Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers.

The cryopreservation of endothelial cell monolayers is an important step that bridges the cryopreservation of cells in suspension to that of tissues. Previous studies have identified clear distinctions in freezing mechanisms between cells in suspension and in monolayers, as well as developed novel protocols for monolayer cryopreservation. Recently, our group has shown that human umbilical vein endothelial cell (HUVEC) and porcine corneal endothelial cell (PCEC) monolayers grown on Rinzl plastic substrate can be cryopreserved in 5% dimethyl sulfoxide, 6% hydroxyethyl starch, and 2% chondroitin sulfate, following a slow-cooling protocol (-1 °C/min) with rapid plunge into liquid nitrogen from -40 °C. However, membrane integrity assessments were done immediately post thaw, which may result in an overestimation of cell viability due to possible delayed injury responses. Here, we show that for the optimal protocol condition of plunge at the -40 °C interrupt temperature, HUVEC and PCEC monolayers exhibited no significant immediate post-thaw injuries nor delayed injury responses during the 24-h post-thaw overnight culture period. HUVEC monolayers experienced no significant impact to their natural growth rate during the post-thaw culture, while PCEC monolayers experienced significantly higher growth than the unfrozen controls. The difference in the low-temperature responses between HUVEC and PCEC monolayers was further shown under high temperature plunge conditions. At these suboptimal plunge temperatures, HUVEC monolayers exhibited moderate immediate membrane injury but a pronounced delayed injury response during the 24-h post-thaw culture, while PCEC monolayers showed significant immediate membrane injury but no additional delayed injury response during the same period. Therefore, we provide further validation of our group's previously designed endothelial monolayer cryopreservation protocol for HUVEC and PCEC monolayers, and we identify several cell-type-specific responses to the freezing process.

Step-by-step protocol for alternative injury models in newt cardiac regeneration.

Although the heart is one of the most important organs for animal survival, its regenerative capacity varies among animal species. Notably, adult mammals cannot regenerate their hearts after damage such as acute myocardial infarction. In contrast, some vertebrate animals can regenerate the heart throughout their lives. Cross-species comparative studies are important to understand the full picture of cardiac regeneration in vertebrates. Among the animal species able to regenerate the heart, some urodele amphibians, such as newts, possess a remarkable capacity for this process. Standardized methods of inducing cardiac regeneration in the newt are needed as a platform for studies comparing newts and other animal models. The procedures presented here describe amputation and cryo-injury techniques for the induction of cardiac regeneration in Pleurodeles waltl, an emerging model newt species. Both procedures consist of simplified steps that require no special equipment. We additionally show some examples of the regenerative process obtained using these procedures. This protocol has been developed for P. waltl. However, these methods are also expected to be applicable to other newt and salamander species, facilitating comparative research with other model animals.

Cryo-injury procedure-induced cardiac regeneration shows unique gene expression profiles in the newt Pleurodeles waltl.

BACKGROUND: Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo-injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo-injury procedures are common in mouse and zebrafish. RESULTS: In the present study, we induced cryo-damage on the ventricle in Iberian ribbed newts using a liquid nitrogen-chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo-injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures. CONCLUSIONS: Notably, we established a cryo-injury procedure for the newt and confirmed that regeneration of the cryo-damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo-injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models.

Optimized addition of nitric oxide compounds in semen extender improves post-thaw seminal attributes of Murrah buffaloes.

Semen dilution and cryopreservation alter the homogeneity of seminal plasma, resulting in a non-physiological redox milieu and consequently poor sperm functionality. Considering the concentration-specific bimodal action of nitric oxide (NO) in the regulation of sperm functions, cryopreservation media supplemented with optimized concentrations can improve the semen attributes. The present study aimed to evaluate the effect of adding an optimized concentration of sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) in an extender on in vitro semen quality. An aliquot of semen samples (n = 32) from Murrah buffalo bulls (n = 8) was divided into control (C) and treatment (T-I: SNP in extender at 1 µmol/L; T-II: L-NAME in extender at 10 µmol/L). Fresh semen quality parameters showed no significant difference at 0 h except for the structural integrity in the T-II group. Post-thaw semen quality parameters and sperm kinematics using computer-aided sperm analysis (CASA) revealed significantly higher (p < 0.05) cryoresistance in the treatment groups. Viability, acrosome integrity, and membrane integrity were significantly higher (p < 0.05) in both treatment groups; however, the results were pervasive in T-II. Lower abnormal spermatozoa were observed in both T-I and T-II. SNP supplementation led to a significant rise (p < 0.05) in NO, whereas L-NAME reduced the NO concentration in post-thawed samples, which was directly correlated with different sperm functionality and associated biomarkers viz. total antioxidant capacity (TAC) and thiobarbituric acid reactive substance (TBARS). It was concluded that the cryopreservation media supplemented with SNP and L-NAME at 1 µmol/L and 10 µmol/L, respectively, lower the cryo-damage and improve post-thaw seminal attributes.

Functional electrical stimulation following nerve injury in a large animal model.

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.

Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization.

Sperm cryopreservation facilitates the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. The cryopreservation process can damage sperm and compromise functionality. Several cryobiological studies have found that the physical and biological factors that affect sperm survival at low temperatures during the cryopreservation process often involve the integrity of sperm membrane. In this review, the behaviour of the sperm membrane against cooling, cold shock, ice crystal formation, oxidative stress, osmotic changes, reorganization of the lipid bilayer and addition of cryoprotective agents (CPA) is discussed. In addition, the phenomenon of reactive oxygen species (ROS) and its relationship with the cryopreservation process is also described. Semen cryopreservation techniques have progressed slowly in past years, and the current performance, measured as post-thawed survival, is not very different compared to past decades. Recent advances in understanding the structure of the cell membrane, its function and metabolism have driven to new conservation systems, including lyophilization and vitrification. However, none of these technologies is commercially available, although its future appears very promising.

Quantitative phosphoproteomics explain cryopreservation-induced reductions in ram sperm motility.

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SIGNIFICANCE: This study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants.

Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox.

Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 µM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for H2O2. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.

Improvements in morphology and membrane stability obtained from TPP-TAB, a cryopreservation medium treated infertile smoker sperm cells - An in vitro study.

BACKGROUND: Plants are the major sources of antioxidants, which maintains oxidant: antioxidant state or is to protect from excessive reactive oxygen species in case of cryo medium selection though maintain sperm stability. BOTTLENECK: Cryo preservation of infertile smokers is challengeable with the available medium due to over production of ROS hitches, were cause loss of sperm physiology. Hence, a novel medium is needed to store/protect sperm cells of infertile smokers where they attending/ongoing IVF or oncogenic surgical treatment. AIM: The aim of this study, is to check the stability of sperm cells by TPP-T.arjuna bark (TAB) (E4) cryo medium preserved infertile smoker's against ROS/cryo injury- as a continuous study. MATERIALS AND METHODS: 42 infertile smoker's subjects with 28 control subjects were selected. Surface morphology (acrosome) of sperm by scanning electronic microscope, sperm membrane proteins by colorimetric method, sperm head and tail defects by CASA method and finally sperm cell stability is checking its zeta electric potential charges, were all done with E4 cryo medium treated frozen/thawed selected study subjects. RESULTS: Sperm morphology and zeta potential shows there is no damage along the stability of cells maintained during E4 medium cryopreservation in infertile subjects. SUMMARY: This is the first study is too established for infertile smokers sperm stability was checked for six months with E4 cryo medium.

CD62p expression and ADP induced re-expression in platelet apheresis concentrates after long-term cryopreservation at -80 ℃ / 第三军医大学学报

0.05). However, the positive rate of re-expressed CD62p declined to (35.2?18.2)% when the platelets stored at -80 ℃ for 48 months, with significant difference with that for 36 months (P