Background and Objectives: We developed a predictive statistical model to identify donor–recipient characteristics related to kidney graft survival in the Chilean population. Given the large number of potential predictors relative to the sample size, we implemented an automated variable selection mechanism that could be revised in future studies as more national data is collected. Materials and Methods: A retrospective multicenter study was conducted to analyze data from 822 adult kidney transplant recipients from adult donors between 1998 and 2018. To the best of our knowledge, this is the largest kidney transplant database to date in Chile. A procedure based on a cross-validated regularized Cox regression using the Elastic Net penalty was applied to objectively identify predictors of death-censored graft failure. Hazard ratios were estimated by adjusting a multivariate Cox regression with the selected predictors. Results: Seven variables were associated with the risk of death-censored graft failure; four from the donor: age , male sex , history of hypertension , and history of diabetes ; two from the recipient: years on dialysis log-transformation  and history of previous solid organ transplantation ; and one from the transplant: number of HLA mismatches . Only the latter is considered for patient prioritization in deceased kidney allocation in Chile. Conclusions: A risk model for kidney graft failure was developed and trained for the Chilean population, providing objective criteria which can be used to improve efficiency in deceased kidney allocation. The shortage of deceased donor kidneys available for transplantation has increased the relevance of allocation mechanisms. To better understand the factors predicting transplant outcomes, several survival analysis methodologies have been applied in the literature ,2,3,4. TIn Chile, the deceased kidney allocation system mainly considers HLA mismatch to prioritize patients with good transplant prognosis. Authors have suggested including factors such as donor–recipient age difference ,8 for loTherefore, we have collected data from multiple transplant centers to construct the largest database of kidney-transplanted patients in Chile, comprising 1459 transplants in total. Using this evidence, we have undertaken an in-depth analysis to objectively identify factors influencing transplant outcomes in Chile. However, this sample size is relatively small compared to the number of factors that can be used to predict graft survival ,9. For tA major data collection effort was conducted in collaboration with five transplant centers in Chile: Hospital Barros Luco Trudeau, Hospital del Salvador, Hospital Las Higueras, Hospital Sótero del Río, and Clínica Santa María, which jointly comprise approximately 14% of the total number of transplants conducted in Chile. To the best of our knowledge, this is the largest kidney transplant dataset that has been collected to date in Chile. This process required the digitalization and standardization of thousands of physical files stored in each medical center. The resulting database was implemented in REDCap and registered 1459 kidney transplant cases. Specifically, it contains pre-transplant information of donor–recipient characteristics and follow-up diagnosis post-transplant, including information on graft failure, with a total of 25 pre-transplant characteristics, which we sought to evaluate as potential predictors of graft survival. Anonymity was preserved for clinical records used in the analysis.To select the final sample for the study, we applied the criteria summarized in Two definitions of graft failure are typically used in survival analysis after a kidney transplant ,13,14. OPreliminary evidence was evaluated through different analyses to better understand the influence between potential predictors and transplant outcomes. First, an exploratory data analysis and a statistical description were computed. Distribution plots for survival and censorship times were examined, and variables were described by their mean and standard deviation or by their frequency and proportion in categories. Second, survival curves were estimated using the nonparametric Kaplan–Meier method for different subsets of patients , and theDespite our significant data collecting effort, the sample size was still too limited to evaluate the predictive power of the 15+ factors that needed to be tested, including alternative functional forms. Consequently, we applied an automatized procedure to objectively identify predictors for death-censored graft failure based on a cross-validated regularized Cox model using the Elastic Net penalty . Regularelations . We conselations . To obtaelations .Predictive accuracy was assessed by computing the C-statistic, which indicates the proportion of comparable pairs ranked correctly by the model. In addition to in-sample predictions, predictive accuracy was also assessed out-of-sample using 76 kidney transplants not included in the training data due to missing information. These transplants had missing data in recipient comorbidities assessed in the variable selection process. Since some of these were not selected in the final model, this sub-sample was used for evaluating the out-of-sample prediction of the final model specification.The proportionality assumption in the model was tested using the scaled Schoenfeld residuals  and consThe statistical analyses were conducted using R , while data processing was carried out using Python . Detailed information about the libraries and functions used can be found in the A visual description of the study cohort is shown in MissForest was used to impute missing values in donor history of diabetes (9.8% missing), donor creatinine > 1.5 mg/dL (9.6%), recipient weight (6.8%), recipient years on dialysis (2.5%), and cold ischemia time (0.9%). The statistical comparison between imputed and pre-existing values showed a reasonable estimation by the machine learning algorithm see .Graft survival at 1, 5, and 10 years from transplant estimated using the Kaplan–Meier method was 93% (95% CI: 91–95%), 89% (95% CI: 87–91%), and 81% (95% CI: 78–85%), respectively. Survival curves were examined in different groups of patients as preliminary evidence and are presented in p = 0.044) at a 10% significance level, while the linear component is not (p = 0.732). Considering this evidence, a functional transformation was included to consider an effect for recipients beyond 50 years old. On the other hand, the rest of examined variables showed a reasonable log-linearity adjustment. Nonetheless, three extra functional forms were incorporated considering graphical evidence and medical criteria: a binary variable that becomes active when maximum recipient PRA is greater than 50%, and log-transformations for recipient weight and recipient years on dialysis. These four functional transformations included for variable selection are shown in The preliminary log-linearity analysis in non-binary variables using penalized splines is shown in The variable selection mechanism is executed, including 25 potential predictors of death-censored graft failure see . Figure p-value for the overall test is 0.4, and when evaluating each variable independently, the lowest p-value is 0.092 (corresponding to the number of HLA mismatches). In addition, graphical evidence shows a reasonable adjustment of the proportionality assumption  . The KDPWe applied the KDRI score with the patient sample analyzed in this study, calculating the C-index to assess its predictive power. Since height, weight, HCV status, and DCD exhibited significant missing values in the data, these factors were set at reference levels to compute the KDRI. The predictive power of this adjusted KDRI score yields a concordance index (C-index) of 0.606, which is lower than the C-index calculated in this study (C-index = 0.659). This comparison provided further support that the methodology developed in this work is useful to improve predictions of graft survival, which can potentially be used to construct a “local” KDPI score in Chile’s kidney allocation system.This study analyzed the largest sample of kidney transplants collected up to date in Chile, comprising 1459 transplants from five transplant centers accounting for 14% of the transplants performed from 1998 to 2018. An automated procedure based on the Elastic-Net-regularized Cox’s regression was applied to objectively select predictors for kidney death-censored graft failure in the Chilean population, and a risk model that provides comprehensible results was developed to aid clinicians and decision markers. It is relevant that the data collection process continues to increase the accuracy of the selected variables and the precision of the estimated effects through the proposed methodology. In this regard, the procedure can be used as more information is collected if proportionality and log-linearity conditions for the Cox model are adequately validated.


SRWC) (75 ± 5)%, as the control; SRWC (55 ± 5)%, mild drought; SRWC (45 ± 5)%, severe drought] were investigated on sucrose-starch metabolism in sweet potato tuberous roots (TRs) by pot experiment. Compared to the control, drought stress increased soluble sugar and sucrose content by 4–60% and 9–75%, respectively, but reduced starch accumulation by 30–66% through decreasing the starch accumulate rate in TRs. In the drought-treated TRs, the inhibition of sucrose decomposition was attributed to the reduced activities of acid invertase (AI) and alkaline invertase (AKI) and the IbA-INV3 expression, rather than sucrose synthase (SuSy), consequently leading to the increased sucrose content in TRs. In addition, starch synthesis was inhibited mainly by reducing ADP-glucose pyrophosphorylase (AGPase), granular starch synthase (GBSS) and starch branching enzyme (SBE) activities in TRs under drought stress, and AGPase was the rate-limiting enzyme. Furthermore, soil drought remarkably up-regulated the IbSWEET11, IbSWEET605, and IbSUT4 expressions in Jishu 26 TRs, while it down-regulated or had no significant differences in Xushu 32 and Ningzishu 1 TRs. These results suggested that the sucrose-loading capability in Jishu 26 TRs were stronger than that in Xushu 32 and Ningzishu 1 TRs. Moreover, IbA-INV3, IbAGPS1, IbAGPS2, IbGBSSI and IbSBEII play important roles in different drought-tolerant cultivars under drought stress.In this study, the influences of long-term soil drought with three levels [soil-relative water content ( At present, more than a third of the world’s arable land faces water shortages. Drought stress has become one of the main environmental factors limiting crop yields in world under the double exacerbation of the greenhouse effect and climate change ,2. How tSUTs) and sucrose-will-eventually-be-exported transporter genes (SWEETs) [IbINH) in sweet potato increased sucrose content and decreased hexose content and Inv activity, but the opposite results were observed in the down-regulated IbINH plants by RNAi technology [Sucrose is the main photosynthate for phloem transport in higher plants. Sucrose transport in plant phloem mainly relies on the involvement of sugar transport genes, such as sucrose transporter ((SWEETs) ,11. Drou(SWEETs) . Yang etchnology . In othechnology . Other schnology , under wchnology , which aAGPase, SSS and GBSS gene expression and their low activities have led to the restriction of starch synthesis [IbSUT3 and IbSUT4 (a homologue of AtSUC2 and AtSUC4); IbSWEET11 (a homologue of AtSWEET12-like) and IbSWEET605 (a homologue of AtSUC4); SuSy2, SuSy3 and SuSy4; AGPase small subunits 1 and 2 (AGPS1 and AGPS2) [SSSI, SSSII, SSSIII and SSSIV [GBSSIa, GBSSIb and GBSSII [SBEI, SBEIIa and SBEIIb [In roots of higher plants, starch biosynthesis is mainly catalyzed by ADP-glucose pyrophosphorylase (AGPase), granular starch synthase (GBSS) and soluble starch synthase (SSS); starch branching enzyme (SBE) and debranching enzyme (DBE) are also involved in amylose and amylopectin synthesis and affect starch’s fine structure ,18,19. Hynthesis ,22. Someynthesis . Therefod AGPS2) ,24; SSSInd SSSIV ; GBSSIa,d GBSSII ; and SBEd SBEIIb ,28. Eachd SBEIIb .Although many studies have been performed on the relationship between sweet potato TR yield/biomass and photosynthesis, or root morphology or N metabolism under drought stress ,32,33,34IbA-INV2 and IbA-INV3; and (3) to clarify the difference among Xushu 32, Ningzishu 1 and Jishu 26 in sucrose transport capacity in response to drought stress.In this study, we want to (1) investigate the changes of SuSy, AGPase, SSS, GBSS, SBE and DBE and their gene expression levels in sweet potato TRs under long-term soil drought stress, and the relationship with starch accumulation; (2) determine the sucrose-cleaving enzyme, invertase, for changes in its activity and its gene expressions, SRWC levels, and long-term soil drought stress raised soluble sugar content and sucrose content (SRWC (75 ± 5)%), the soluble sugar content and sucrose content in Ningzishu 1 TRs were improved by 38–60% and 33–75%, and by 4–9% and 9–10% in Jishu 26 TRs, under long-term soil drought treatment (SRWC (55 ± 5)% and SRWC (45 ± 5)%), respectively. For Xushu 32 TRs, the soluble sugar content and sucrose content were raised by 33–40% and 20–42% in 2018, and by 5–9% and 22–66% in 2019, respectively.The soluble sugar content and sucrose content in sweet potato TRs reduced or first improved and then decreased with increasing DAT for the three  content . CompareSRWC decreasing  was decreased, as well as the maximum rate (maxV), in the SRWC (45 ± 5)%-treated TRs for all three cultivars. However, SRWC (45 ± 5)% treatment prolonged the starch rapid accumulation period in Xushu 32 and Ningzishu 1 TRs by 6 d and 14 d, respectively, averaged over two years. Moreover, the coefficient of variation of TV was larger than that of T and mT.The starch dynamic accumulation in TRs with increasing DAT were simulated using Logistics Formulas (1)–(3) . CompareSRWC decreasing. Compared to the control, long-term drought stress increased SuSy activity in TRs of Xushu 32, Ningzishu 1 and Jishu 26 by 43–77%, 30–65% and 36–67%, respectively, averaged over two years. Nevertheless, the activity of AKI and AI in TRs declined with the decrease in SRWC.With increasing DAT, the activities of SuSy, AI and alkaline invertase (AKI) in TRs showed a single peak curve . SuSy acSRWC decreasing %) at 90 DAT, the expression levels of IbSUT4 were obviously up-regulated by 88% for Xushu 32, but down-regulated by 36% for Ningzishu 1; in 2019, the expression levels of IbSUT4 were observably increased by 69% for Jishu 26, but there were no significant differences between the control and SWRC (45 ± 5)% treatment in IbSUT4 for Xushu 32. However, the expression level of IbSUT3 for Jishu 26 was better than for Xushu 32 and Ningzishu 1 at 50 DAT. Under SWRC (45 ± 5)% treatment, the expression levels of IbSWEET11 and IbSWEET605 had increased by 2.7- and 0.4-fold at 90 DAT for Jishu 26; however, there were no significant differences between the control and SWRC (45 ± 5)% treatment in IbSWEET11 and IbSWEET605 for Xushu 32.In 2018 , under lIbSuSy20, IbINH and IbA-INV2 in TRs at 50 DAT, but increased the expression level of IbSuSy60 in TRs, with average increases of (0.6–2.9)-fold. In addition, SWRC (45 ± 5)% treatment reduced the expression level of IbA-INV3 by 48% in Ningzishu 1 TRs at 90 DAT, but it had no obvious effect on IbA-INV3 expression in Xushu 32 and Jishu 26 TRs between the control and SWRC (45 ± 5)% treatment. In the drought-treated TRs, the expression levels of IbAGPS1, IbAGPS2, IbSSS SPSS67, IbGBSSI and IbSBEII were significantly down-regulated at 50 DAT for Xushu 32 and Jishu 26, while the expression levels of these genes were up-regulated for Ningzishu 1. Nevertheless, at 90 DAT, these genes for the three cultivars had an opposite expression trend.Moreover, the related genes in sucrose and starch metabolism were also investigated . CompareTV and maxV, as well as Al, AKI, AGPase, GBSS and SBE  [Sucrose is the main photosynthate for phloem transport in higher plants, and also the primary source for starch synthesis. Sucrose transport in plant phloem mainly relies on the involvement of sugar transport genes, such as and SUTs ,11. In tu 32 TRs , which smulation , in acco max L.) ,11. It wIbSuSy60 in TRs, but decreased AI and AKI activities and the expression levels of IbINV2 . Healthy and uniform sweet potato seedings with five leaves were cut from TRs and transplanted into pots, with one plant per pot.−1 organic matter, 0.6 and 0.5 g kg−1 total nitrogen and 85.7 and 84.6 mg kg−1 available potassium, 32.7 and 35.1 mg kg−1 available phosphorus. In each year, 4.8 g of N in the form of urea, 1.6 g of P2O5 in the form of superphosphate and 3.2 g of K2O in the form of potassium chlorate were applied to each pot at basal application. The bulk density and field water holding capacity of the soil were 1.42 g cm2 and 0.29 g g−1, respectively.The soil at the experiment site was sandy, and collected from the 0–30 cm depth layer in the farmers’ fields. A total of 30 kg of soil was filled into the pots . In 2018 and 2019, the soil contained, respectively, 12.4 and 13.9 g kgSRWC (75 ± 5)%, well-watered, as the control), SRWC (55 ± 5)% (mild drought) and SRWC (45 ± 5)% (severe drought). After the slow seedling is over (about 7–10 days), soil water treatments were set until the end of harvest stage. Sweet potato plants were watered well before the set of the soil water treatments. Soil water content was determined about every 2–4 days at around 18:00, using a soil sampler to collect the soil from 0–25 cm depth in 2018. Three pots were sampled per plot, and the sample holes were filled with soil from empty pots. The soil samples were measured for the fresh weight, and then were dried at 105 °C in an oven for 8 h. The next day, sweet potato plants would be watered to the upper limit of the appropriate soil moisture limit. In 2019, the soil relative water content was measured by TDR (AZS-100 Handheld Meter), and the soil relative volume content was recalculated and converted to SRWC.Two rows of pots were placed together. Each plot consisted of 36 pots of plants, and the experiment was a randomized complete block design. Each treatment had three replications. Three water treatment were designed as follows: (75 ± 5)% of soil relative water content  in 2018, and at 10, 35, 50, 70, 95 and 115 DAT in 2019 from each treatment. The fresh TRs were washed with ddHSoluble sugar, sucrose and starch (amylose and amylopectin) were determined according to the methods of our laboratory’s previous studies ,40. Solu2, following an added cool-extraction buffer. The supernatant after centrifugation was used to analyze SuSy, AI, AKI, AGPase, SSS, SBE and DBE activities. In addition, the extraction buffer was added to the precipitate for GBSS analysis. The SuSy, AI, AKI, AGPase, DBE and SBE activities were measured as previous described methods [The sucrose-starch metabolizing enzymes were determined using the methods of our laboratory’s previous studies ,40. In d methods ,42,43,44GAPDH, and the relative expression levels related in sucrose-starch metabolism were analyzed using the 2−ΔΔCT method [Total RNA from the TRs at 50 DAT and 95 DAT were extracted using RNA plant kits , and then reverse transcribed, and the synthetic cDNA was used for qPCR. In this study, each qPCR result had three biological replications. The primers in this study were shown in T method ,46.S (g per plant) is starch accumulation in TRs, DAT (d) is days after transplanting, maxS (g) is the maximum accumulation, and a and b are parameters.Starch dynamic accumulation in TRs is simulated by logistic model:From Formula (1):DAT = DAT0:DAT1 is the days after transplanting when the rapid increase in starch accumulation was started, DAT2 is the days after transplanting when the rapid increase in starch accumulation was terminated, mDAT is the days after transplanting when the maximum rate of starch accumulation was reached, T(d) = DAT2 − DAT1 is the rapid starch increase period, TV and maxV are the average and maximal rates of starch accumulation.The maximum rate of starch accumulation in TRs was when Weather data in Xuzhou were collected from the National Metaoralogical Information Center. The average temperature and solar radiation from June to October in 2018 and 2019 were shown in The data were recorded and analyzed using Microsoft Excel 2019, Origin 2018 and SPSS 23.0. One-way analysis of variance (ANOVA) was conducted followed by Tukey HSD test at 0.05 and 0.01 levels to examine the differences of measurements under different water treatments within each cultivar. In addition, the statistical analysis of the results was separate for each year.IbA-INV3 expression, rather than SuSy, consequently leading to the increased sucrose content in TRs. In addition, starch synthesis was inhibited primarily by reducing the activities of AGPase, GBSS and SBE in TRs under long-term soil drought stress, and AGPase was the limiting-rate enzyme in starch synthesis in TRs. Furthermore, the capacity of sucrose loading in Jishu 26 TRs were stronger than that in Xushu 32 and Ningzishu 1 TRs through the up-regulation of IbSWEET11, IbSWEET605 and IbSUT4 under long-term drought stress, and IbA-INV3, IbAGPS1, IbAGPS2, IbGBSSI and IbSBEII play important roles in different drought-tolerant cultivars under long-term soil drought stress. In conclusion, the efficient sucrose transport, higher starch accumulation average and maximum rates in TRs may be important functional traits for improving drought tolerance.Although long-term soil drought stress reduces tuber yield by carbon metabolism in sweet potato TRs, little is reported on how it influences starch metabolism process in TRs. Our results showed that long-term soil drought stress inhibited starch accumulation in TRs by a reduced average and maximum rate. In the drought-treated TRs, the restriction of sucrose decomposition was attributed to the declined activities of AI and AKI and


Pueraria montana starch (PMS) was modified with octenyl succinic anhydride (OSA) and evaluated as an emulsifier to prepare emulsions. The FTIR, SEM, and XRD results showed that PMS was successfully modified. Furthermore, the emulsification index (EI), mean droplet size, and ζ-potential values showed that modified starch with a higher degree of substitution (DS) enhanced the storage stability of emulsions. Similarly, the retention degree and encapsulation efficiency results of puerarin proved the assumption after storage of 16 d. The Pickering emulsions also helped in the controlled release of microencapsulated puerarin in vitro. The study outcomes proved that Pickering emulsions stabilized with OSA-modified PMS have promising applicability in functional foods as efficient food-grade delivery systems, enhancing oral supplementation and accessibility of puerarin.Puerarin is a bioactive flavonoid isolated from Kudzu roots that possesses numerous health benefits. However, its poor bioavailability and existing complex delivery systems with safety issues are challenging tasks for its incorporation into functional foods. Preparing modified-starch-stabilized Pickering emulsions containing microencapsulated puerarin with improved bioaccessibility was the key objective of the present research work. Acid-hydrolyzed high-amylose  Puerarin is the phytoestrogen and main biologically active isoflavone chiefly present in the roots of Pueraria lobata , which i−2 mol/L) and gastrointestinal fluids, making its oral bioavailability approximately 7%, which is very low for harvesting its beneficial effects [Due to all these potential benefits, puerarin possesses great interest in being incorporated into functional foods as a health-improving nutraceutical . Accordi effects ,16. The  effects ,17,18.Furthermore, according to some studies, orally administered puerarin with a dose of 10mg/kg has a shorter half-life of approximately 0.80–1.00 h, a lower partition coefficient of 1.95, and a Cmax value of 140–230 μg/L in vivo ,19. OwinIn recent years, the formulations for oral administration with improved absorption of isoflavones have attracted widespread acceptability and adaptability. Finding a novel efficient formulation and a safe administration method for puerarin delivery is critical. This challenging task could be overcome by microencapsulating the nutraceuticals within food colloids. This technique provides protection to unstable bioactive compounds and increases their bioavailability by retaining them within a microencapsulating membrane or matrix . EmulsifParticle-stabilized or Pickering emulsions have gained significant scientific and industrial attention due to their higher stability against destabilization mechanisms  . The coaPueraria montana starch as a non-conventional substance has been studied for OSA modification and production of highly stabilized O/W Pickering emulsions.These emulsions are stabilized by a variety of particles, such as modified starches, protein nanogels, kaolinite, nanocellulose, and kafirin. Starches are natural polymers and have extensive utilization in Pickering emulsions as emulsifiers, with several advantages, such as low cost, FDA approval, toxicity, bioavailability, biocompatibility, mucoadhesiveness, gastro resistance, pH responsiveness, and degradability by colonic microbiota . NaturalAdditionally, acid hydrolysis has also been used as a pretreatment of starch before OSA modification to obtain unique properties for better stabilization of the emulsions ,43. AcidPueraria montana starch were studied. Additionally, the physicochemical characteristics of microencapsulated puerarin in Pickering emulsions, including loading capacity, microencapsulation efficiency, in vitro release, pH, and storage stabilities, were investigated. Keeping in view the above background, the study proposes the development of OSA-modified-starch-based Pickering emulsions for microencapsulation of puerarin as a dietary supplement for oral administration. These emulsions could be utilized in functional foods for better biological activities by enhancing the bioavailability of puerarin and compensating for its deficiency in daily intake. Moreover, the effects of acid hydrolysis on the DS and emulsifying characteristics of OSA esterification with different concentrations on hydrolyzed Pueraria montana starch was procured from the Key Laboratory Functional Foods, Guangdong Academy of Agricultural Sciences, Guangzhou. MCT oil was obtained from Jarrow Formulas, Los Angeles (CA 90035-4317), USA. Octenyl succinic anhydride with 97% purity was purchased from Sigma Aldrich, Co., 3050 Spruce Street, St. Louis, MO, USA, whereas puerarin with a purity of ≥97% was procured from the Aladdin industrial corporation, Shanghai, China. MilliQ water was used to prepare solutions and emulsions. All other chemicals were of analytical grade.Pueraria montana starch  was pretreated with 1.0 M HCl in a 250 mL beaker. Incubation of the mixture was carried out in a water bath at 50 °C for 2, 4, 6, 8, 12, and 24 h. The mixtures were stirred with magnetic stirrers after specific intervals. Then, 5% NaOH was used to neutralize the incubated samples. Afterward, the samples were centrifuged at 4000× g for 5 min and washed with MilliQ water to obtain a neutral pH. Then, absolute ethanol was used twice to wash the samples by centrifuging for 5 min at 4000× g. Finally, the hydrolyzed starch samples were freeze-dried for 72 h. The dried starch precipitates were ground finely to pass through a 100-mesh sieve. The sieved starch powders were put into screw-capped tubes and kept in a desiccator for further use.Acid hydrolysis of the PMS was carried out before OSA modification. The previously described method in  was usedPueraria montana starch was subjected to OSA modification using the method described in [w/w) of PMS were prepared using MilliQ water. The solutions were continuously mixed with a magnetic stirrer. Then, using the 0.5 M NaOH solution, the pH of the starch slurries was adjusted to 8.0 and maintained at 8.0 ± 0.2 during the whole reaction time. Afterward, the OSA with contents of 2%, 4%, 6%, and 8% of starch was diluted five times with absolute ethanol. The diluted OSA was mixed drop by drop for 2 h in the previously prepared starch mixtures. The process was performed at 35 °C for 3 h. After finishing the modification procedure, the pH of starch mixtures was adjusted to 6.5 using 0.5 M HCl. Then, washing of the suspension was completed twice using MilliQ water and twice with 70% ethanol by centrifuging at 3300 rpm for 30 min. After centrifugation, the precipitates were freeze-dried for 73 h, grounded, sieved through 100-mesh size, labeled, and stored for further analyses. After acid hydrolysis pretreatment, the ribed in . AqueousThe average number of OH groups substituted per glucose unit is termed the degree of substitution (DS). A slightly modified titration method in  was usedsampleV is the NaOH volume (L) used for sample titration, whereas blankV is the NaOH volume (L) used for blank titration. M represents the molarity of NaOH, W is the weight (g) of starch, and 210 denotes the molecular weight of the octenyl succinate group.Then, Equation (2)  was usedModified and native starch samples were analyzed for particle size using a laser particle size analyzer .The characterization of OSA-modified PMS and native starch powders for XRD patterns was completed by using the X’Pert³ Powder diffractometer . The range of diffraction scattering angles (2θ) was 5°–60°. Measurement of the relative crystallinity was conducted using the software of Jade 6.0 .−1 using 32 scans at a resolution of 4 cm−1. The spectra were analyzed using Thermo Scientific OMNIC FTIR software .FTIR spectroscopic analysis was carried out using the method of  with theThe morphological features of the modified and native starch samples were studied by using the ZEISS Merlin Field Emission Scanning Electron Microscope. The starch samples were fixed with carbon tape on aluminum stubs, gold plated, and 10 µm images were taken under 1.00 Kx resolution and 5 kV accelerated voltage.OSA-modified PMS samples were used as specific emulsifiers for preparation of O/W Pickering emulsions. Further, 5% modified starch samples were gelatinized at 90 °C for 20 min to prepare a continuous phase, whereas 5 mg/L puerarin was mixed in MCT (10%) and used as a dispersed phase. First of all, puerarin-loaded coarse emulsions were prepared using a rotor-stator  at 10,000 rpm for 5 min. The MCT and puerarin mixture were poured drop by drop during homogenization. Then, these coarse emulsions were homogenized in a high-pressure homogenize machine , completing 3 cycles at 150 MPa.The emulsions were stored for 16 d at 4 °C, and the preparation day was taken as the first day of storage. The emulsification index of the emulsions was recorded on 1, 8, and 16 d by using the method as described by Zheng et al. , followiThe distributions of droplet size of Pickering emulsions were determined on days 0, 8, and 16 using a laser size analyzer . The droplet size distribution was determined immediately after preparing the emulsions. Then, 3 mL of each emulsion was put into 5 mL centrifuge tubes and stored at room temperature. During the storage period, droplet size distributions (DSDs) of emulsions were measured again to assess their stability . The results of the DSDs were also used to calculate the median droplet diameter values  . AdditioMeasurement of the ζ-potential of emulsions was determined by a Zetasizer Nano Series . After diluting the samples with MilliQ water, the emulsions’ pH  was set using HCl and NaOH. Then, the samples were filled into capillary cells to measure zeta potential. The samples were diluted with deionized water and filled into the test cell before measurement. This experiment was performed to check the stability of emulsions on different pH as well. Each sample took three readings with triplicate measurements.The retention degree of the emulsions was determined after 16 d of storage, whereas the microencapsulation efficiency of puerarin was determined after the freshly prepared emulsions. The dissolved puerarin in MCT was taken as control (puerarin–MCT) for the comparison of retention degree after 16 d. Puerarin was extracted using the OSA couple liquid–liquid extraction method. In short, we took 2 mL ethyl acetate in a 5 mL centrifuge tube and added 500 μL emulsions, then sonicated for 20 min and centrifuged for 5 min at 8000 rpm. Then, the residue of (500 μL) supernate was dried with a nitrogen gas stream, and the dried residues were redissolved into acetonitrile. Then, the filtration of the samples was conducted using a 0.45 μm membrane for HPLC analysis. Retention degree and MEE were determined with the following equations.actualC is the amount of puerarin loaded in the lipid droplets of the emulsions, whereas addedC is the amount of total puerarin used during the preparation process, and 10dC is the amount of puerarin retained in the emulsion after storage of 16 d.In these equations, v/v) with continuous magnetic stirring (200 rpm) at 37 °C, and 1 mL of aliquots was taken out and replaced with an equal volume of freshly prepared PBS at specific time intervals of 2, 4, 6, 8,10, 20, 30, 40, and 50 h. Extraction of the released puerarin from the emulsions was conducted and analyzed with HPLC.The dialysis bag diffusion method was used to determine the in vitro release profile measurements of puerarin from the emulsion samples by following the slightly modified method . Concisev/v), and the mobile phase was degassed before running the column, maintaining the temperature at 25 °C. The run time was set for 20 min, with an injection volume of 20 µL, a flow rate of 1 mL/min, and a wavelength of 254 nm.An Agilent HPLC system (1260 series) equipped with Hypersil GOLD-C18 column  was used to perform the quantitative analysis of puerarin by following the method as described in . The pH p < 0.05. Values of means and standard errors were considered and shown in charts as coordinate pairs with error bars.The data were compiled by completing entire analyses in triplicate. Statistics 8.1  and the Tukey method were used for comparison of means and ANOVA . The difference was taken as significant statistically when Esterification of the starch was carried out with 2% OSA after acid hydrolysis pretreatment for 2, 4, 6, 8, 12, and 24 h . The resPueraria montana (Kudzu) starch after acid hydrolysis. The results for DS are shown in Different OSA concentrations  were used to modify the high-amylose The diverse polymorphic forms of starch categorized as A, B, and C types are based on the semi-crystalline structure and X-ray patterns of the starch granules. According to , the priXRD patterns of the native and acid-hydrolyzed high-amylose PMS (Kudzu) starches are presented in The SEM morphological analysis of the native and modified starch granules can be viewed in −1 were studied, as shown in −1, which was attributable to the O-H stretching vibration absorption, decreased. The intensity of the band associated with the O-H stretching vibration absorption decreased with increased additions of OSA at 3488 cm−1 compared to DE. This might be due to the esterification of the O-H band to the carbonyl group. The antisymmetric stretching of C-H in the -CH2 and CH3 groups caused the absorption peak at about 3000 cm−1. Additionally, new distinguishing peaks were given to the spectra of the OSA-modified starch as compared to the native starch at around 1609 cm−1 and 1565 cm−1 [The impact of esterification with various OSA additions on the structure of PMS starch was investigated using infrared spectra analysis. The spectra between 500 and 4000 cm565 cm−1 ,63,64. T565 cm−1 , which dThe median granular size (D50) of high-amylose PMS was 8.14 μm in the beginning, but it was decreased after the pretreatment and OSA modification, as shown in w/w, the emulsifying index of the hydrolyzed and OSA-modified starches was also increased. The increasing degree of substitution (DS) also increased the oleophilic groups, which resulted in an improvement in the emulsion index. The improved EI of OSA-modified and hydrolyzed starches with higher DS resulted in easier adsorbance to the oil–water interfaces. Due to this phenomenon, higher steric hindrance and denser interface film have occurred between the emulsion droplets [The prepared Pickering emulsions were stored for 1, 8, and 16 days, and their emulsification index (EI) values are given in droplets ,71.The state of the emulsion depicted in Zeta potential, which is used to assess how strongly particles repel or attract one another, is a key criterion for evaluating the dispersion stability of colloids . Figure Additionally, the five emulsions were also investigated for their stability behavior at a pH range of 3, 6, and 9, and As it was found that Un-2% OSA-starch-based emulsions were highly unstable, the puerarin was microencapsulated using other modified starches. w/w), the emulsifying stability of OSA-based starch emulsions was also increased. The increase in the oleophilic group with increased DS may be responsible for the improvement in emulsion stability, making it easier for higher-DS OSA starch to adsorb to the oil–water interface, resulting in a higher steric hindrance and a denser interface layer between the droplets in the emulsion [The results showed that Pickering emulsions could be employed as an efficient delivery strategy for puerarin encapsulation since the emulsion stabilized by 8% OSA-modified starch with greater DS had superior stability. With an increasing OSA addition from 4 to 8%  starch in combination with acid hydrolysis could make it an effective emulsifier to produce stabilized Pickering emulsions. Meanwhile, these emulsions could be successfully employed as a novel strategy to microencapsulate and deliver puerarin, which is a hydrophobic bioactive isoflavone. The details of the starch modification, preparation, and characterization of Pickering emulsions containing microencapsulated puerarin have thoroughly been provided in this research work. According to the findings, the stabilized Pickering emulsion with 24 h H-8% OSA starch showed better resilience against harsh conditions, superior storage stability, and microencapsulation efficiency (MEE) of puerarin. Furthermore, the controlled release of puerarin in vitro was successfully achieved in the emulsions containing 24 h H-8% OSA starch. The present research will be helpful in establishing the high-amylose PMS granules as alternative particle stabilizers to be incorporated in the stabilization of the Pickering emulsions. Additionally, this research will be helpful in expanding the applicability of puerarin and related bioactive compounds in the functional food industry by exploiting and fabricating novel ingredients to introduce new food-grade, economical, and orally administered delivery systems.OSA modification of high-amylose


Clinical Neuroradiology. From the first issue in 2006, I took care of the processes of this journal, founded in 1991, with the support of my younger colleagues as members of the editorial board. In these past 17 years, we have succeeded in turning a German language journal, which consequently had a limited readership only, into a widely read international scientific journal with a respectable Impact Factor.In 2005, I took on the role of Editor-in-Chief of Without your support as readers, reviewers, and authors of our journal, this would not have been possible. The current position of the journal among the scientific neuroradiological journals was only achieved because we received a high number of scientifically excellent submissions from you and because you continuously supported the journal with reviews of high quality, which enabled us to select the papers to be published in the journal.I would like the journal to remain on the cutting edge scientifically. For a neuroradiologist who is no longer active in his profession and who no longer interacts with patients on a day to day basis, who therefore no longer receives professional “updates” and “upgrades” directly, this is not possible while maintaining the quality desired. For this reason, I am passing on the position of Editor-in-Chief at the end of this year. I am glad that we were able to find someone from the current editorial board as successor, who already has an in-depth experience with the journal. Martin Bendszus will take over the position of Editor-in-Chief on 1 January 2023.Clinical Neuroradiology, the members of the editorial board, and also the publisher Springer Nature for their support over the past 17 years. Please continue to support Clinical Neuroradiology with excellent scientific papers. The editorial board still relies on your high-quality reviews, so please continue to support the journal with your expertise if at all possible. Support the journal, as it is the official journal of the German, Austrian and Swiss Societies for Neuroradiology and thus also a figurehead of German-speaking neuroradiology.As I bid farewell, I would like to thank the readership of László SolymosiEditor-in-Chief


As long‐standing wound researchers, we were sad to hear of the passing of a wound‐care legend, Professor Hugo Partsch. Hugo for many is regarded as the grandfather of compression science. As an Austrian dermatologist, he contributed significantly to the research in phlebology and compression.International Wound Journal, 13(3), 317–325.Harding, K. G., Vanscheidt, W., Partsch, H., Caprini, J. A., & Comerota, A. J. (2016). Adaptive compression therapy for venous leg ulcers: a clinically effective, patient‐centred approach. Harding, K. G., Vanscheidt, W., Partsch, H., & Caprini, J. A. (2014). Actitouch System Improves Quality of Life; High Adherence to Therapy and High Satisfaction in Patients with Venous Leg Ulcers.International Wound Journal, 5(3), 389–397.Partsch, H. (2008). Intermittent pneumatic compression in immobile patients. International Wound Journal, 10(5), 516–526.Flour, M., Clark, M., Partsch, H., Mosti, G., Uhl, J. F., Chauveau, M., … & Schingale, F. (2013). Dogmas and controversies in compression therapy: report of an International Compression Club (ICC) meeting, Brussels, May 2011. International Wound Journal, 13(3), 317–325.Harding, K. G., Vanscheidt, W., Partsch, H., Caprini, J. A., & Comerota, A. J. (2016). Adaptive compression therapy for venous leg ulcers: a clinically effective, patient‐centred approach. Here is his contribution to our journal in the 20 years of its existence and as a co‐author with our Editor‐in‐Chief:However, his contribution was more significant that the short list above with his ResearchGate profile having more than 500 research items. In 2005, he founded the International Compression Club (ICC), a forum that links medical experts with industry and aims to promote the science behind compression therapy.VASA, Journal for Vascular Diseases. A former Professor of Dermatology at the University of Vienna and the former Head of the Dermatological Department at the Wilhelminen‐Hospital (Vienna), he has published more than 360 papers in scientific medical journals and contributed to books.” Witnessing him receiving this award among the “best of the best” in wound care cemented his “rock star” status in my mind.Personal reflection (DQ): In 2008, I had the honour of meeting Hugo for the first and only time in my long wound‐care career. He was presented with a Lifetime Achievement Award for his contribution to wound care. This was the citation of his award—“Professor Partsch is the Co‐Editor of Personal reflection (KGH)—Hugo was an excellent clinician, a global leader in research into leg ulcers, an excellent and inspiring educator but also was a true gentleman. He always had time to talk and put you in contact with people. He was admired and respected by all he met. Even in his latter years, when we met up at conferences he would be the first to appear for breakfast—usually after an early morning run—and would be a fantastic source of ideas, contacts, and data. I was fortunate to have the honour to publish with Hugo as a co‐author and without doubt, it was one of the privileges and highlights of my academic career.His passing while sad reminds us all of the extensive influence of Hugo Partsch in the wound‐care area and he will leave a legacy in this area for other clinicians and scientists to benefit from. His legacy will live on through the continued work of many others.Keith Harding, Editor‐in‐Chief, IWJDouglas Queen, Editor, IWJ


White matter injury (WMI) significantly affects neurobehavioral recovery in intracerebral hemorrhage (ICH) patients. Gut dysbiosis plays an important role in the pathogenesis of neurological disorders. Oxymatrine (OMT) has therapeutic effects on inflammation‐mediated diseases. Whether OMT exerts therapeutic effects on WMI after ICH and the role of gut microbiota involved in this process is largely unknown.Neurological deficits, WMI, gut microbial composition, intestinal barrier function, and systemic inflammation were investigated after ICH. Fecal microbiota transplantation (FMT) was performed to elucidate the role of gut microbiota in the pathogenesis of ICH.CST) region after ICH. ICH induced significant and persistent gut dysbiosis, which was obviously regulated by OMT. In addition, OMT alleviated intestinal barrier dysfunction and systemic inflammation. Correlation analysis revealed that gut microbiota alteration was significantly correlated with inflammation, intestinal barrier permeability, and neurological deficits after ICH. Moreover, OMT‐induced gut microbiota alteration could confer protection against neurological deficits and intestinal barrier disruption.OMT promoted long‐term neurological function recovery and ameliorated WMI in the peri‐hematoma region and distal corticospinal tract  in ICH. ICH induced severe WMI and neurological deficits, accompanied by persistent gut dysbiosis. OMT promoted better long‐term neurological function recovery and ameliorated WMI after ICH by modulating gut microbial composition and alleviating intestinal barrier dysfunction. The severity of ipsilateral CST injury was closely associated with the activity of daily living impairment in ICH patients.CST at the cervical enlargement, which could prolong for at least 5 weeks.Intracerebral hemorrhage (ICH) is the second most common subtype of stroke (10%–15%) with high morbidity and mortality, remaining a major cause of death and disability globally.CST injury of the cervical enlargement, and alleviate neurological deficits after ICH.Accumulating evidence implicates intestinal bacteria is a crucial factor for the bidirectional communication between the gut and central nervous system.Oxymatrine (OMT) is a main quinolizidine alkaloid extracted from the traditional Chinese herb Sophorae Flavescentis Radix. Emerging studies demonstrated that OMT conferred neuroprotective effects on several neurological disorders, such as ischemic stroke and spinal cord injury.CST injury in cervical enlargement, and the role of gut microbiota in the underlying mechanism in an ICH model. We hypothesized that OMT alleviated ICH‐induced WMI and neurobehavioral deficits by modulating gut microbiota.Therefore, the present study aimed to investigate whether OMT could attenuate hemorrhage‐induced WMI, especially secondary 22.1The experimental protocols were approved by the Institutional Ethics Committee of Zhujiang Hospital of Southern Medical University (LAEC‐2020‐227) and conducted according to the guidelines of the National Institute of Health Laboratory Animal Care and Use Guidelines.2.2Animals were randomly assigned into three groups, namely Sham, ICH + Vehicle, and ICH + OMT group Figure . The mic2.3t‐test was used to compare the means of two groups. Comparisons among multiple groups were analyzed using one‐way analysis of variance (ANOVA) with a post hoc analysis with the Bonferroni method. Continuous neurological function scores were analyzed by two‐way ANOVA adjusted by Tukey's post hoc test. Regarding non‐normal distribution variables, the Kruskal–Wallis H‐test and Mann–Whitney U‐test were conducted for statistical analysis between different groups. Correlation analysis between bacterial species and inflammatory markers was evaluated by Spearman's rank correlation. GraphPad Prism Software version 6.0 (GraphPad Prism) was applied to visualize analysis results. The p‐Value <0.05 was considered statistically significant. Other materials and methods are described in the Appendix Statistical analysis was performed using the SPSS software version 20.0 (SPSS). Normality was evaluated using Shapiro–Wilk's test. For normal distribution variables, the student 33.1OMT by oral administration for 3 days reduced hematoma volume and brain water content  compared with that of vehicle‐treated mice after ICH Figure . Then, wThe mNSS test showed that neurological function impairment reached the peak on Day 1 and thereafter gradually declined from Day 3 after ICH. Instead, OMT partially alleviated ICH‐induced neurological deficits Figure . Similar3.2The integrity of white matter was assessed by immunostaining with MBP (a marker of myelin sheath) and NF200 (a marker of axons) Figure A. Our reCST region of cervical enlargement to further clarify secondary WMI. The results showed that the expression of NF200 was dramatically decreased after ICH, consistent with that in the peri‐hematoma region, which indicated axonal Wallerian degeneration may extended to the CST region of cervical enlargement. The MFI of NF200 was significantly increased after OMT administration  is an extracellular matrix compound, derived from reactive astrocytes, which is the major inhibitor of axonal growth. A mass of activated astrocytes, detected by the colocalization of GFAP and CSPG, aggregated at the peri‐hematoma region after ICH Figure B,C. The n Figure D, suggesn Figure B–E. Our 3.3Rikenellaceae and S24_7 was significantly higher but those of Bacteroides, Parabacteroides, Coprococcus, and Proteobacteria were significantly lower in the ICH + Vehicle group compared with the Sham group. While OMT significantly increased the relative abundance of Bacteroides, Parabacteroides, and Proteobacteria and decreased those of Rikenellaceae after ICH , including the ratio of F/B. On the other hand, Bacteroides, Parabacteroides, Helicobacter, and Akkermansia were more enriched in the ICH + OMT group compared with the ICH + Vehicle group  was significantly decreased in the ICH + OMT group , serum concentration of FD4 and behavioral scores, but negatively correlated with the mRNA levels of intestinal tight junction proteins (Tjs). While there was a negative correlation between microbial species from Firmicutes and Proteobacteria phylum, specially Lachnospiraceae, Desulfovibrionaceae, Ruminococcaceae, Oscillospira, and Desulfovibio and those above markers except intestinal Tjs expression  injury also plays a pivotal role in the functional outcome. In stroke patients, the integrity of the CST was a determining factor for the proportional recovery of motor impairment.CST fiber pathways in ICH patients with favorable outcomes.CST axon injury and demyelination at cervical enlargement, which is perhaps due to the protection of OMT against WMI in the primary lesion, or its inhibition of astrocytes/microglia activation and inflammatory response. Prolonged activation of microglia induced by myelin debris has long been implicated in aggravating inflammatory action by secreting pro‐inflammatory cytokines.CST injury. Furthermore, OMT significantly alleviated ICH‐induced neurological deficits both in the acute phase and the chronic phase. Perhaps slighter primary WMI in the peri‐hematoma region and secondary CST injury account for better neurobehavioral performance after OMT treatment.The severity of WMI is closely related to neuromotor dysfunction in ICH patients.Ruminoclostridium, Lachnospiraceae, and Ruminococcaceae.Gut microbiota is involved in the development and progression of ischemic stroke, through the microbiota–gut–brain axis.Intestinal injury, especially the gastrointestinal bleeding, is a common critical complication in ICH patients and an important risk factor for poor outcomes.Although our study has provided evidence that oral administration of OMT significantly alleviated ICH‐induced neurological deficits, gut dysbiosis, intestinal barrier dysfunction, and systemic inflammation, and FMT confirmed that gut microbiota plays an important role in these pathological processes, there were still several limitations in the present study. Firstly, medicine metabolism by bacteria in the intestine usually affects their pharmacokinetics and efficacy.5CST region of the cervical enlargement, improved ICH‐caused gut dysbiosis, promoted the intestinal barrier reconstruction, and inhibited the systemic inflammatory response. The role of intestinal flora in the effects of OMT on alleviating WMI induced by ICH still needs to be further studied.In conclusion, our study is the first time to reveal that OMT treatment effectively reduced WMI both at the striatum and distal The work presented here was carried out in collaboration among all authors. Haitao Sun conceived and designed the study. Hongwei Zhou, Wen Yuan, and Junshan Liu provided support of essential laboratory resources for this study. Jing Li conducted this study and drafted this manuscript; Jianhao Liang, Meiqin Zeng, Kaijian Sun, Yunhao Luo, Huaping Zheng, and Feng Li analyzed the data. Haitao Sun revised the manuscript. All authors reviewed and approved the final manuscript.This work was supported by Basic and Applied Basic Research Foundation of Guangdong Province (2020A1515010038), Key‐Area Research and Development Program of Guangdong Province (2018B030340001), Presidential Foundation of Zhujiang Hospital, Southern Medical University (yzjj2018rc03), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (GDHVPS2018), Young Elite Scientists Sponsorship Program by CACM (2019‐QNRC2‐C14), and Scientific research project funded by health Commission of Guangxi Zhuang Autonomous Region, China (Z20211397).The authors declare no conflict of interest.Not applicable.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Figure S4.Click here for additional data file.Appendix S1.Click here for additional data file.


Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment. Stroke remains the second leading cause of mortality and disability on a global scale, resulting in approximately 11.6 million deaths and a loss of 116 million disability-adjusted life years in 2019 .At present, recombinant tissue plasminogen activator (rtPA) is the only FDA-approved therapeutic intervention for ischemic stroke. Timely initiation of rtPA treatment is a critical aspect of stroke management and has demonstrated markedly improved outcomes for suitable patients. However, it is crucial to initiate rtPA treatment within a limited 4.5 h window from the onset of stroke symptoms. Beyond this time window, the potential for complications such as intracerebral hemorrhage significantly escalates. Furthermore, delayed rtPA treatment is correlated with an increased risk of hemorrhagic transformation and the deterioration of stroke outcomes  (PEG2000 PE); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG); and cholesterol  at a molar ratio of 43:28:6:8:15. Xe-liposomes were prepared by the previously described pressurization-freeze method [®) was obtained from Genentech Inc., San Francisco, CA, USA.Liposomes were composed of 1,2-dipalmitoyl-e method . The rtPTo evaluate the effect in vitro, porcine clots were incubated with rtPA either in the presence or absence of Xe-liposomes, and the weight loss of the clots was measured for comparison. The thrombolytic activity of rtPA was assessed using a modified version of the well-established in vitro clot-weight method . Fresh pBased on our previous dose-finding study , the optAll animal experiments were approved by the Animal Welfare Committee at The University of Texas Health Science Center in Houston. Male Sprague Dawley rats  were randomly selected as the animal model. Young males were exclusively used in this study considering potential sex differences in the outcome of embolic stroke, which could be influenced by the hormonal cycling of females in response to the Xe treatment. The animals were individually housed in cages in a temperature-controlled room (21–25 °C) with a 12-h light/dark cycle and 45–50% humidity, with free access to food and water. All experimental procedures were conducted in accordance with the approved protocols of the Animal Care and Use Committee of The University of Texas Health Science Center in Houston. Treatments and behavioral tests were conducted in a randomized and blinded manner.Cerebral ischemia was induced by occluding the right MCA using a clot. The reperfusion was achieved through rtPA-induced clot lysis. On the day of surgery, rats were anesthetized with 5 vol% isoflurane for induction and maintained at 2 vol% throughout the procedure. Rats were placed in a prone position on a rodent surgical monitor , which provided surgical warming and vital monitoring of body temperature, ECG, and heart rate. Body temperature was closely monitored and maintained at 37 °C throughout the entire experiment. A hole was carefully drilled at coordinates 1.7 mm posterior and 5.5 mm lateral from the bregma. A laser Doppler flowmetry (LDF) probe  was positioned onto the right parietal bone to monitor changes in cerebral blood flow (CBF) and confirm the successful induction of cerebral ischemia. A catheter was inserted into the femoral vein for the administration of rtPA. Subsequently, a midline neck incision was performed to expose the right common carotid artery (CCA) under an operating microscope. Ligation of the external carotid artery (ECA) was performed near its distal end, while isolation and separation of the internal carotid artery (ICA) from surrounding tissues were carried out. Incisions were made in the proximal branches of the ECA to facilitate the introduction of a PE-10 catheter. In order to produce homologous blood clots, 200 μL of rat arterial blood was mixed with 10 μL of 0.2 U thrombin and 20 mM CaCl2, followed by withdrawal into a 30 cm PE-50 tubing and incubation at 37 °C for 2 h. The clots were then washed, stored in sterile saline at 4 °C for 22 h, and transferred to distilled water for a 5-min rinsing period. A modified PE-10 catheter was used to draw a clot fragment measuring 12 × 0.35 mm. The catheter, containing the clot, was gently inserted into the right ECA until the tip reached a position just proximal to the origin of the MCA. The clot in the catheter was then injected into the MCA using 80 μL of saline. Regional blood flow was recorded over time using a LDF meter . Cases with CBF less than 45% and exceeding a 65% reduction in CBF were strictly excluded from the study design.n = 9 per group): sham control, eMCAO with no treatment control, eMCAO with free rtPA, and eMCAO with Xe-liposomes + free rtPA. Behavioral outcomes were assessed on days 1, 2, and 3 after eMCAO, and infarct size was determined through triphenyltetrazolium chloride (TTC) staining on day 3. In order to assess the potential impact of Xe-liposomes on rtPA thrombolytic activity in vivo, reperfusion of eMCAO was induced by IV administration of rtPA (10 mg/kg) and the alteration in blood flow was measured as an indicator of rtPA clot lytic activity for the two treatment groups .The aim of this experiment was to evaluate the potential cytoprotective effect of Xe-liposomes in combination with early IV administration of rtPA (10  mg/kg at 2 h) in an eMCAO rat model . Xe-lipon = 9 per group): sham control, eMCAO with no treatment control, eMCAO with free rtPA, eMCAO with Xe-liposomes + free rtPA. Behavioral outcomes were assessed on days 1, 2, and 3 following eMCAO, while infarct size, neuronal cell death, and hemorrhage analyses were performed on day 3 after stroke onset.This experiment aimed to evaluate the potential synergistic effect of Xe-liposomes in combination with late IV administration of rtPA (10  mg/kg at 4 h) on behavior, histology, and infarct size at days 3 post-eMCAO . Xe-lipoLaser Doppler flowmetry is a method of real-time monitoring of blood flow in the area of the brain supplied via the MCA. Monitoring the blood flow change can ensure the accuracy and reproducibility of the occlusion. The MCA regional blood flow was continuously monitored using a straight-needle LDF probe  connected to a standard laser Doppler monitor  . A burr 2) was applied over the CCA during Xe-liposome administration for a duration of 5 min to trigger Xe release from Xe-liposomes into the brain as previously described [A total of 600 μL of Xe-liposomes (10 mg/mL) were IV administered through the tail vein, 5 min prior to IV administration of rtPA. Continuous-wave ultrasound . A dose of 10 mg/kg of rtPA was IV infused for 60 min. The selected rtPA dose of 10 mg/kg was motivated by the fact that the clinical dose of rtPA (0.9 mg/kg) demonstrated no recanalization in the rat embolic stroke model due to the rat fibrinolytic system being ten times less sensitive than the human .All behavioral tests were conducted in a quiet and low-lit room by an observer blinded with respect to the treatment groups. Motor function and neurological outcomes were evaluated on post-surgery days 1, 2, and 3 using beam walking and grid walking tests . Limb plOn day 3 post eMCAO, brain tissue samples were collected. The dissected brains were sectioned coronally into 2 mm thick slices using a Jacobowitz brain slicer. These brain sections were then stained with 2% 2,3,5-triphenyltetrazolium chloride  in phosphate-buffered saline at room temperature for 30 min. TTC is a water-soluble dye that shows a color change in the presence of living cells. In living cells, the mitochondrial enzyme succinate dehydrogenase reduces TTC to form a water-insoluble, light-sensitive compound known as formazan, which imparts a deep red stain to healthy tissue. Conversely, damaged or dead tissue lacks this enzyme and formazan is not produced, resulting in the tissue remaining white. This color alteration is used to identify infarction and penumbra areas in stroke studies.Following fixation in phosphate-buffered 4% paraformaldehyde solution, the stained sections were subjected to morphometric analysis. Infarct size was quantified using the freely available image processing software, Image-J (Version 1.51), developed by the National Institutes of Health (NIH). Infarct volume was calculated for each slide by multiplying the slide thickness (2 mm), and then summing the volume of all slides to determine the total infarct volume. Subsequently, the normalized infarct volume was determined as a percentage of the whole brain volume.Immunostaining of brain coronal cryostat sections (10 µm) was performed by utilizing antibodies against MMP9  at 4 °C overnight. As a secondary antibody, Alexa Fluor 488 Goat anti-rabbit IgG was used . In order to counterstain the cerebral artery, the sections were incubated with α-smooth muscle (α-SM) actin antibody against rat microvascular smooth muscle antigens . Alexa Fluor 555 Goat anti-mouse IgG  was used as the secondary antibody. The fluorescently labeled slides were then analyzed under a fluorescence microscope .To detect DNA fragmentation in degenerating neurons, coronal brain sections (5 μm) were obtained using a cryotome and subjected to the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay . In brief, the sections were fixed in 4% paraformaldehyde for 20 min at room temperature and then permeabilized by incubating with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min at 4 °C. For TUNEL assay, each slide was incubated with 50 μL of TUNEL reaction mixture at 37 °C for 1 h. Additionally, the slides were stained with 4′,6-diamidino-2-phenylindole  to visualize the nuclei. Images were captured using a Nikon ECLIPSE Ti fluorescence microscope equipped with a CoolSNAP photomatrics camera. TUNEL-positive cells were photographed at 100× magnification from three randomly selected fields close to the infarct border. The number of dead cells was counted using NIS-Elements BR 3.2 diagnostic software .p < 0.05 was considered significant.In this preclinical animal study, due to the small sample sizes within the experimental groups, nonparametric statistical analysis methods were used to assess differences between the groups. Pairwise comparisons of spontaneous Xe release from Xe-ELIP in PBS or HSP and release with or without ultrasound were performed by the Wilcoxon rank sum test for two groups. For comparisons of multiple groups, the Kruskal–Wallis analysis of variance of ranks and median test was used to assess if there were global differences between the groups. This was followed by post hoc multiple comparisons of mean ranks for all groups by computing normal z-values for each comparison followed by probabilities adjusted for the number of comparisons for two-sided tests of significance. Data were plotted as means and standard deviation. Neurological outcomes between the treatment groups were reported as medians and quartiles. Statistica  software was utilized for the statistical analyses. A All sample size determinations were made at α = 0.05. A sample size of more than seven per group was determined to ensure a statistical power of at least 90% to detect the expected difference. Survival, change of regional blood flow in response to occlusion and treatment, and gross hemorrhage were continuously monitored and recorded for both studies of Xe-liposomes in combination with early rtPA administration  and lateIn the early rtPA treatment group, 39 animals successfully underwent eMCAO surgery with eligible occlusion rates . A totalIn the late rtPA treatment group, 40 animals successfully underwent eMCAO surgery with eligible occlusion rates . A totalIn order to investigate the thrombolytic efficiency of rtPA in the presence of Xe-liposomes, an in vitro clot lysis assay was conducted . Free rtp = 0.96 vs. free rtPA; The therapeutic efficacy of Xe-liposomes in conjunction with IV rtPA was assessed in a rat eMCAO model. Male Sprague Dawley rats were subjected to eMCAO by injecting a 12 mm long blood clot into the MCA to induce ischemia. Continuous monitoring of MCA regional blood flow was performed to verify the success of occlusion and to assess the effects of rtPA-induced reperfusion. Rats with eMCAO resulting in regional blood flow reduction within the range of 50–60% were selected for inclusion in the experimental group. In order to determine if Xe-liposomes interfere rtPA activity, regional blood flow was monitored before and after the rtPA treatment with the presence and absence of Xe-liposomes. The MCA regional blood flow change compared to the baseline for eMCAO after free rtPA treatment was 21 ± 7.9%, while the eMCAO after Xe-liposomes combined with rtPA treatment was 23 ± 5.5% (p < 0.05 vs. no treatment). When Xe-liposomes were administered in combination with rtPA, a further reduction in infarct size to 2.4 ± 0.9% (p < 0.05 vs. rtPA treatment) was observed. The mortality rates for embolic strokes with no treatment, treated with rtPA alone, and treated with the Xe-liposome + rtPA combination were 59%, 27%, and 27%, respectively (The therapeutic effect of Xe-liposomes in combination with early IV rtPA (10 mg/kg) was evaluated . The untectively . There wp < 0.05 vs. no treatment group). Animals subjected to Xe-liposomes along with rtPA treatment demonstrated further enhanced neurological improvement across all tests, surpassing both the untreated control and rtPA-only groups.The impact of combining Xe-liposomes with early rtPA treatment on the neurological disability of animals with ischemia due to eMCAO occlusion was investigated b–d. Untrp < 0.001 vs. no treatment). Xe-liposomes in combination with the rtPA treatment further reduced the infarct size to 2.0 ± 1.4% of the brain volume (p = 0.032 vs. rtPA treatment).In the late rtPA treatment group, rtPA was administrated at 4 h after stroke onset . The untBehavioral deficits were found to correlate with infarct volume outcomes b–d. Anim2) was quantified to evaluate cell death. In the affected tissue around the ischemic core, the Xe-liposomes + rtPA treatment group exhibited 155 ± 81 cells/mm2 with TUNEL positivity, significantly lower than the rtPA-only group , the untreated eMCAO group , and the sham control group   b.Given the well-known association between the MMP9 pathway and BBB integrity, as well as its role in hemorrhagic transformation following cerebral ischemia ,31, the The efficacy of current treatments using cytoprotective drugs for acute stroke remains limited. Many studies have focused solely on the administration of these drugs without considering their integration with IV rtPA treatment. At present, the primary strategy in treating acute ischemic stroke involves vascular reperfusion facilitated by thrombolysis (rtPA) and endovascular thrombectomy. The aim of cytoprotection is to forestall or mitigate brain cell death and minimize infarct size resulting from stroke. To achieve this goal, it is crucial to ensure that neuroprotective agents are compatible with rtPA treatment.In our earlier investigations, we presented compelling evidence for the efficacy of Xe-liposomes in preserving brain tissue from ischemic injury and maintaining neurological function in a transient MCAO rat model. To further translate our findings to potential clinical application, we investigated the compatibility of Xe-liposomes with IV rtPA treatment and assessed whether this combination could offer comparable benefits in the treatment of stroke.In the present study, we examined the effects of Xe-liposome cerebroprotection combined with rtPA on rtPA thrombolytic activity. Our findings demonstrated that Xe-liposomes did not inhibit rtPA thrombolytic activity in vitro or in vivo. The transformation of plasminogen to plasmin, a fibrinolytic enzyme crucial for clot dissolution, is catalyzed by rtPA. In a previous in vitro clot lysis study, human plasminogen (1 μg/mL) was used instead of porcine plasminogen due to the markedly reduced activation of porcine plasminogen by rtPA compared to that of human plasminogen . The typRemarkably, animals treated with the combination of Xe-liposomes and rtPA exhibited superior cognitive function compared to those receiving rtPA alone. An intriguing trend was also observed, revealing a decrease in rtPA-induced hemorrhagic transformation during late rtPA treatment when combined with Xe-liposomes. We postulate that this effect could be attributed to the stabilization of the BBB by rtPA. These findings demonstrate the potential of Xe-liposomes as a beneficial cytoprotective agent when combined with rtPA for the treatment of ischemic stroke.Prior studies have provided diverse insights into the impact of Xe on rtPA activity. David et al. investigated the influence of different Xe concentrations on the thrombolytic efficiency of rtPA . Their fCombining Xe-liposome administration with both early and late rtPA therapy resulted in improved neurological outcomes. The proposed mechanisms underlying the cerebroprotective effects of Xe-liposomes involve pleiotropic pathways including neuroprotection, anti-apoptosis, anti-inflammation, and protection against ischemic-reperfusion injury ,39. ReceDespite the efficacy of rtPA in clot lysis, its administration at the later stages of stroke is associated with an increased risk of hemorrhagic transformation. Consequently, there is a growing need to develop novel therapies aimed at improving stroke outcomes by stabilizing brain tissue. In the present study, the effect of Xe-liposomes on reducing infarct size and behavioral impairments was demonstrated. Moreover, Xe-liposomes showed potential for modulating MMP9 and stabilizing the BBB. A parallel study focused on validating the detailed mechanism underlying the effect of Xe on the BBB and MMP9 is currently underway, and its results will be published independently. Nonetheless, the crucial aspect is that Xe does not exacerbate rtPA-induced hemorrhagic transformation. This valuable safety and efficacy data will help future research aimed at validating the therapeutic potential of Xe-liposomes in larger animal models and clinical trials.Several limitations need to be acknowledged in the present study. Infarct size assessment on day 3 without factoring in edematous effects might lead to an underestimation of the full extent of ischemic injury. Edema, comprising cytotoxic and vasogenic components, is a common reaction to cerebral infarction and leads to brain tissue swelling. This swelling can extend beyond the boundaries of the initial infarcted area due to vasogenic edema. The most substantial increment in brain tissue swelling is noted at the 24-h mark post-injury . FailingOur study built upon previous research that demonstrated that the co-administration of Xe-liposomes and rtPA effectively reduced infarct volume and improved neurological function in thrombotic stroke animal models. We extensively investigated the compatibility of Xe-liposomes with rtPA in a clinically relevant embolic stroke model and confirmed no interference with the thrombolytic activity of rtPA both in vitro and in vivo. The combination treatment of rtPA with Xe-liposomes demonstrated superior outcomes, maintaining the thrombolytic activity of rtPA while minimizing the risk of hemorrhagic transformation. Our study suggests that the combined therapy of Xe-liposomes and rtPA may provide cerebroprotection and improve neurological function recovery in cases of acute stroke. The implications of these findings underscore the need for further investigation to determine the feasibility of translating this approach from pre-clinical studies to human clinical trials. In a clinical setting, Xe-liposomes could be pre-administered, co-administered, or post-administered with rtPA to maximize treatment efficacy.


This study evaluated myocardial protection and clinical outcomes when usinglactated Ringer’s solution as the base solution for del Nido cardioplegiacompared with histidine-tryptophan-ketoglutarate (HTK) solution in valvularsurgery.From January 2017 to May 2018, 71 adult patients who underwent valvularsurgery with del Nido cardioplegia (n=37) or HTK cardioplegia (n=34) wereretrospectively analyzed.vs. 55.88%; P<0.001), lower totalvolume of cardioplegia administered , shorter hospital stay , and lesspostoperative red cell transfusion . There is no difference in aorticcross-clamping time, postoperative change in left ventricular ejectionfraction, intensive care unit stay, duration of inotropic support, new onsetof atrial fibrillation, in-hospital mortality, complications, and three-yearoverall survival rate.Patients’ characteristics were comparable between groups. Postoperative peaktroponin T levels were similar. The del Nido group had a decreased incidenceof ventricular fibrillation after aortic cross-clamp removal (13.51Lactated Ringer’s-based del Nido cardioplegia can be safely used for valvularsurgery with acceptable clinical outcomes compared to HTK cardioplegia. Recent studies have demonstrated its usefulnessand safety for myocardial protection in single cardiac valve surgery. In addition,theoretical advantages and outcome studies have led to the increased popularity ofsingle-dose cardioplegia in complex valve procedures and minimally invasive valvesurgery,4,5,6.Cardioplegia is one of the critical components of myocardial protection duringcardiac surgery. Traditional cardioplegia is usually administered repetitively every20 minutes. On the other hand, single-dose cardioplegia is typically administeredonce or with extended dosing intervals, allowing for fewer disruptions and improvingthe surgical workflow, which can lead to shorter aortic occlusion periods or reducedoperative time. It providesequivalent clinical outcomes compared to traditional multidosecardioplegia,8. In addition, it is also used inorgan preservation  for transplantation. At ourinstitution, HTK cardioplegia has been the standard single-dose cardioplegia formyocardial protection in both pediatric and adult cardiac surgery for a decade.Histidine-tryptophan-ketoglutarate (HTK) solution  and del Nido cardioplegia are single-dosecardioplegia available in many cardiac centers. HTK cardioplegia was first describedby Bretschneider. Recently, del Nido cardioplegia has become more commonlyused in adult cardiac surgery. Since Plasma-Lyte™ A , the base solution fordel Nido cardioplegia, is unavailable in many countries, our institution hasutilized lactated Ringer’s solution as a base solution for del Nido cardioplegiawith excellent results compared to our standard blood cardioplegia,11. To evaluate the safety and efficacy of this modified delNido cardioplegia, we compared the clinical outcomes associated with modified delNido cardioplegia and HTK cardioplegia in valvular heart surgery.Del Nido cardioplegia has been used exclusively in pediatric cardiac centers formyocardial protection during cardiac surgeryPatients aged 18 years or older who had elective cardiac surgery for valvularheart disease using single-dose cardioplegia, either HTK or del Nidocardioplegia, were eligible for inclusion in the study. The cardiac procedurewas performed through standard median sternotomy, including isolated mitralvalve surgery, isolated aortic valve surgery, and multiple valve surgery. Theexclusion criteria were emergency surgery, concomitant coronary bypass surgery,concomitant aortic surgery, and concomitant maze procedure. From January 2017 toMay 2018, a total of 71 patients (37 in the del Nido group and 34 in the HTKgroup) were retrospectively reviewed. All patients were followed up until June2021.The primary outcome was the highest postoperative troponin T level within 24hours. Immediately postoperative, 12-hour postoperative, and 24-hourpostoperative samples were routinely obtained.Secondary outcomes were postoperative outcomes, intraoperative outcomes, andassessments of additional measures of impaired myocardial protection.Postoperative outcomes consisted of the intensive care unit (ICU) stay, hospitalstay, mortality, occurrence of postoperative complications, and the need forpostoperative red cell transfusion. Intraoperative outcomes included the totalvolume of cardioplegia administered, the number of doses, total cardiopulmonarybypass (CPB) time, and aortic cross-clamping time. Assessments of additionalmeasures of impaired myocardial protection included the incidence of ventricularfibrillation after aortic cross-clamp removal, postoperative changes in leftventricular ejection fraction (LVEF), duration of inotrope/ vasopressorrequirement, incidence of new-onset postoperative atrial fibrillation orflutter, and requirement for intra-aortic balloon pump (IABP) support.Before and after the procedure, LVEF was evaluated by intraoperativetransesophageal echocardiography. Postoperative changes in LVEF were defined asthe difference between postoperative LVEF and preoperative LVEF. Patientcharacteristics variables and postoperative outcomes  were asdefined by the STS Adult Cardiac Surgery Database.,9,10,11.The compositions of HTK and del Nido cardioplegias are described in In patients receiving HTK cardioplegia, approximately 1,500-2,000 mL of HTKsolution was infused with hydrostatic pressure (from approximately 2 m height)over 6-8 minutes through an aortic root catheter or directly through thecoronary ostia depending on the degree of aortic valve insufficiency. Generally,redosing was not required unless there was electrical activity. Additional dosesof 100-200 mL were administered as needed. HTK solution was delivered at atemperature of 4-8°C.All patients received ultrafiltration during the operation.The study protocol was approved by the institutional review board of RamathibodiHospital , with patient consentwaived.. To detect the statistical difference in a two-sided testwith 5% alpha error and 90% power, 26 patients were required in each group.The sample size for the primary outcome was calculated by the difference between24-hour postoperative troponin levels of the HTK and del Nido cardioplegiagroups reported by Talwar et al.t-test or the Mann-Whitney U test. Categorical variableswere expressed as frequencies and percentages and were analyzed usingchi-squared or Fisher’s exact test. Survival analyses were estimated by theKaplan-Meier method. STATA version 14  was used for statistical analysis. A P-value < 0.05is considered a statistical significance.Continuous variables were expressed as mean (standard deviation) or median(interquartile range) and were compared using an independent samplevs. 20.59 %, P=0.042), and preoperativeLVEF was lower in the del Nido group  than in the HTK group   .vs. 55.88%,P<0.001) compared with the HTK group. Postoperativechanges in LVEF were similar  between the groups. The total cardioplegia volumein the del Nido group was significantly lower than that in the HTK group . The number of doses, the aortic cross-clampingtime, and total bypass time were similar between groups  and a shorter hospital stay  than the HTKgroup  months. Three deaths occurredduring the follow-up period (one in the del Nido group and two in the HTKgroup). At 36 months, the cumulative survival rate was 96.97% and 96.88% for thedel Nido and the HTK group, respectively. The Kaplan-Meier survival analysis ofthe del Nido group P=0.564).This present study on adult valvular surgery suggested equivalent myocardialprotection and clinical outcomes with lactated Ringer’s-based del Nido cardioplegiaas compared to HTK cardioplegia. Assessment of myocardial injury, such as peaktroponin T levels, the incidence of new-onset postoperative atrial fibrillation orflutter, duration of inotrope/vasopressor support, the requirement for IABP support,and postoperative change in LVEF were similar between the cardioplegia protocols.Notably, the incidence of ventricular fibrillation after aortic cross-clamp removalwas lower in del Nido cardioplegia. Additionally, the use of del Nido cardioplegiawas associated with a lower total volume of cardioplegia, shorter length of hospitalstay, and lower incidence of postoperative red cell transfusion.. Malposition and dislodging ofthe coronary sinus catheter can occur during cardiac procedure and lead toundesirable complications such as rupture of the coronary sinus. Moreover,retrograde cardioplegia could not always be achieved, especially in a minimallyinvasive approach. Thus, a single-dose cardioplegia is an attractive option formyocardial protection during valvular heart surgery. Its longer redosing intervalprovides an uninterrupted, streamlined surgical workflow. Additionally, fewermanipulations potentially reduce the risk of serious complications.Administration of cardioplegia during the exposure of the valvular structure invalvular heart surgery is challenging. In mitral valve surgery, over-traction of themitral retractor can distort the aortic valve. Therefore, the mitral retractor mustbe periodically dismounted during the administration of antegrade cardioplegia toensure aortic valve competency and appropriate delivery to the coronary arteries. Inaortic valve surgery, the cardioplegia must be delivered through the coronary ostia.Repetitive manipulation around the coronary ostia may result in a coronary ostialinjury. In addition, cardioplegia distribution may not be sufficient from theunbalanced coronary ostial infusion. These maneuvers usually interrupt the surgicalworkflow when used in conjunction with traditional multidose cardioplegia. The useof retrograde cardioplegia may be a suitable option in this situation. However, itis associated with insufficient protection of the right ventricle,3,5,8,11. However,very few studies have a face-to-face comparison between the two cardioplegiaprotocols. In pediatric patients who underwent elective surgical correction fortetralogy of Fallot, the randomized study by Talwar et al. demonstrated lower postoperativetroponin release in the del Nido group than in those who received HTK solution. Leeet al. also reported similarresults in minimally invasive cardiac surgery patients. The generally acceptabletime limit for a singledose infusion of del Nido cardioplegia is 60-90minutes,10,11,14. However, ifnecessary, additional doses can be administered after 90 minutes of the initialdose. In comparison, the additional dose of HTK cardioplegia may not be requiredwithin 180 minutes of the initial dose unless there is a spontaneous return ofcardiac electrical activity. Most of these previous studies were performed inlow-risk, non-complex, single-valve patients with preserved ventricular function.Therefore, the aortic cross-clamping time is usually < 90 minutes. In our study,the mean aortic cross-clamping time in the del Nido and HTK groups was longer thanin other studies  which could beexplained by a more complex procedure  included in our study. Our study’s prolonged aortic cross-clamping timecould have been sufficient to produce higher troponin release postoperatively inboth groups. Therefore, there was no difference in postoperative troponin levels.Similar results for prolonged aortic-cross clamping time were also observed in Duanet al. complex valve surgerystudy.Myocardial injury, represented by peak postoperative troponin T levels in this study,was similar between cardioplegia protocols. Both del Nido and HTK cardioplegias havebeen reported to have similar or superior myocardial protection compared to theblood cardioplegia strategy, at the messengerribonucleic acid (mRNA) expression level of associated markers, HTK cardioplegiacaused more damage to the myocardium compared to blood cardioplegia. In addition, asignificant increase in ventricular fibrillation after aortic cross-clamp removalwith HTK cardioplegia has been consistently reported,12. Ourstudy also demonstrated a higher incidence of ventricular fibrillation in the HTKgroup compared to the del Nido group. This finding may indirectly indicate bettermyocardial protection with del Nido cardioplegia. However, these findings had noimpact on outcomes, as demonstrated by similar postoperative troponin levels andclinical outcomes. On the other hand, another interesting finding is that the use ofdel Nido cardioplegia is associated with a lower incidence of ventricularfibrillation after aortic cross-clamp removal,11,12,14. One of the ingredients of delNido cardioplegia, lidocaine, may be responsible for these findings.Ventricular fibrillation can occur when the heart muscle is damaged or stressed. Thiscan happen when the heart is exposed to ischemia, hypoxia, or other forms of stress.Therefore, the occurrence of ventricular fibrillation may be an indicator of poormyocardial protection during surgical procedures. According to the finding fromElcik et al.,4,11,12. In our study, del Nidocardioplegia was also associated with a lower total volume of cardioplegia and alower incidence of postoperative red cell transfusion. Although the perfusedsolution can be removed from the coronary sinus to reduce hemodilution, theseunnecessary procedures added on to the operation, including additional venouscannulation, total caval occlusion, and right atrial incision, can prolong theoperation and lead to undesirable outcomes.The total volume of HTK cardioplegia is generally higher than of del Nidocardioplegia. Additionally, HTK is crystalloid cardioplegia, whereas del Nidocardioplegia is 1:4 blood cardioplegia. Therefore, more hemodilution may occur inpatients who receive HTK cardioplegia. Many del Nido cardioplegia studies havereported a reduction in the total cardioplegia volume. The reduction in the totalvolume of cardioplegia may result in less hemodilution and a lower incidence ofpostoperative red cell transfusion. Incontrast, lactated Ringer’s solution has calcium in a range of 1.5-3 mEq/L. Ourmodification to utilize lactated Ringer’s as a based solution leads to highercalcium concentration in the delivered cardioplegia. Theoretically, this may increase intracellularcalcium accumulation, resulting in poor myocardial recovery. Although the calciumconcentration in this modified version of del Nido cardioplegia was a concern, therewas no evidence of impaired outcomes with our cardioplegia strategy in bothshort-term and midterm follow-ups.Plasma-Lyte™, the base solution of original del Nido cardioplegia, has nocalcium. The final calcium concentration of the delivered cardioplegia can beconsidered as trace after mixing one part of oxygenated pump blood with thecrystalloid componentThis study was limited by its retrospective, non-randomized, andsingle-institution design. We included various procedure types in thisinvestigation, although the proportion of operation types was not statisticallydifferent. In addition, we could not control confounding variables due to thesmall sample size. As the goal of this study was to compare our modified delNido cardioplegia with HTK cardioplegia, the findings in this single-institutionstudy may not be generalizable or applicable to other cardiac centers becausethe variations in cardioplegia protocol may not provide comparable results. Tovalidate our findings, a larger sample size or randomized controlled study tocompare our modified del Nido cardioplegia and HTK cardioplegia is required.The use of lactated Ringer’s-based del Nido cardioplegia is a safe and effectivealternative to HTK cardioplegia for myocardial protection in valvular surgery. Ourfindings demonstrated that the modified del Nido cardioplegia provided comparableoutcomes with several advantages, including a decreased incidence of ventricularfibrillation after aortic cross-clamp removal, lower total cardioplegia volume,shorter length of hospital stay, and a lower incidence of postoperative red celltransfusion. However, further investigation in a large-scale or randomizedcontrolled study is warranted to confirm our findings.