Development of a hepatic cryoinjury model to study liver regeneration.

The liver is a remarkable organ that can regenerate in response to injury. Depending on the extent of injury, the liver can undergo compensatory hyperplasia or fibrosis. Despite decades of research, the molecular mechanisms underlying these processes are poorly understood. Here, we developed a new model to study liver regeneration based on cryoinjury. To visualise liver regeneration at cellular resolution, we adapted the CUBIC tissue-clearing approach. Hepatic cryoinjury induced a localised necrotic and apoptotic lesion characterised by inflammation and infiltration of innate immune cells. Following this initial phase, we observed fibrosis, which resolved as regeneration re-established homeostasis in 30 days. Importantly, this approach enables the comparison of healthy and injured parenchyma with an individual animal, providing unique advantages to previous models. In summary, the hepatic cryoinjury model provides a fast and reproducible method for studying the cellular and molecular pathways underpinning fibrosis and liver regeneration.

Development of a hepatic cryoinjury model to study liver regeneration.

The liver is a remarkable organ that can regenerate in response to injury. Depending on the extent of injury, the liver can undergo compensatory hyperplasia or fibrosis. Despite decades of research, the molecular mechanisms underlying these processes are poorly understood. Here, we developed a new model to study liver regeneration based on cryoinjury. To visualise liver regeneration at cellular resolution, we adapted the CUBIC tissue-clearing approach. Hepatic cryoinjury induced a localised necrotic and apoptotic lesion characterised by inflammation and infiltration of innate immune cells. Following this initial phase, we observed fibrosis, which resolved as regeneration re-established homeostasis in 30 days. Importantly, this approach enables the comparison of healthy and injured parenchyma with an individual animal, providing unique advantages to previous models. In summary, the hepatic cryoinjury model provides a fast and reproducible method for studying the cellular and molecular pathways underpinning fibrosis and liver regeneration.

L-Proline as a Cryoprotective Agent for the Preservation of Galea Spixii Skin Fibroblasts.

Somatic cell biobanking is a promising strategy for developing reproductive techniques. Although cryopreservation, a technique used for creating biobanks, has been performed on Galea spixii, structural and physiological damage to its cells highlight the need to optimize the cryoprotective solution being used. Therefore, the osmoprotective activity of 5 mM L-proline was evaluated as an alternative cryoprotectant for G. spixii fibroblast conservation. The concentration was defined based on previous studies conducted on mammalian cells. Cells derived from the skin of six individuals were cultured until the fifth passage were cryopreserved under the following treatments: (i) control (non-cryopreserved); (ii) a solution with 10% dimethyl sulfoxide (Me2SO), 10% fetal bovine serum (FBS), and 0.2 M sucrose; (iii) a solution with 10% Me2SO, 10% FBS, and 5 mM L-proline; and (iv) a solution with 10% Me2SO, 10% FBS, 0.2 M sucrose, and 5 mM L-proline. Tests were conducted to analyze cell morphology, viability, metabolism, proliferation, and apoptosis; reactive oxygen species (ROS) levels; and mitochondrial membrane activity (ΔΨm). A reduction in the number of viable cells (72.3% ± 1.2%) was observed in the sucrose-containing group compared to the control (86.7% ± 2.0%) and L-proline (88.4% ± 1.8% and 87.8% ± 2.1%) groups. After apoptotic analysis, a reduction in the number of viable cells was observed in the group with sucrose alone (74.6% ± 4.1%) compared to the control group (88.2% ± 1.1%). The ROS levels (1.03 ± 0.5 and 1.07 ± 0.5, respectively) and ΔΨm values (0.99 ± 0.42 and 1.22 ± 0.73, respectively) observed in the groups with L-proline were similar to that observed in the control group (1.00 ± 0.5 and 1.00 ± 0.4, respectively). Moreover, no difference was observed between groups for cell morphology, metabolism, or proliferation. Thus, L-proline is a cryoprotectant agent that can be used during G. spixii fibroblast cryopreservation, alone or with sucrose. In addition, we developed an adequate biobank for G. spixii, whereby stored cells could be used for reproductive techniques.

The Effect of κ-Carrageenan on Porcine Sperm Cryo-Survival.

κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen-thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress.

DNMT1 driven by mouse amniotic fluid mesenchymal stem cell exosomes improved corneal cryoinjury via inducing microRNA-33 promoter DNA hypermethylation modification in corneal epithelium cells.

Severe corneal cryoinjury can cause permanent corneal swelling and bullous keratopathy, one of the main reason for loss of sight. Mouse amniotic fluid mesenchymal stem cells (mAF-MSCs) can repair corneal damage caused by freezing; however, whether the exosomes derived from mAF-MSCs have the same repair effect is unknown. In this study, the mAF-MSC-exosomes were transplanted into the eyeballs of corneal cryoinjured mice. Histopathological examination showed that the mAF-MSC-exosomes improved the corneal structure and status of corneal epithelial cells in corneal cryoinjured mice. RRBS-sequencing showed that compared with the control group, four genes (Rpl13-ps6, miR-33, Hymai, and Plagl1), underwent DNA hypermethylation modification after mAF-MSC-exosomes treatment. The result of FISH indicated that miR-33-3p hybridization signals were enhanced in corneal epithelial cells from mice treated with mAF-MSC-exosomes. Semi-quantitative PCR and western blotting indicated that mAF-MSC-exosomes contained high levels of DNMT1 mRNA and protein. Additionally, luciferase report assays indicated that miR-33-3p overexpression in NIH-3T3 mouse embryonic fibroblast cells inhibited the activity of luciferase carrying a sequence from the 3' untranslated region of Bcl6. Moreover, BCL6 mRNA and protein levels in corneal tissues from mice treated with mAF-MSC-exosomes were higher than those in the control group. Therefore, our results suggested that mAF-MSC-exosomes could repair corneal cryoinjury by releasing DNMT1, which induced hypermethylation of the miR-33 promoter in corneal epithelial cells. Consequent downregulated miR-33 transcription upregulated Bcl6 expression, ultimately achieving the repair of corneal cryoinjury in mice.

Preventive and therapeutic vascular photobiomodulation decreases the inflammatory markers and enhances the muscle repair process in an animal model.

Photobiomodulation therapy (PBM) has shown positive effects when applied locally to modulate the inflammatory process and facilitate muscle repair. However, the available literature on the mechanisms of action of vascular photobiomodulation (VPBM), a non-invasive method of vascular irradiation, specifically in the context of local muscle repair, is limited. Thus, this study aimed to assess the impact of vascular photobiomodulation (VPBM) using a low-level laser (LLL) on the inflammatory response and the process of skeletal muscle repair whether administered prior to or following cryoinjury-induced acute muscle damage in the tibialis anterior (TA) muscles. Wistar rats (n = 85) were organized into the following experimental groups: (1) Control (n = 5); (2) Non-Injury + VPBM (n = 20); (3) Injured (n = 20); (4) Pre-VPBM + Injury (n = 20); (5) Injury + Post-VPBM (n = 20). VPBM was administered over the vein/artery at the base of the animals' tails (wavelength: 780 nm; power: 40 mW; application area: 0.04 cm2; energy density: 80 J/cm2). Euthanasia of the animals was carried out at 1, 2, 5, and 7 days after inducing the injuries. Tibialis anterior (TA) muscles were collected for both qualitative and quantitative histological analysis using H&E staining and for assessing protein expression of TNF-α, MCP-1, IL-1ß, and IL-6 via ELISA. Blood samples were collected and analyzed using an automatic hematological analyzer and a leukocyte differential counter. Data were subjected to statistical analysis (ANOVA/Tukey). The results revealed that applying VPBM prior to injury led to an increase in circulating neutrophils (granulocytes) after 1 day and a subsequent increase in monocytes after 2 and 5 days, compared to the Non-Injury + VPBM and Injured groups. Notably, an increase in erythrocytes and hemoglobin concentration was observed in the Non-Injury + VPBM group on days 1 and 2 in comparison to the Injured group. In terms of histological aspects, only the Prior VPBM + Injured group exhibited a reduction in the number of inflammatory cells after 1, 5, and 7 days, along with an increase in blood vessels at 5 days. Both the Prior VPBM + Injured and Injured + VPBM after groups displayed a decrease in myonecrosis at 1, 2, and 7 days, an increase in newly-formed and immature fibers after 5 and 7 days, and neovascularization after 1, 2, and 7 days. Regarding protein expression, there was an increase in MCP-1 after 1 and 5 days, TNF-α, IL-6, and IL-1ß after 1, 2, and 5 days in the Injured + VPBM after group when compared to the other experimental groups. The Prior VPBM + Injured group exhibited increased MCP-1 production after 2 days, in comparison to the Non-Injury + VPBM and Control groups. Notably, on day 7, the Injured group continued to show elevated MCP-1 protein expression when compared to the VPBM groups. In conclusion, VPBM effectively modulated hematological parameters, circulating leukocytes, the protein expression of the chemokine MCP-1, and the proinflammatory cytokines TNF-α and IL-1ß, ultimately influencing the inflammatory process. This modulation resulted in a reduction of myonecrosis, restoration of tissue architecture, increased formation of newly and immature muscle fibers, and enhanced neovascularization, with more pronounced effects when VPBM was applied prior to the muscle injury.

Is cryoablation a valid option for renal cell carcinomas in direct contact with critical organs?

PURPOSE: This study aimed to assess the outcomes of percutaneous cryoablation (PCA) for renal cell carcinomas (RCCs) contacting critical organs without intervening fat tissue. MATERIAL AND METHODS: Twenty-three patients with 24 RCCs (mean size, 28.8 mm) contacting critical organs on preprocedural images were included. The organ displacement techniques, technical success, efficacy, and adverse events per Clavien-Dindo classification were retrospectively reviewed. RESULTS: The organs contacting the RCCs included the colon (n = 16), pancreas (n = 3), duodenum (n = 3), small intestine (n = 1), and stomach (n = 1). In all procedures, hydrodissection was conducted, and probe traction was additionally utilized in one to displace organs. Two procedures were terminated with an insufficient ice-ball margin (<6 mm) due to recurring proximity of the colon or thermal sink effect by renal hilar vessels, yielding a technical success rate of 91.6% (22/24). No severe adverse events were noted. All patients were alive without any metastases during a median follow-up of 34.4 months. The primary and secondary technical efficacy rates were 91.6% (22/24) and 95.8% (23/24) of tumors, respectively. CONCLUSION: PCA can be a valid option for RCCs contacting critical organs with a good safety profile and sufficient technical efficacy.

Cryoablation with KCl Solution Enhances Necrosis and Apoptosis of HepG2 Liver Cancer Cells.

Cryoablation has become a valuable treatment modality for the management of liver cancer. However, one of the major challenges in cryosurgery is the incomplete cryodestruction near the edge of the iceball. This issue can be addressed by optimizing cryoablation parameters and administering thermotropic drugs prior to the procedure. These drugs help enhance tumor response, thereby strengthening the destruction of the incomplete frozen zone in liver cance. In the present study, the feasibility and effectiveness of a thermophysical agent, KCl solution, were investigated to enhance the cryodestruction of HepG2 human liver cancer cells. All cryoablation parameters were simultaneously optimized in order to significantly improve the effect of cryoablation, resulting in an increase in the lethal temperature from - 25 °C to - 17 °C. Subsequently, it was found that the application of KCl solution prior to freezing significantly decreased cell viability post-thaw compared to cryoablation treatment alone. This effect was attributed to the eutectic effect of KCl solution. Importantly, it was found that the combination of KCl solution and freezing was less effective when applied to LO2 human liver normal cells. The data revealed that the ratio of mRNA levels of Bcl-2 and bax decreased significantly more in HepG2 cells than in LO2 cells when cryoablation was used with KCl solution. In conclusion, the results of this study demonstrate the effectiveness of KCl solution in promoting cryoablation and describe a novel therapeutic model for the treatment of liver cancer that may distinguish between cancer and normal cells.

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.

An updated review on the application of proteomics to explore sperm cryoinjury mechanisms in livestock animals.

This comprehensive review critically examines the application of proteomics in understanding sperm cryoinjury mechanisms in livestock animals, in the context of the widespread use of semen cryopreservation for genetic conservation. Despite its global adoption, cryopreservation often detrimentally affects sperm quality and fertility due to cryoinjuries. These injuries primarily arise from ice crystal formation, osmotic shifts, oxidative stress, and the reorganization of membrane proteins and lipids during freezing and thawing, leading to premature capacitation-like changes. Moreover, the cryopreservation process induces proteome remodeling in mammalian sperm. Although there have been technological advances in semen cryopreservation, the precise mechanisms of mammalian sperm cryoinjury remain elusive. This review offers an in-depth exploration of how recent advancements in proteomic technologies have enabled a detailed investigation into these molecular disruptions. It presents an analysis of protein-level alterations post-thaw and their impact on sperm viability and functionality. Additionally, it discusses the role of proteomics in refining cryopreservation techniques to mitigate cryoinjury and enhance reproductive outcomes in livestock. This work synthesizes current knowledge, highlights gaps, and suggests directions for future research in animal reproductive science and biotechnology.

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.

Time-Sequential Monitoring of the Early Mesothelial Reaction in the Pleura after Cryoinjury.

(1) Background: An early mesothelial reaction of the pleura, leading to fibrosis, has been reported in animals after chemical or heavy metal exposure. However, the visual monitoring of early time-sequential mesothelial reaction-associated cryoinjury has not been fully investigated. Therefore, this study aimed to evaluate and visualize the early mesothelial reactions seen following cryoinjury using rabbit pleura. (2) Methods: We monitored the early mesothelial reaction in rabbit pleurae after cryoinjury using optical coherence tomography (OCT), in real-time, which was then compared with pathological images. Due to the penetration limit of OCT, we made a thoracic window to image the parietal and visceral pleurae in vivo. We also used an innovative technique for capturing the microstructure in vivo, employing a computer-controlled intermittent iso-pressure breath hold to reduce respiratory motion, increasing the resolution of OCT. We organized three sample groups: the normal group, the sham group with just a thoracic window, and the experimental group with a thoracic window and cryotherapy. In the experimental group, localized cryoinjury was performed. The mesothelial cells at the level of pleura of the cryotherapy-injured site were visualized by OCT within the first 30 min and then again after 2 days at the same site. (3) Results: In the experimental group, focal thickening of the parietal pleura was observed at the site of cryoinjury using OCT after the first injury, and it was then confirmed pathologically as focal mesothelial cell proliferation. Two days after cryoinjury, diffuse mesothelial cell proliferation in the parietal pleura was noted on the reverse side around the cryoinjured site in the same rabbit. In the sham group, no pleural reaction was found. The OCT and pathological examinations revealed different patterns of mesothelial cell reactions between the parietal and visceral pleurae: the focal proliferation of mesothelial cells was found in the parietal pleura, while only a morphological change from flat cells to cuboidal cells and a thickened monolayer without proliferation of mesothelial cells were found in the visceral pleural. (4) Conclusions: An early mesothelial reaction occurs following cryoinjury to the parietal and visceral pleurae.

Granulocyte-Macrophage Colony-Stimulating Factor Cytokine Addition After the Freeze-Thawing Process Improves Human Sperm Motility and Vitality in Asthenoteratozoospermia Patients.

The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 µL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.

New strategies for germ cell cryopreservation: Cryoinjury modulation.

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

Engineered heart tissue maturation inhibits cardiomyocyte proliferative response to cryoinjury.

The cellular and molecular mechanisms that are responsible for the poor regenerative capacity of the adult heart after myocardial infarction (MI) are still unclear and their understanding is crucial to develop novel regenerative therapies. Considering the lack of reliable in vitro tissue-like models to evaluate the molecular mechanisms of cardiac regeneration, we used cryoinjury on rat Engineered Heart Tissues (rEHTs) as a new model which recapitulates in part the in vivo response after myocardial injury of neonatal and adult heart. When we subjected to cryoinjury immature and mature rEHTs, we observed a significant increase in cardiomyocyte (CM) DNA synthesis when compared to the controls. As expected, the number of mitotic CMs significantly increases in immature rEHTs when compared to mature rEHTs, suggesting that the extent of CM maturation plays a crucial role in their proliferative response after cryoinjury. Moreover, we show that cryoinjury induces a temporary activation of fibroblast response in mature EHTs, similar to the early response after MI, that is however incomplete in immature EHTs. Our results support the hypothesis that the endogenous maturation program in cardiac myocytes plays a major role in determining the proliferative response to injury. Therefore, we propose rEHTs as a robust, novel tool to in vitro investigate critical aspects of cardiac regeneration in a tissue-like asset free from confounding factors in response to injury, such as the immune system response or circulating inflammatory cytokines.

Bax/Bcl-2 Expression Ratio Analysis of Rat Ovary Vitrified with Date Juice Concentrate as a Natural Extracellular Cryoprotectant.

Background: The use of extremely low temperatures in vitrification is known to cause cryoinjury so that it can trigger the activation of the intrinsic apoptotic pathway, which can damage the structural integrity of the pre-antral follicle. Based on that, it is necessary to use an appropriate cryoprotectant to protect the preserved cell. Aims: This study aimed to identify the potential use of date juice concentrate (DJC) as a natural extracellular cryoprotectant to suppress the rate of apoptosis after vitrification. Settings and Design: This experimental research uses 24 samples of ovarian rats. Rats were fed and drank an ad libitum. Materials and Methods: Ovaries were isolated in the proestrus phase, then processed into slides for immunohistochemistry (IHC) staining using anti-Bax and anti-Bcl-2 antibodies. IHC results were evaluated for the brown colour using ImageJ IHC Profiler. The results were analysed as an optical density and displayed in the Bax/Bcl-2 ratio. Statistical Analysis Used: All data were statistically analysed with either parametric (analysis of various) or non-parametric (Kruskal-Wallis) tests. Results: The combination of EG 7.5% + DJC 15% (KP2) showed the lowest Bax/Bcl-2 ratio in primordial and primary follicles. Meanwhile, the lowest Bax/Bcl-2 ratio in secondary follicles is found in KP4 (EG 15% + DJC 15%). The DJC is known to contain a dominant amount of glucose. The DJC shows antioxidant activity and contains antioxidant compounds, phenols and flavonoids. Conclusion: The sugar content and antioxidant compounds of DJC can protect against follicle membrane damage, so the rate of intrinsic apoptosis pathway is also suppressed initially with Bax protein suppression in the mitochondrial membrane.

Comparative iTRAQ proteomics identified proteins in fresh and frozen thawed yak spermatozoa.

The changes in semen and cryodamage after the cryopreservation process negatively affect sperm function and motility. However, possible proteomic alterations of yak semen during cryopreservation have not yet been achieved. In this study, we compared proteomes of fresh and frozen thawed yak sperm using iTRAQ combined with LC-MS/MS proteome approach. Totally, 2064 proteins were quantitatively identified, including 161 in fresh sperm that showed significant differences compared to frozen thawed sperm. According to the Gene ontology (GO) enrichment analysis, differentially expressed proteins (DEPs) are predominantly associated with spermatogenesis, tricarboxylic acid cycle, ATP synthesis, and differentiation biological process. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPs were mainly involved in metabolic pathways related to pyruvate metabolism, carbon metabolism, glycolysis/gluconeogenesis, together with the citrate (TCA) cycle. In the analysis of the protein-protein interaction (PPI) network, 15 potential proteins (PDHB, DLAT, PDHA2, PGK1, TP5C1, etc.) that could be related to the sperm quality of the yaks were obtained. Furthermore, 6 DEPs were validated by parallel reaction monitoring (PRM), confirming that the iTRAQ data were reliable. These results indicate that cryopreservation alters the proteome of yak sperm, which is possibly related to cryodamage and fertilization ability.

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.

Cryopreservation: A Comprehensive Overview, Challenges, and Future Perspectives.

Cryopreservation is the most prevalent method of long-term cell preservation. Effective cell cryopreservation depends on freezing, adequate storage, and correct thawing techniques. Recent advances in cryopreservation techniques minimize the cellular damage which occurs while processing samples. This article focuses on the fundamentals of cryopreservation techniques and how they can be implemented in a variety of clinical settings. The article presents a brief description of each of the standard cryopreservation procedures, such as slow freezing and vitrification. Alongside that, the membrane permeating and nonpermeating cryoprotectants are briefly discussed, along with current advancements in the field of cryopreservation and variables influencing the cryopreservation process. The diminution of cryoinjury incurred by the cell via the resuscitation process will also be highlighted. In the end application of cryopreservation techniques in many fields, with a special emphasis on stem cell preservation techniques and current advancements presented. Furthermore, the challenges while implementing cryopreservation and the futuristic scope of the fields are illustrated herein. The content of this review sheds light on various ways to enhance the output of the cell preservation process and minimize cryoinjury while improving cell revival.

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.