[Risk factors associated with in-hospital mortality in patients requiring extracorporeal membrane oxygenation in the perioperative period of heart transplantation].

Objective: To explore risk factors associated with in-hospital mortality in patients requiring extracorporeal membrane oxygenation (ECMO) in the perioperative period of heart transplantation. Methods: The data of ECMO cases in the perioperative period of heart transplantation from the Chinese Society of Extracorporeal Life Support (CSECLS) between January 2017 and December 2021 were retrospectively analyzed. These patients were divided into the survival group and non-survival group according to their outcomes at discharge. The demographics, indications and complications of ECMO between the two groups were compared, and the related risk factors of poor prognosis were analyzed. Results: A total of 77 patients were included in the study, including 67 males and 10 females, with a median age [M(Q1, Q3)] of 48 (36, 59) years. Sixty-three patients (81.8%) were successfully withdrawn from the ECMO and 46 patients (59.7%) survived to discharge. The median ECMO time was 139 (92, 253) hours. Compared with the survival group, the non-survival group (n=31) had more patients with chronic kidney disease before surgery [22.6% (7/31) vs 4.3% (2/46), P=0.034], and a higher proportion of continuous renal replacement therapy (CRRT) during ECMO [74.2% (23/31) vs 50.0% (23/46), P=0.034]. Moreover, the non-survival group had longer duration of extracorporeal circulation [262 (195, 312) vs 201 (155, 261) min, P=0.056] and higher lactate value in the first 24 hours of ECMO support [2.7 (2.1, 4.7) vs 2.3 (1.4, 3.8) mmol/L, P=0.060], but the differences were not statistically significant. Multivariate logistic regression analysis showed that perioperative application of CRRT was an independent risk factor for poor prognosis in ECMO patients during heart transplantation (OR=19.345, 95%CI: 1.209-309.440, P=0.036). Conclusion: CRRT treatment during ECMO is a risk factor for in-hospital mortality in patients undergoing heart transplantation.

[Comparison of extracorporeal membrane oxygenation applicated in critical patients with COVID-19 and novel influenza A (H1N1) virus pneumonia].

Objective: To compare the clinical characteristics and prognosis of critical patients with COVID-19 and novel influenza A (H1N1) virus pneumonia (influenza pneumonia) applied with extracorporeal membrane oxygenation (ECMO). Methods: A total of 24 patients with influenza pneumonia treated with ECMO in respiratory intensive-care unit (ICU) of Beijing Chaoyang Hospital from March 2016 to December 2019 and 12 patients with COVID-19 hospitalized from February 1, 2020 to March 31, 2020 in 5 government designated infectious hospitals of Beijing and Hebei Province that applied with ECMO were enrolled. The demographic data, clinical manifestations, and ECMO related information were described and analyzed and all numerical variables are described as M (P25, P75). Results: The age of COVID-19 patients was 77 (66, 79) years old, which was older than influenza pneumonia patients [46 (32, 62) years old], P<0.05; acute lung injury score and respiratory ECMO survival prediction (RESP) score before ECMO application were 3.3 (3.0, 3.5) and 1 (0, 2), respectively, which were lower than influenza pneumonia patients [3.8 (3.5, 4.0) and 4 (2, 6), respectively], all P values<0.05. Thrombotic complications, bleeding complications, and ventilator-associated pneumonia occurred in ECMO applied COVID-19 patients were 4, 10 and 5 cases, respectively, which were more than that among influenza pneumonia patients (1, 9, and 2 cases, respectively), all P values<0.05. The length of ICU stay of COVID-19 patients was 31 (28, 75) d, which was longer than that of influenza pneumonia patients [27 (18, 39) d], P<0.05. The cases with successful decannulation of ECMO among COVID-19 and influenza pneumonia patients were 6 and 14 cases, respectively and mortality during ICU stay were 8 cases and 11 cases, respectively, and the difference were not statistically significant (all P values>0.05). Conclusion: COVID-19 patients applied with ECMO have more ECMO-related complications and a longer stay in the ICU than patients with influenza pneumonia.

[Application of pump-controlled retrograde trial off in weaning from veno-arterial extracorporeal membrane oxygenation in adult patients].

Objective: To Summarize the experience of pump-controlled retrograde trial off (PCRTO) in the process of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) withdrawal in adult patients. Methods: Adult patients who received ECMO assistance in Intensive Care Unit for Cardiac Surgery from March to July 2019 were collected. According to our strategies, PCRTO was used if the patients can wean from VA-ECMO and hemodynamic indexes were recorded during the process. The statistics data was collected, including the 48 hours survival rate, ECMO re-assistance rate, thrombus complications, Intensive Care Unit (ICU) stay time and hospital stay time after weaning from VA-ECMO. The patients who failed in the test were continued to be assisted by ECMO. Results: There were 46 patients assisted by VA-ECMO in our center. In total, 21 adults who met the offline test standard underwent 26 PCRTOs, including 10 male adults (47.6%), with an age of 65 (55, 68) years old. Eighteen adults passed the withdrawal test. No new thrombus was found in the arteriovenous ultrasound of the lower extremity after weaning from ECMO, and no pulmonary embolism was found in the chest X-ray. The success rate of weaning from ECMO was 69.23%(18/26). The D-dimer decreased [584(348,2 107)µg/L vs 1 440(631,2 916)µg/L, P=0.014] and the left ventricular ejection fraction (LVEF) increased (51.4%±8.5% vs 46.9%±10.6%, P=0.013) on the next day after weaning. There were significant differences in heart rate (HR), central venous pressure (CVP), oxygenation index and lactate (Lac) during the PCRTO in the group which involved the cases of the 8 failed experiments (all P<0.05). Compared with the failure group, there were significant differences in age, blood flow rate, CVP before the test, HR, pulse oxygen saturation(SpO(2)), CVP, Lac and oxygenation index after the test, and the variations of SpO(2), CVP and Lac. Conclusion: PCRTO is a simple, reversible, safe and effective weaning method. It can be used in the process of VA-ECMO withdrawal in adult patients.

Parthenogenetic activation of rat oocytes and their development (in vitro).

The present study was performed to determine suitable methods for parthenogenetic activation and subsequent development of rat oocytes in vitro. In the first series of experiments, the ability of electrical pulses, strontium, ethanol and ionomycin to activate Sprague-Dawley (SD) rat oocytes was examined. The synergistic effect of strontium and cycloheximide or puromycin was also examined in the second series of experiments. In the third series of experiments, the development of F1 hybrid (SD x Dark Agouti) parthenotes activated with different concentrations of strontium (10-0.08 mM) was compared with that of SD parthenotes. The effect of the timing of activation (10 min and 2, 4 and 6 h after cervical dislocation) was also assessed in a fourth series of experiments. The oocytes activated by strontium showed higher pronuclear formation and cleavage rates than those in the other groups (P < 0.05). Higher blastocyst development was obtained from parthenotes activated by strontium and strontium-cycloheximide compared with the strontium-puromycin group (P < 0.01). However, the total cell number of blastocysts from the strontium-cycloheximide activation group was higher than that of other groups (P < 0.05). With strontium (2.5-10 mM) treatment, 40.9% of blastocysts were obtained from F1 hybrid oocytes, whereas 22.9% were obtained from SD (P < 0.01). The oocytes activated 10 min or 2 h following cervical dislocation showed higher blastocyst development than those of the 4 and 6 h groups (P < 0.01). These results suggest that strontium-cycloheximide produces the highest parthenogenetic activation rate in the rat and that oocytes must be activated by 2 h after cervical dislocation.

Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/microl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/microl and then decreased at 10 ng/microl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/microl to maximise integration and efficiency rates in pigs.

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses.

The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm(-1) DC pulses of 60 micros duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.

Developments in transgenic techniques in pigs.

Manipulation of the pig genome is currently restricted to the random insertion of new DNA using pronuclear microinjection. This method suffers from a number of inherent limitations, the majority of which result from the inability to control the site at which the transgene becomes integrated. These drawbacks, together with the need to be able to target existing genes, will result in the replacement of pronuclear injection by new methods that have the capability to direct insertion to a particular genomic site that does not influence expression. Currently, it is possible to control the site of insertion in mice using embryonic stem (ES) cell and homologous recombination technologies. However, pluripotent ES cells have yet to be isolated in pigs. The possibility of using nuclear transfer to reprogramme early differentiated embryonic cells as well as somatic cells from adult animals may provide an alternative method for generating precise genetic modifications. Methods that allow these changes to be carried out in situ are also likely to be developed in the future.

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.