Tetrazolium Chloride Research Articles

Ubiquitin-specific protease 11 Aggravates Ischemia-reperfusion-induced Cardiomyocyte Pyroptosis and Injury by Promoting TRAF3 Deubiquitination

In myocardial ischemia-reperfusion injury, myocardial damage is aggravated when blood perfusion is restored in myocardial infarction. Ubiquitin-specific protease 11 (USP11), a deubiquitinating enzyme, could remove the ubiquitination of substrate proteins and regulate protein stability, thereby affecting multiple pathological processes. This study aimed to investigate the potential function of USP11 in myocardial ischemia-reperfusion injury and its underlying mechanisms. In vivo and in vitro experimental study. The ischemia-reperfusion rat model in vivo was evolved, wherein the left anterior descending coronary artery was ligated for 30 min, followed by ligature release for 120 min. Meanwhile, H9C2 cells were brought to hypoxia for 6 h and then reoxygenated for 18 h to establish a cell hypoxia-reoxygenation (H/R) injury in vitro. Then, the loss-of-function experiments of USP11 were performed. Triphenyltetrazolium chloride and hematoxylin and eosin staining were performed to observe myocardial injury. The MTT assay was utilized to detect H9C2 cell viability. Pyroptosis was analyzed by TUNEL staining and flow cytometry. Pyroptosis-related protein expression and TRAF3 were analyzed via Western blot. The content of inflammatory factors was examined by enzyme-linked immunoassay. Co-immunoprecipitation and ubiquitination assays were performed to analyze for USP11 interacting with TRAF3. USP11 was upregulated in the ischemic heart tissue. Ischemia-reperfusion and H/R injuries increased USP11 expression. USP11 loss-of-function assays showed that USP11 knockdown alleviated ischemia-reperfusion- and H/R-induced myocardial cell damage, pyroptosis, pro-inflammatory factor secretion, and IKKβ/NF-κB pathway activation. In H9C2 cells, USP11 stabilized TRAF3 by deubiquitination. Furthermore, rescue experiments revealed that TRAF3 overexpression reversed the protection of silencing USP11 on H/R-induced H9C2 cell injury. This study confirmed that USP11 knockdown ameliorated myocardial ischemia-reperfusion injury by downregulating TRAF3, suggesting that USP11 silencing can be a novel target of myocardial infarction.

PO-04-071 PULSE FIELD ABLATION AND LESION FORMATION USING A UNIQUE INTEGRATED MULTIELECTRODE FOCAL, LINEAR, AND LOOP ABLATION CATHETER

Pulse field ablation (PFA) is a promising energy modality for cardiac ablation due to its speed, tissue selectivity, and potential ability to create consistent transmural lesions Evaluate PFA lesions created using an innovative catheter design combining focal, linear and pulmonary vein (PV) ablation PEF ablation was performed using an 8F bidirectional linear catheter with a 2mm tip and 4 ring electrodes through which a 3.5F loop catheter (6 to 9 rings, 3mm length on 19mm diameter) can be inserted and deployed beyond the tip (Figure, A). In 8 swine, R-wave gated biphasic pulse trains of voltage between 600 and 700V were delivered sequentially and simultaneously to the loop or linear catheter electrode pairs. The linear catheter is compatible with any commercially available 8.5F transseptal sheath. Porcine intracardiac tissue was ablated in vivo targeting the superior vena cava (SVC), right atrium (RA), RA appendage (RAA), cavotricuspid isthmus, left atrium, PVs, and right ventricle. Electrogram (EGM) amplitudes/morphology and pre- and post-ablation pacing thresholds were assessed. Triphenyl tetrazolium chloride (infused 20 minutes before euthanasia) or tetrazolium blue stain was used to demarcate and measure the lesions. Histopathologic analysis of all cardiac tissues was performed, esophageal pre and post ablation phrenic nerve stimulation were done. EGM attenuation and pacing thresholds increase were documented (see Table). Focal: PFA was delivered from the distal electrode pair of the linear catheter. Lesion measured 10.3±2.6 mm diameter, 3.15±1.56 mm depth (Figure, B). Linear: PFA was delivered to 5 electrodes of the linear catheter (Figure, C). Lesion dimension range: width: 4 - 20mm, length: 12 to 20mm. Loop: PFA was delivered sequentially via loop catheter electrode pairs to PVs resulting in transmural lesions with 2.02 ± 0.94mm depth measured at the thickest area of affected myocardium (Figure, D). Simultaneous biphasic PEF was delivered by loop electrode pairs to the RAA and SVC, resulting in transmural lesions. RAA lesion measured 132mm2 surface area with average EGM attenuation 87% ± 2% and pacing thresholds increased post ablation (p=0.00), 4.0 ± 2.1 mA pre, 18.5 ± 4.2 mA post (Figure, E) No esophageal or phrenic injuries were documented. The combined linear, focal, and circular PFA ablation device demonstrated acute lesion success and each configuration has the potential to create effective transmural lesions in the atrium.Tabled 1TableEGM % Reduction*Pacing Threshold (mA)*Lesion DimensionsFocal lesion in atrial tissue88% ± 3.8% (N=7)Pre-ablation: 2.23 ± 1.83 mAPost-ablation: 19.3 ± 2.23 mA (N=9)Diameter: 10.3 ± 2.6 mm (N=15)Surface area: 87.6 ± 41.8 mm2 (N=15)Depth: 3.15 ± 1.56 mm (N=11)**10/11 lesions transmural (1 lesion was not assessed).Linear lesions in RA and LA95% ± 5% (N=7)Pre-ablation: 2.02 ± 1.78 mAPost-ablation: 15.1 ± 5.92 mA (N=29)Width: 8.86 ± 4.93 mm (N=9)Length: 17.17 ± 3.71 mm (N=9)Surface area: 156 ± 103 mm2 (N=9)Loop lesions in SVC and PV88% ± 5% (N=53)Pre-ablation: 3.5 ± 2.8 mAPost-ablation: 18.3 ± 3.2 mA (N=80)Transmural lesions,Depth: 2.02 ± 0.94mm (N=10) Open table in a new tab

PO-02-181 THE CHARACTERISTICS OF TEMPERATURE-CONTROLLED ABLATION: WHAT’S OLD IS NOW NEW

More than 20 years ago, temperature was used to guide ablative intervention. This was subsequently replaced by power to drive intervention. Recently, temperature-guided ablation has been reintroduced into the ablation armament. But the anatomic and electrical characteristics of currently available temperature-controlled ablation is unclear. To verify the DiamondTemp temperature-controlled ablation system in the ventricle Both ventricles of canines (33.6kg [32.4-39.4]) were ablated using the open-irrigated temperature-controlled DiamondTemp ablation system controlling power to maintain temperature set-point and followed for one month. The temperature set points for the ablation were set at 50 and 60°C and the ablation duration was set at 30-90 sec. After ablation of the right and left ventricles, gadolinium and 8-10% triphenyl tetrazolium chloride were administered intravenously; postmortem delayed contrast-enhanced magnetic resonance (MR) imaging was performed on a 3 Tesla device, fixed with 10% formalin solution, and pathological examination was performed. 8 animals underwent evaluation of temperature-guided ablation with 65 lesions created. Lesions were well distributed throughout the myocardium. The lesion sites and number for the set temperature are as shown in the table. The power used to create these lesions is also as shown in the table. As can be seen, there is appreciable variability in actual power required to achieve and maintain a particular temperature set point. Power required to maintains the set point is 28.3±10.4(W) in set point 50°C and 42.1±8.7(W) in set point 60°C. Lesion size is also as noted in the table where the 50°C group produced smaller lesion than the 60°C group (p<0.001), the gross pathological lesion volumes were smaller than the MR lesion volumes (p<0.001) and the RV lesions tended to be smaller than the LV lesions due to LV wall thickness. (p=0.095). In looking at all lesions, the maximum depth was 8.1±2.1 mm, the depth at maximum width was 3.1±1.9mm and the maximum width at any site was 8.4±2.1mm. At a set point of 50°C, transmural lesions were seen in 12.9% in 50.0 (%) at a set point 60°C (p=0.001). These data demonstrate that in in situ deliveries, the biophysics of ablation using a temperature-controlled RF ablation system can be effective and accurate in safely creating lesions of required size. The lesion size with a set point of 60°C was more effective than the set point 50°C, with good control.

PO-02-076 AN INVESTIGATION OF LESION EFFICACY AND SAFETY WITH THE TACTIFLEX™ SE ABLATION CATHETER

Treatment of cardiac arrhythmias by the creation of myocardial lesions utilizing RF current is well established as an effective mode of therapy in the clinical electrophysiology laboratory. Efficacy, however, should be evaluated in concert with safety considerations, as passive resistance in the cardiac tissue creates heating that can lead to adverse events (e.g., steam pops). A bench top bovine tissue model allows for comparative testing of RF ablation with the TactiFlex™ SE Ablation Catheter (test device) and a commercially available solid-tip RF ablation catheter with uniform irrigation profile (control device), analyzed with respect to both efficacy and safety. To generate comparative statistics for lesion dimension (maximum width and depth) and steam pop likelihood for the TactiFlex™ SE and control RF catheter designs. In this bench ablation model, recently harvested bovine ventricles were sectioned and placed in a body-temperature saline solution (∼37oC). A catheter in a holding fixture was placed in contact with the tissue at a predefined contact force using an independent force gauge. RF energy was applied to the tissue; lesions (N=288 trials) were created at three different power levels (30W at 30s or 50s, 40W at 12s or 20s, 50W at 6s and 10s), steam pop occurrences were noted. Applied contact force levels were fixed at either 5g or 20g. After ablation, the tissue was stained with 1% Triphenyltetrazolium chloride (TTC). Lesions were measured with respect to maximum width and depth. A linear regression model was applied for lesion width and depth vs. catheter design, power, duration, contact force and orientation. The trials did not result in significantly different widths between catheter designs (p = 0.137); refer to Figure 1 for dimensions vs. RF power and duration. Trials with the TactiFlex™ SE yielded shallower lesions (p=0.018) compared to the control device, but with a regression bias of only 0.2mm. Logistic regression demonstrated that steam pops were significantly less likely with the TactiFlex™ SE (p=0.002). Steam pops occurred in only 1.4% of trials with the TactiFlex™ SE Ablation Catheter (2/144 trials) versus 9.7% of trials with the control catheter design (14/144 trials). RF lesions can be created with the TactiFlex™ SE Ablation Catheter that are clinically similar in size to those created with the control device, with a significant reduction in steam pop likelihood.

Determination of Pollinator Characteristics of Some Hazelnut Genotypes

This research was carried out in Karasu, Kocaali and Arifiye districts of Sakarya Province to determine the suitable pollinators for Çakıldak hazelnut cultivar (Corylus avellana) in 2021-2022. In the study, 27 genotypes that were late male flowering, formed a large amount of catkins, had high pollen quality, had round nut shape and short husk length were examined. Pollen viability was detected according to 2, 3, 5,-Triphenyl Tetrazolium Chloride (TTC). Pollen viability of the genotypes was ranged from 22.3% to 93.7% according to the TTC method. According to the agar method, pollen germination rate was determined between 15.6% and 78.1% at 20% sucrose concentration containing 1% agar. 89% of the genotypes were round, 11% were in the oblong nut group. It was determined that most of them amount of male inflorescences (catkins) depending on the tree crown volume and age. T-22KRS02, T-22KRS03, T-22KRS07, T-22KRS08, T-22KRS09, T-22KRS10, T-22KRS11, T-22KCL11, T-22KCL14, T-22KCL16 and Mincane were selected to be evaluated in the second phase of the study.

The tetrazolium test: reviving an old but useful stress marker.

Adverse environmental conditions often compromise plant survival. Thus, stress monitoring with accurate and cost-effective technologies must be a priority to anticipate environmental threats, but processing time and the associated costs are still important limitations. Revisiting research technologies such as biochemical markers can provide straightforward data to foresee the effects of plant stressors. The tetrazolium test is based on the ability of living tissues to reduce a colorless salt, 2,3,5-triphenyl tetrazolium chloride (TTC), into the red compound formazan. It is not only useful for seed viability tests, but also for tissue damage assessments, as initially proposed by Larcher et al. (1969).

Protective effect and underlying mechanism of muscone on acute cerebral ischemia-reperfusion injury in rats.

Musk is a widely used traditional Chinese medicine, which has resuscitation, activating blood, and disperse swelling effects. Musk is commonly used in the prevention of myocardial infarction and ischemic stroke, and muscone is its main active component. The effect and mechanism of muscone to improve the condition of ischemic stroke is not clear, accordingly, we verified its efficacy in ischemia-reperfused rats, and investigated its mechanism by PC12 and THP-1cells. A transient middle cerebral artery occlusion (tMCAO) rat model was established for in vivo experiments. 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining was used to calculate infarct rate. Neuroprotection and angiogenesis were assessed by Hematoxylin-eosin (HE) staining, nissl staining, immunofluorescence staining, and quantitative real-time PCR (qRT-PCR). Oxygen glucose deprivation-reperfusion (OGD/R) model of PC12cells was established for neuroprotection analysis, where CCK-8 assay was used to measure cell viability, flow cytometry and Hoechst 33258 staining were used to demonstrate apoptosis, and protein levels were detected by Western blot. For angiogenesis analysis, enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were used to detect angiogenic factors expressed by THP-1. Cell viability assay, scratch wound assay, and tube formation assay were used to evaluate angiogenic effect of HUVECs treated with medium of THP-1. And the angiogenic pathway in HUVECs was detected by Western blot. According to the results, in cerebral ischemia-reperfusion rats, the infarct rate and tissue damage were significantly reduced by muscone, and the expression of neurotrophic factors and angiogenesis-related factors were all elevated. In OGD/R-PC12cell models, muscone could increase cell viability and inhibit apoptosis via Bax/Bcl-2/Caspase-3 pathway. In THP-1-mediated angiogenesis of HUVECs, muscone promoted the secretion of angiogenesis-related factors in THP-1 and thus indirectly promoted the proliferation, migration and tube formation of HUVECs, and then regulated phosphorylation of VEGFR2 and Akt in HUVECs. Our study indicated that muscone may be a potential neuroprotective and proangiogenic agent in cerebral ischemia.

PO-02-178 ABLATION TISSUE HISTOLOGY AND CORRELATION WITH MAGNETIC RESONANCE IMAGING WITH A DIAMOND-BASED, TEMPERATURE-CONTROLLED RADIOFREQUENCY ABLATION

A new catheter has been developed which uses diamonds embedded in the catheter tip to enable temperature-guided ablation. The degree to which subsequent Magnetic Resonance (MR) findings correlate with actual tissue pathology has never been assessed. To evaluate the correlation between MR imaging and lesions assessed by Gross pathology. 20 canines underwent evaluation, after ablation. 4 were studied acutely 4-6 hours after ablation, 8 animals were studied at a time of chronic assessment 4 weeks later, and 8 were studied at a time of chronic assessment 4 weeks later myocardial infarction creation. MR lesions were followed by intravenous gadolinium and 8-10% triphenyl tetrazolium chloride administration. Ex-vivo delayed contrast enhancement MR imaging on a 3 Tesla scanner (Signa HDxt, GE Healthcare) was performed followed by pathology examination after fixation in 10% formalin solution. Total of 111 lesions were created in LV and RV tissue. In the acute animals 30 deliveries were made to a temperature set-point of 50 or 60°C. The target temperatures were set at 60°C with 32 deliveries to healthy myocardium, and 8 to post-infarcted myocardium. Lesion size and characteristic are shown in table 1. The correlation between the actual lesion size and the MR contoured size is shown in Fig 1. In all animals taken together, MR proved to be an exceptional means of determining actual lesion size. While tissue histology, seen in the table, is more useful and accurate. Overall, the correlation R2 value is 0.72±0.11 (p<0.0001) in all groups. In individual groups, the correlation was 0.94±0.54 (p<0.0001) for acute, 0.48±0.13 (p<0.0001) for chronic healthy, and 0.97± 0.35 (p=0.0006) in infarcted tissue. Even with infarctions, the MR correlation remained consistent but lesions were smaller in infarcted myocardium. These data showed the characteristics of lesion size of correlation to energy delivered. MR with gadolinium is a very useful means of identifying actual lesion size in situ. This correlates well with actual tissue findings and disclose that heat penetration in infarcted tissue is less robust as seen with other catheter systems.

Electrical auricular tragus stimulation induces release of circulating cardioprotective factors in healthy human volunteers.

Background: Vagal activation during remote ischemic conditioning (RIC) with subsequent release of cardioprotective factors from the spleen into the circulation reduces infarct size. These humoral cardioprotective factors can be transferred with plasma-dialysate from donors undergoing RIC to isolated recipient hearts where they reduce infarct size. Electrical auricular tragus stimulation (ATS) has been demonstrated to reduce the cardiac injury biomarker release and the frequency of ventricular arrhythmias and to improve contractile function in patients with acute myocardial infarction. Aim: To determine whether ATS, as RIC, induces release of cardioprotective factors into the circulation. Methods: Healthy volunteers (3 females, 7 males, 26±5 years) were randomized to receive ATS or RIC, respectively, with an interval of at least two weeks between protocols. ATS was induced by bipolar electrical rectangular pulses of 200μs width at a frequency of 30 Hz (Curamed TEN630, CURAmed, Shenzen Dongdixin Technology, Shenzhen, China). The amplitude (5.5 ± 0.81 mA) was set for each volunteer to not be perceived as uncomfortable over the total stimulation period. A microprocessor-controlled circuit periodically interrupted the stimulation signal after 5 s of stimulation for 5 s. ATS was continued over 30 min. RIC served as a reference to induce the release of cardioprotective factors by 3 x 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation. Venous blood samples were taken before and 60 min after ATS or RIC, respectively, and used to prepare plasma-dialysates (1:10 dialysis against buffer for 24 h, cut-off 12-14 kDa). Male Lewis rats were sacrificed and their hearts isolated and perfused at constant pressure with buffer. Plasma-dialysates from samples taken before and 60 min after ATS or RIC, respectively, were infused for 8 min followed by 2 min washout into rat hearts before global 30 min ischemia/ 120 min reperfusion. Infarct size was demarcated by triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Results: With infusion of plasma-dialysate before ATS infarct size was 30.9±4.5% of ventricular mass and not different from that with infusion of plasma-dialysate before RIC (35.8±5.8%). Infusion of plasma-dialysate after ATS reduced infarct size to 18.3±3.9%. This cardioprotective effect was similar to that seen with infusion of plasma-dialysate after RIC (20.1±3.5%). Conclusion: ATS induces release of cardioprotective factors into the circulation of healthy volunteers. Plasma-dialysates after ATS or RIC, respectively, reduce myocardial infarct size by the same magnitude in ex vivo isolated perfused rat hearts with global ischemia/ reperfusion. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PO-04-063 EFFECT OF PULSED FIELD ABLATION ON THE BRONCHUS: PRECLINICAL INSIGHTS

The relative tissue selectivity of pulsed field ablation (PFA) allows for the safe delivery of ablation lesions even when in close proximity to collateral tissues such as the esophagus and phrenic nerve. Frequent coughing spells during PFA of the left atrium can be frequently seen during clinical procedures; there is little insight into the mechanism and effects on the bronchi. To assess the effect of PFA on the bronchus in swine. Microsecond pulses were delivered in a single swine under general anesthesia with fluoroscopic guidance. A standard ablation catheter (Blazer II, Boston Scientific, 7Fr and 4mm tip) and a commercially available microsecond pulse generator (ECM 830, BTX) were used. Bipolar, biphasic mode of microsecond pulse delivery (750kV) was applied with 5 repetitions of ∼1 second-long applications within the bronchus. Tetrazolium chloride staining was performed and the swine was humanely sacrificed after 2 days. Histological assessment was performed after formalin fixation with Hematoxylin and Eosin staining. A total of 7 PFA lesions were created in both the right and left bronchi. Only 2 of 7 lesions were identified at necropsy (Figure). The lesions were oval in shape and pink-white in the center surrounded by a zone of hyperemia. Histology demonstrated disappearance of cilia and mucosal cells at the site of the PFA lesions compared to the adjacent normal tissue. The layer of respiratory epithelium was denuded. Structures beneath the epithelium, including lamina propria, smooth muscles, mucosa glands, and cartilage were spared without evidence of any hemorrhage or inflammation. This exploratory sampling of bronchial susceptibility to PFA suggests minimal damage to the epithelium. Detailed post-PFA bronchoscopy studies would be necessary required to assess if such lesions are seen during left atrial ablation procedures.

Vitamin B12 alleviates myocardial ischemia/reperfusion injury via the SIRT3/AMPK signaling pathway

AimTo examine the protective effect of vitamin B12 against myocardial ischemia/reperfusion (I/R) injury and elucidate its underlying mechanism of action. MethodsMice were subjected to myocardial I/R injury by left anterior descending coronary artery (LAD) occlusion followed by 24 h reperfusion. Cardiac function and injury were evaluated by echocardiography, triphenyl tetrazolium chloride (TTC) and cardiac troponin T (cTnT) staining, and measuring lactate dehydrogenase (LDH) levels. In addition, various molecular and biochemical methods, as well as RNA sequencing were used to determine the effects and mechanism of action of vitamin B12 on I/R injury. ResultsWe found that high doses of vitamin B12 inhibited myocardial I/R injury. Furthermore, our data indicated that vitamin B12 supplementation alleviated cardiac dysfunction and injury by mitigating oxidative stress and apoptosis through downregulation of Nox2, the Ac-SOD2/SOD2 and Bax/Bcl-2 ratios and cleaved caspase-3 expression, and upregulation of SIRT3 expression and AMPK activity. However, these effects were largely reversed following treatment with the SIRT3 inhibitor, 3-TYP. Our RNA-sequencing data further demonstrated that vitamin B12 supplementation reduced inflammation during I/R injury. ConclusionHigh doses of vitamin B12 supplements improved myocardial I/R injury by suppressing the accumulation of reactive oxygen species and apoptosis of myocardial tissue through modulation of the SIRT3/AMPK signaling pathway, while reducing inflammation. Our findings suggested that vitamin B12 administered at high doses could be a potential therapy for myocardial I/R damage.

Liensinine ameliorates ischemia-reperfusion-induced brain injury by inhibiting autophagy via PI3K/AKT signaling.

The current study aimed to explore the role of autophagy in cerebral ischemia-reperfusion injuries (CIRI) and elucidate the efficacy of liensinine treatment. An in vitro ischemia-reperfusion (I/R) neuronal cell model was established and pretreated with liensinine or rapamycin (RAPA). Cell proliferation and survival were detected using a cell counting kit-8 (CCK-8) assay, while cell damage and apoptosis were detected using the lactate dehydrogenase (LDH) leakage rate and flow cytometry. Autophagy activity was detected using monodansylcadaverine (MDC) staining. Thereafter, I/R models were established in vivo in rats and the presence of neurological deficits was examined. Hematoxylin-eosin (HE) and triphenyl tetrazolium chloride (TTC) staining was used to detect pathological damage in brain tissue and the volume ratio of the cerebral infarction. The levels of PI3K/AKT pathway-related proteins and autophagy-related proteins (mTOR, LC3, P62, and TSC2) were detected using Western blot. The findings showed that liensinine treatment increased cell viability, decreased cell injury and apoptosis, and inhibited autophagy. The addition of RAPA to promote autophagy inhibited cell viability and enhanced cell injury and apoptosis. The I/R rats in the model group exhibited deficient neurological function, while those in the liensinine treatment group showed restoration of normal neural function and reduction of the necrotic area and infarct volume ratio in the brain tissue. Furthermore, liensinine treatment also inhibited the PI3K/Akt pathway activity and autophagy. However, addition of RAPA reversed the effects of liensinine treatment and aggravated brain tissue injury. Therefore, liensinine can play a neuroprotective role in CIRI by inhibiting autophagy through regulation of the PI3K/Akt pathway.

Geraniol protects hippocampal CA1 neurons and improves functional outcomes in global model of stroke in rats.

Geraniol (GE), an acyclic monoterpene, is a chief constituent of essential oils of herbs and fruits. It possesses diverse pharmacological actions like antioxidant, anti-inflammatory, anti-apoptotic, and anti-parkinson. However, its neuroprotective potential in stroke is yet to be explored at large. The present study evaluated the neuroprotective potential of GE against the global model of cerebral ischemia/reperfusion (I/R)-injury in rats. Bilateral common carotid artery (BCCA) occlusion for 30 min followed by 7 days of reperfusion caused varied biochemical/enzymatic alterations viz. increase in levels of lipid peroxidation (LPO), nitric oxide (NO), xanthine oxidase (XO), and decrease in the levels of cerebroprotectives like superoxide dismutase (SOD), catalase (CAT), total thiols, and glutathione (GSH). GE-pretreatment markedly reversed these changes and restored the levels of protective enzymatic and non-enzymatic antioxidants near to normal compared to I/R group. Besides, GE treatment showed marked improvement in anxiety-related behavior and neuronal deficits in animals subjected to I/R injury. Moreover, 2,3,5-triphenyl tetrazolium chloride (TTC)-stained rat brain coronal sections and histopathological studies revealed neuronal protection against I/R-injury, as evidenced by a reduction in infarct area (%) and an increase in hippocampal CA1 neuronal density in the GE-treated groups. The results of this study revealed that GE exhibited potential neuroprotective activity by reducing oxidative stress and infarction area, and protecting hippocampal CA1 neurons against I/R-injury in the global stroke model in rats.

CircHECTD1 promoted MIRI-associated inflammation via inhibiting miR-138-5p and upregulating ROCK2.

Myocardial ischemia-reperfusion injury (MIRI) was often observed after surgeries, causing a lot of suffering to patients. Inflammation and apoptosis were critical determinants during MIRI. We conveyed experiments to reveal the regulatory functions of circHECTD1 in MIRI development. The Rat MIRI model was established and determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We analyzed cell apoptosis using TUNEL and flow cytometry. Proteins expression was evaluated by western blot. The RNA level was determined by qRT-PCR. Secreted inflammatory factors were analyzed by ELISA assay. To predict the interaction sequences on circHECTD1, miR-138-5p, and ROCK2, bioinformatics analysis was performed. Dual-luciferase assay was used to confirm these interaction sequences. CircHECTD1 and ROCK2 were upregulated in the rat MIRI model, while miR-138-5p was decreased. CircHECTD1 knockdown alleviated H/R-induced inflammation in H9c2 cells. Direct interaction and regulation of circHECTD1/miR-138-5p and miR-138-5p/ROCK2 were confirmed by dual-luciferase assay. CircHECTD1 promoted H/R-induced inflammation and cell apoptosis by inhibiting miR-138-5p. miR-138-5p alleviated H/R-induced inflammation, while ectopic ROCK2 antagonized such effect of miR-138-5p. Our research suggested that the circHECTD1-modulated miR-138-5p suppressing is responsible for ROCK2 activation during H/R-induced inflammatory response, providing a novel insight into MIRI-associated inflammation.

Diazoxide is a powerful cardioprotectant but is not feasible in a realistic infarct scenario.

Diazoxide is a powerful cardioprotective agent that activates mitochondrial ATP-dependent K-channels and stimulates mitochondrial respiration. Diazoxide reduced infarct size in isolated rodent heart preparations and upon pretreatment in juvenile pigs with coronary occlusion/reperfusion. We aimed to study the use of diazoxide in a more realistic adult pig model of reperfused acute myocardial infarction when diazoxide was administered just before reperfusion. In a first approach, we pretreated anaesthetised adult Göttingen minipigs with 7 mg kg-1 diazoxide (n = 5) or placebo (n = 5) intravenously over 10 min and subjected them to 60 min coronary occlusion and 180 min reperfusion; blood pressure was maintained by use of an aortic snare. The primary endpoint was infarct size (triphenyl tetrazolium chloride staining) as a fraction of area at risk; no-reflow area (thioflavin-S staining) was the secondary endpoint. In a second approach, diazoxide (n = 5) was given from 50 to 60 min coronary occlusion, and blood pressure was not maintained. There was a significant reduction in infarct size (22% ± 11% of area at risk with diazoxide pretreatment vs. 47% ± 11% with placebo) and area of no-reflow (14% ± 14% of infarct size with diazoxide pretreatment vs. 46% ± 20% with placebo). With diazoxide from 50 to 60 min coronary occlusion, however, there was marked hypotension, and infarct size (44% ± 7%) and area of no-reflow were not reduced (35% ± 25%). Cardioprotection by diazoxide pretreatment was confirmed in adult pigs with reperfused acute myocardial infarction but is not feasible when diazoxide is administered in a more realistic scenario before reperfusion and causes hypotension.

Pulling wire to avoid misinsertion of filament and vascular damage improves ischemic stroke models' success rate and repeatability

Background: Middle cerebral artery occlusion (MCAO) ischemia-reperfusion in rats is a commonly used in vivo model in stroke research, but the problems of long time-consuming, unstable neurological injury and high mortality are not conducive to an experimental animal study. In this study, the success rate of the MCAO model in rats was improved by changing the surgical approach and giving preoperative as well as postoperative care support. Methods: Place A-wire below the common carotid artery (CCA) to ligate the proximal end of the CCA, B-wire to fix the filament, and C-wire to pull the filament through the bifurcation of the internal carotid artery (ICA) and the external carotid artery (ECA). After temporarily clamping the ICA, insert the filament into the CCA incision, then drag the C-wire to reduce the angle between the CCA and the pterygoid artery (PPA) to avoid filament insertion into the PPA. Push forward the head of the filament to the middle cerebral artery (MCA), ventral thalamic artery (VTA), and anterior choroidal segment to block the blood supply. Neurological function scoring, triphenyltetrazolium chloride (TTC) staining and Nissl staining were applied to evaluate the model success or failure, the infarct area and neuro damage. Results: Compared with the traditional Zea longa mapping method, our method significantly reduced modeling time and surgical trauma and improved postoperative survival rate and success rate. Furthermore, our method is more consistent in terms of infarct area, and there was a significant reduction in Nissl bodies. Conclusions: The wire-lifting method pulls the vessel, reduces the angle between the CCA and the PPA, facilitates the insertion of the filament into the MCA to avoid the accidental insertion of the filament into the PPA, reduces the surgical trauma, and saves the model preparation time, which can effectively improve the survival rate and modeling success rate.

Attenuation of stretch-induced arrhythmias following chemical ablation of Purkinje fibres, in isolated rabbit hearts.

Purkinje fibres (PFs) play an important role in some ventricular arrhythmias and acute ventricular stretch can evoke mechanically-induced arrhythmias. We tested whether Purkinje fibres, play a role in these arrhythmias. Pseudo-ECGs were recorded in isolated, Langendorff-perfused, rabbit hearts in which the left ventricular endocardial surface was also irrigated with Tyrode, via an indwelling catheter placed in the left ventricular lumen. The number and period of ectopic activations was measured during left ventricular lumen inflation via an indwelling fluid-filled balloon (500μL added over 2s and maintained for 15s in total). Mechanically-induced arrhythmias occurred in 70% of balloon inflations: they were maximal in the first 5s and ceased within 15s. Brief, (10s) irrigation of the left ventricular lumen with Lugol solution (IK/I2), via the indwelling catheter, reduced inflation-induced ectopics by 98% (p < 0.05). Ablation of endocardial PFs by Lugol was confirmed by Triphenyltetrazolium Chloride staining. Optical mapping revealed the left ventricular epicardial activation patterns of ectopics could have PF-mediated and focal sources. In silico modelling predicted ectopic sources originating in the endocardial region propagate to and through the Purkinje fibres network. Acute distention-induced ectopics are multi-focal, their attenuation by Lugol, their activation patterns and in silico modelling indicate a participation of Purkinje fibres in these arrhythmias.

Determination of Self-Compatibility of the 'Arsel' Olive Cultivar Obtained by Hybridization Breeding

This study was aimed to determine the fertilization biology of the 'Arsel' olive cultivar obtained by hybridization breeding. For this reason, treatments of open pollination, cross pollination, and self-pollination were performed for 2 years and self-compatibility and appropriate pollinators of this new cultivar were investigated. Among the varieties included as pollinators (‘Memecik’, ‘Gemlik’, ‘Uslu’, ‘Ayvalık’ and ‘Eğriburun Nizip’), the highest fruit set was achieved with ‘Memecik’ and ‘Gemlik’ varietes in both years (2.64%, 1.37% and 1.75%, 1.39%). For this reason, ‘Memecik’ and ‘Gemlik’ cultivars were the best pollinator for ‘Arsel’ olive was thought. On the other hand, considering the statistical analyzes and productivity index values, ‘Arsel’ variety was found to be self-incompatible. However, the data also indicated that cross pollination was effective in increasing fruit set. Therefore, it is thought that the use of pollinator cultivar in orchard establishment would be beneficial in terms of yield. Pollen viability and germination tests were performed by using 2,3,5 Triphenyl Tetrazolium Chloride (TTC) and agar in petri (15% sucrose + 1% agar + 100 ppm H3BO3) methods in the study. The highest pollen viability rate was observed in ‘Memecik’ cultivar in 2020 and there isn’t statistical differences between olive cultivars in 2018. The highest pollen germination rate was observed in ‘Arsel’ cultivar in 2018. Accordingly, differences were determined between pollen viability and germination rates of examined olive cultivars in terms of years.

Cardioprotective Effect of Empagliflozin and Circulating Ketone Bodies During Acute Myocardial Infarction.

SGLT2i (sodium-glucose cotransporter-2 inhibitors) improve clinical outcomes in patients with heart failure, but the mechanisms of action are not completely understood. SGLT2i increases circulating levels of ketone bodies, which has been demonstrated to enhance myocardial energetics and induce reverse ventricular remodeling. However, the role of SGLT2i or ketone bodies on myocardial ischemia reperfusion injury remains in the dark. The objective of this study is to investigate the cardioprotective potential of empagliflozin and ketone bodies during acute myocardial infarction (MI). We used a nondiabetic porcine model of ischemia reperfusion using a percutaneous occlusion of proximal left anterior descending artery for 45 minutes. Animals received 1-week pretreatment with either empagliflozin or placebo prior to MI induction. Additionally, a third group received intravenous infusion of the ketone body beta-hydroxybutyrate BOHB (beta-hydroxybutyrate) during the MI induction. Acute effects of the treatments were assessed 4-hour post-MI by cardiac magnetic resonance and histology (thioflavin for area at risk, triphenyltetrazolium chloride staining for MI size). All animals were euthanized immediately postcardiac magnetic resonance, and heart samples were collected. The area at risk was similar in all groups. Empagliflozin treatment increased BOHB levels. Empagliflozin-treated animals showed significantly higher myocardial salvage, smaller MI size (both by cardiac magnetic resonance and histology), less microvascular obstruction, and improved cardiac function (left ventricle ejection fraction and strain). Furthermore, empagliflozin-treated animals demonstrated reduced biomarkers of cardiomyocyte apoptosis and oxidative stress compared with placebo. The BOHB group showed similar results to the empagliflozin group. One-week pretreatment with empagliflozin ameliorates ischemia reperfusion injury, reduces MI size and microvascular obstruction, increases myocardial salvage, preserves left ventricle systolic function, and lowers apoptosis and oxidative stress. Periprocedural intravenous infusion of BOHB during myocardial ischemia also induces cardioprotection, suggesting a role for BOHB availability as an additional mechanism within the wide spectrum of actions of SGLT2i.

Relationship between Flower Phenotypic Traits and Fruit Yields in Tea (Camellia sinensis L.) Varieties

In order to provide early selection indicators for the breeding of plants used for producing tea seed oil or harvesting tea, we investigated the relationships between flower morphology and fruit yields in tea plants. We analyzed 106 tea varieties to determine the relationships between flower morphological traits and fruit yields. Notably, the homogeneity of flower traits within the same tea plant variety was found to be very high. The average length and width measurements of certain phenotypic traits of tea plants, including pistil length, stamen length, stamen bundle inner width, stamen bundle outer width, and stigma width, were 11.8, 10.9, 2.5, 15.0, 3.7 mm, respectively. In this study, the flower traits that affect fruit yield appear to be related to the difficulty of pollination by insects (e.g., bees), in terms of their contacting the stigma. In 2013, three phenotypic trait variables showed significant effects on yield; namely, the stamen bundle outer width (negative), stigma width (positive), and stigma width minus the stamen bundle inner width (positive). In 2015, only the stamen bundle outer width had a significant negative effect on yield. Regarding pollen viability, in the TTC (2,3,5-triphenyl tetrazolium chloride) staining test, about 84% of the considered tea varieties presented pollen viability exceeding 70%. This indicates that most tea pollen has the ability to germinate normally after contact with the cross-pollinated stigma. The yields of all of the tea varieties exhibited a positively skewed distribution in 2013 and 2015. Although our results indicate that flowers in the anther superior group tend to produce fewer fruits than flowers in the stigma superior group in 2013, in the analysis of the effect of traits on yield, there were no significant differences in the relative positions of stigmas and anthers. In conclusion, we determined that the main trait affecting fruit yield is stamen bundle outer width, while the secondary trait affecting fruit yield is stigma width. However, the efficacy of the stigma width may also be affected by the position of the stigma relative to the anther and the stamen bundle inner width. These two traits have the potential to be used as reference indicators for early selection in future breeding programs.