorders that impair memory, cognition, movements, and general functioning. This deterioration is mostly due to inflammation triggered by aberrant protein deposition, oxidative stress and modification in lipid pathways. Because of these multifactorial aspects, the development of multi-target directed ligand (MTDL) could represent a potential strategy for the treatment of NDDs.Recently, the thyronamine-like analog SG-2, originally developed as a synthetic TAAR1 agonist, has revealed to efficiently reprogram lipid metabolism and to produce memory-enhancement in mice1. Long-term potentiation (LTP) is one of the basic mechanisms of memory. LTP is inhibited by beta-Amyloid oligomers (Aβ), and in the early stage of AD it is selectively impaired in the entorhinal cortex (EC).In the present study, to further expand our knowledge on the potential of this novel analog to act as a neuroprotective agent, we investigated if administration of SG-2 has any effect on LTP in EC of a transgenic model of AD (hAPP-J20 mouse).Extracellular in vitro recordings were performed in EC slices from 2 month-old APP-J20 mice: field potentials were evoked in layer II after stimulation of the same layer and LTP was elicited by high frequency stimulation (HFS), consisting of three trains of 100 pulses at 100Hz. SG-2 (1 or 5μM) was administered for 10 minutes, starting 5 minutes before the delivery of HFS.LTP cannot be elicited by HFS in mhAPP slices perfused with artificial cerebrospinal fluid (ACSF) alone. When we tried to rescue LTP in mhAPP slices using SG2 at the lowest concentration (1µM), 10 min perfusion with SG2 was not effective. In contrast, a higher concentration of SG2 (5 μM), rescued LTP to a level that was significantly higher than that observed in mhAPP slices alone (n=6; p=0.046), as well as in mhAPP slices perfused with SG2 1 μM (n=5; p=0.043). Our results suggest that SG-2 plays a neuroprotective effect, rescuing Aβ-induced neuronal dysfunction and might open new perspective in the study of AD. Metabolic reprogramming and neuroprotective functions for the histone deacetylase SIRT6 are well known, and a reduction of SIRT6 expression has been observed in patients with AD. Exposure of human neuroblastoma (SH-SY5Y) cells to SG-2 (1 or 10 μM) resulted in significant (p=0.044) over-expression of SIRT6, and concomitant activation of AMPK leading to the inhibition of mTOR phosphorylation, further underlying potential for SG-2 as a multi-target neuroprotective ligand.Reference: (1) Bellusci et al. Frontiers in Pharmacology 2017; 8: 905.

A127 THE ROLE OF ENDOGENOUS OPIOIDS IN MITIGATING OPIOID TOLERANCE IN A PRE-CLINICAL MODEL OF IBD

Abstract Background While opioid drugs are used to treat pain in IBD patients, escalating doses can induce tolerance and paradoxical increased pain signaling. Endogenous opioids are released by immune cells during chronic colitis and have an analgesic action but it is unknown whether they can mitigate the development of tolerance to opioid drugs in IBD. Aims To evaluate whether endogenous opioids released during chronic inflammation prevent the development of tolerance and/or increased pain signaling following chronic morphine treatment. Methods Three groups of C57BL/6 mice were studied: 1) control, 2) chronic colitis induced with 3 cycles of 2% DSS (cDSS mice) and 3) chronic DSS mice injected with escalating doses of morphine on the last 7 days of DSS (10, 20, 30 mg/kg for the first 3 days, then 40mg/kg on 4 to 7 day) (cDSS-morphine mice). Ex vivo afferent nerve recordings were obtained from flat-sheet distal colon preparations (day 31) to determine their mechanosensitivity by probing with a 1g von Frey hair before and after acute morphine (1μM, 10 min perfusion). In parallel experiments, supernatants were obtained from the proximal colon and incubated overnight with isolated dorsal root ganglia (DRG) neurons from control mice. Neuronal excitability was then examined by measuring the rheobase using perforated patch clamp. Results In afferent nerve recordings, acute application of 1μM morphine inhibited the colonic afferent response to probing in control mice (17.86 vs 12.31 Hz; p&amp;lt;0.05), whereas no effect of morphine was seen in cDSS mice. In cDSS-morphine mice acute morphine perfusion paradoxically increased the mean afferent response to probing (13.99 vs 17.28 Hz, p&amp;lt;0.05). Single units were analyzed to identify how many mechanosensitive responses were increased, decreased or not affected by acute morphine perfusion. Compared to control mice, cDSS mice exhibited increased numbers of unaffected units (6/18 vs 1/14) whereas cDSS-morphine mice had decreased number of inhibited units (1/17 vs 11/14) and increased number of excited units (8/17 vs. 2/14) (Figure 1). In patch clamp recordings, colonic supernatants from cDSS mice reduced the excitability of DRG neurons (rheobase increased 28%; p&amp;lt;0.05), however, this effect was lost in neurons incubated with cDSS-morphine supernatants. Conclusions Endogenous opioids released during cDSS colitis do not mitigate tolerance to chronic morphine exposure and may diminish the response to acute morphine. Moreover, the paradoxical hyperexcitable response to acute morphine induced by chronic morphine was not blocked by endogenous opioids. Our patch clamp data suggest that endogenous opioid actions are lost following chronic morphine exposure and could result from inhibition of their release from immune cells. Funding Agencies CCC

A54 DELTA OPIOID RECEPTOR SIGNALING MEDIATES OPIOID INDUCED TOLERANCE AND HYPERALGESIA IN COLONIC SENSORY NERVES

Abstract Background While opioid drugs are widely used for treating abdominal pain, prolonged exposure to opioids can induce tolerance and paradoxically increase pain. We previously showed in colonic afferent nerves that chronic morphine treatment causes tolerance and paradoxical hyperalgesia, however the mechanisms are unknown. Aims To evaluate the role of delta opioid receptor (DOR) signaling in opioid induced tolerance and hyperalgesia in colonic nerves during chronic morphine exposure. Methods C57BL/6 mice were injected (i.p.) twice daily for 7 days with escalating doses of morphine (10, 20, 30 mg/kg for the first 3 days, then 40mg/kg on 4th to 7th day); a subset of mice were also injected with 2.5 mg/kg naltrindole (NTI), a DOR antagonist. The analgesic response in vivo was monitored daily using the tail-flick test. To assess the effect of chronic morphine exposure, mice were euthanized on day 8, and the dorsal root ganglia (DRGs) and colons were harvested. Isolated DRG neurons were incubated with 1μM morphine (30 min) and neuronal excitability was determined by measuring the rheobase (amount of current required to elicit an action potential) using perforated patch clamp. To assess changes in colonic afferent nerve excitability, ex vivo afferent nerve recordings were obtained from flat-sheet colon preparations to determine the effects of 1μM morphine (10 min perfusion) on mechanosensitivity by probing with 1g von Frey hair. Results The tail flick test showed that the DOR antagonist NTI inhibited (30%) the maximal antinociceptive effect of morphine and reduced morphine tolerance. In patch clamp recordings, 1μM morphine paradoxically increased the excitability of small DRG neurons from morphine mice (rheobase decreased 28%; p&amp;lt;0.05, 2-way ANOVA), whereas this excitatory effect was absent on neurons from NTI+morphine mice. Acute application of 1μM morphine had no effect on afferent nerve responses to probing in morphine mice (14.9 vs 16.7 Hz; p=ns, paired t test, n=15) whereas it inhibited the afferent nerve response to probing in NTI+morphine mice (15.2 vs 9.9 Hz; p&amp;lt;0.001, paired t test, n=16). Furthermore, most single units from morphine mice had an increased response to probing following acute morphine application (7/15 vs 2/16 units excited), whereas most units form NTI-morphine mice were inhibited (14/16 vs. 3/15 units inhibited, P&amp;lt;0.001, Chi-square). Conclusions These findings suggest that DOR signaling plays a significant role in the development of opioid tolerance and hyperalgesia in colonic afferent nerves induced by chronic morphine treatment. Thus, antagonists of DOR may help to mitigate these side effects induced by opioids. Funding Agencies CCC

Mechanical Postconditioning Promotes Glucose Metabolism and AMPK Activity in Parallel with Improved Post-Ischemic Recovery in an Isolated Rat Heart Model of Donation after Circulatory Death

Donation after circulatory death (DCD) could improve donor heart availability; however, warm ischemia-reperfusion injury raises concerns about graft quality. Mechanical postconditioning (MPC) may limit injury, but mechanisms remain incompletely characterized. Therefore, we investigated the roles of glucose metabolism and key signaling molecules in MPC using an isolated rat heart model of DCD. Hearts underwent 20 min perfusion, 30 min global ischemia, and 60 minu reperfusion with or without MPC (two cycles: 30 s reperfusion—30 s ischemia). Despite identical perfusion conditions, MPC either significantly decreased (low recovery = LoR; 32 ± 5%; p < 0.05), or increased (high recovery = HiR; 59 ± 7%; p < 0.05) the recovery of left ventricular work compared with no MPC (47 ± 9%). Glucose uptake and glycolysis were increased in HiR vs. LoR hearts (p < 0.05), but glucose oxidation was unchanged. Furthermore, in HiR vs. LoR hearts, phosphorylation of raptor, a downstream target of AMPK, increased (p < 0.05), cytochrome c release (p < 0.05) decreased, and TNFα content tended to decrease. Increased glucose uptake and glycolysis, lower mitochondrial damage, and a trend towards decreased pro-inflammatory cytokines occurred specifically in HiR vs. LoR MPC hearts, which may result from greater AMPK activation. Thus, we identify endogenous cellular mechanisms that occur specifically with cardioprotective MPC, which could be elicited in the development of effective reperfusion strategies for DCD cardiac grafts.

EFFECT OF PULSATILE FLOW ON RENAL FUNCTION IN ELDERLY PATIENTS UNDERGOING CORONARY ARTERY BYPASS SURGERY

The incidence of acute kidney injury (AKI) after on-pump coronary artery bypass graft (CABG) varies among studies but can be reduced if pulsatile flow is used. The aim of this study is to evaluate pulsatile flow impact on renal function of elderly patients undergoing CABG. Over one year (April 2014 to April 2015) 48 patients above the age of 65 underwent on-pump CABG in our institute. Patients were divided into two groups; pulsatile flow (PFG) and non-pulsatile flow (NPFG) groups. Serum creatinine (S.Cr), creatinine clearance (Cr.Cl) and per-perfusion urine output (UO) were measured. AKI Network criteria were adopted for diagnosis. Mean age was 68 in PFG and 69 in NPFG. Males constituted 83.3% of PFG and 79.2% of NPFG. Although 37.5% of PFG and 41.7% of NPFG were hypertensive, all patients had normal ejection fraction (EF). Both groups had nearly 3 coronary anastomoses, cardiopulmonary bypass (CPB) time of 90 min, cross clamp time of 71 min and mean perfusion pressure of 70 mmHg. Mean S.Cr was the same (0.8 mg/dl) in both groups on 1st postoperative day (POD) but UO was significantly larger (708 ml in PFG vs. 648 ml in NPFG). On 3rd POD, S. Cr didn’t change in PFG but it has significantly increased in NPFG (from 0.76 to 1.0 mg/dl). Moreover, Cr.Cl has significantly improved in PFG (81 vs. 72 ml/min in NPFG). Seven of 48 patients (14.6 %) developed AKI (6; 25% in NPFG). In conclusion; Pulsatile perfusion technique is a simple and safe measure to minimize AKI in the elderly. Keywords: Coronary artery bypass graft, elderly, cardiopulmonary bypass, pulsatile flow, non-pulsatile flow, acute kidney injury, serum creatinine, creatinine clearance

Storage-Induced Micro-Erythrocytes Are Rapidly Cleared from Recipient Circulation and Predict Transfusion Recovery

Background Hypothermic storage of red blood cell (RBC) concentrates for up to 42 days is associated with biochemical, molecular, morphological, and mechanical modifications. This "storage lesion" increases with storage duration and is associated with increased clearance of transfused storage-damaged RBCs from the recipient's circulation in the first few hours post-transfusion. This rapid clearance reduces transfusion efficacy, but how it occurs is not fully elucidated. RBCs with reduced surface area called "storage-induced micro-erythrocytes" (SMEs) were recently described. Their proportion increases from 2% to 23% during storage. Their reduced surface-to-volume ratio is expected to induce rapid mechanical clearance by the spleen. We aimed to evaluate whether SMEs can be used as a marker of transfusion efficacy, if this subpopulation of RBCs is preferentially cleared by the spleen after transfusion, and if so, by which mechanisms. Methods We evaluated the proportion of SMEs in stored RBC concentrates in vitro using ImageStream and correlated it to the 51Chromium-labeled 24h post-transfusion recovery (24hPTR) in vivo in 31 healthy human volunteers. We then investigated the fate of SMEs during 8 ex vivo perfusions of human spleens (16 RBC concentrates stored for 35-42 days). Finally, we developed a mouse transfusion model to assess the fate of SMEs in vivo and determine their main mechanisms of clearance. Results The proportion of SMEs in RBC concentrates at day 42 of storage correlated negatively with 24hPTR in healthy volunteers (r=-0.42, P&amp;lt;0.01). When perfused ex vivo into human spleens, 15% of stored RBCs (35-42 days of storage) were cleared during the first 40 min of perfusion in a 2-step process: 7% of circulating RBCs disappeared in the first 2 min (1-2 passages through the spleen) while 8% were cleared between 10 and 40 min after initiating perfusion (&amp;gt;5 passages through the spleen). The percentage of SMEs correlated with splenic retention rate ex vivo (r=0.46, p&amp;lt;0.05). Morphological analysis of 6 stored RBC concentrates showed a mean decrease in the proportion of SMEs from 20.2% to 7.8% between the beginning and end of splenic perfusions. In our mouse transfusion model, SMEs accumulated during RBC storage. The 24hPTR also decreased with storage duration (64% on Day 14 vs. 95% on Day 1). The decrease in 24hPTR of long-stored RBCs was mostly due to clearance of the SME subpopulation. SME and morphologically normal long-stored RBC subpopulations displayed clearances of 83% and 13%, respectively. Stored RBCs accumulated predominantly in the spleen post-transfusion, and were mainly ingested by macrophages. In macrophage-depleted mice, 24hPTR improved (from 64% to 79%), splenic accumulation and clearance of SMEs were delayed, and the proportion of inflammatory monocytes increased and mediated clearance. In splenectomized mice, clearance of SMEs was not delayed, but increased accumulation was observed in the liver and bone marrow, and increased erythrophagocytosis by inflammatory monocytes was also observed. Conclusions We show that the proportion of SMEs correlates with 24hPTR in healthy human volunteers and with retention in human spleens perfused ex vivo. In vivo mouse data confirms these findings, showing that SMEs are cleared from the recipient circulation during the 24h following transfusion. Clearance of SMEs is delayed in macrophage-depleted mice, suggesting a central role of macrophages in this process. The human spleen is also likely to clear SMEs from the recipient's circulation, as suggested by experiments with human spleens perfused ex vivo. However, the spleen is not required, because SME clearance is not affected in splenectomized mice. This suggests that other organs may compensate to remove SMEs and highlights the importance of eliminating these morphologically-altered RBCs. Finally, quantification of SMEs is an operator-independent, reproducible marker of transfusion efficacy. It can be used to assess the potential of new processes to prepare and store RBC concentrates. Pre-transfusion quantification of SMEs could benefit chronically transfused patients, for whom improved transfusion efficacy is expected to reduce transfusion-induced iron overload. Disclosures Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

Transient hyperthermia during oxygenated rewarming of isolated rat livers.

Pretransplant machine perfusion of donor grafts has gained clinical appreciation to improve graft function and survival after transplantation. This study was aimed as pilot investigation to evaluate the additive potential of a transient ex vivo heat shock treatment of the isolated organ during machine perfusion to further protect the graft from subsequent reperfusion injury. Rat livers were retrieved after 20min of cardiac arrest and preserved for 18h by cold storage in HTK solution. Prior to reperfusion, livers were subjected to 2h of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35°C. In half of the livers (n=7), a brief hyperthermic impulse (10min perfusion at 42°C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Induction of heat shock protein 70 was followed on the mRNA and protein level. Chaperone induction by transient hyperthermia was associated with a significant improvement of bile production upon reperfusion and significantly reduced enzyme loss of mitochondrial GLDH. Heat shock treatment further affected pro-inflammatory upregulation in the graft in significantly reducing gene expression as wells as protein release of TNF-alpha. It is concluded, that graft conditioning by controlled hyperthermia ex vivo may represent a feasible and useful tool to improve liver recovery after preservation.