Melatonin in preservation solutions prevents ischemic injury in rat kidneys.

Transplantation is lifesaving and the most effective treatment for end-stage organ failure. The transplantation success depends on the functional preservation of organs prior to transplantation. Currently, the University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) are the most commonly used preservation solutions. Despite intensive efforts, the functional preservation of solid organs prior to transplantation is limited to hours. In this study, we modified the UW solution containing components from both the UW and HTK solutions and analyzed their tissue-protective effect against ischemic injury. The composition of the UW solution was changed by reducing hydroxyethyl starch concentration and adding Histidine/Histidine-HCl which is the main component of HTK solution. Additionally, the preservation solutions were supplemented with melatonin and glucosamine. The protective effects of the preservation solutions were assessed by biochemical and microscopical analysis at 2, 10, 24, and 72 h after preserving the rat kidneys with static cold storage. Lactate dehydrogenase (LDH) activity in preservation solutions was measured at 2, 10, 24, and 72. It was not detectable at 2 h of preservation in all groups and 10 h of preservation in modified UW+melatonin (mUW-m) and modified UW+glucosamine (mUW-g) groups. At the 72nd hour, the lowest LDH activity (0.91 IU/g (0.63-1.17)) was measured in the mUW-m group. In comparison to the UW group, histopathological damage score was low in modified UW (mUW), mUW-m, and mUW-g groups at 10, 24, and 72 hours. The mUW-m solution at low temperature was an effective and suitable solution to protect renal tissue for up to 72 h.

Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts.

BACKGROUND: Warm reperfusion after previous cold storage has been shown to have a negative impact on mitochondrial function of organ grafts. Here, we wanted to investigate whether a more controlled warming up of the cold graft by ex vivo machine perfusion with gradually elevated temperature from cold to normothermia (including comparison of two warming up protocols) prior to implantation would be effective in preventing mitochondrial dysfunction upon reperfusion. METHODS: All experiments were conducted on porcine kidneys retrieved 15 min after cardiac arrest. After 18 h of cold storage in HTK solution (CS, n = 6), kidneys (n = 6) were subjected to 2 h of reconditioning machine perfusion starting with a hypothermic period followed by a gradual increase in perfusion temperature up to 35 °C (controlled oxygenated rewarming-COR). For a second group (n = 6), the slow warming up was begun instantly after connecting the graft onto the machine (iCOR). Functional recovery of all grafts was then observed upon normothermic reperfusion in vitro. At the conclusion of the experiments, tissue specimens were taken for immediate isolation and analysis of renal mitochondria. RESULTS: COR resulted in a significantly and more than 3-fold increased glomerular filtration rate upon reperfusion, along with a significant higher tubular sodium reabsorption and lesser loss of glucose in comparison to the controls. Enzyme release (AST) was also massively reduced during the reperfusion period. Specific analysis at the mitochondrial level revealed significantly better coupling efficiency and spare respiratory capacity in the COR group compared to the cold storage group. Interestingly, additional experiments revealed that the omission of a hypothermic perfusion period did not deteriorate any of the results after COR, provided that the instant temperature increase from 10 to 35 °C was effectuated in the same controlled manner. CONCLUSION: Controlled rewarming after extended cold preservation effectively improves mitochondrial recovery upon reperfusion and early functional outcome of kidney grafts.

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution.

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Human cornea conservation in coconut water solution / Conservação de córneas humanas em solução de água de coco

ABSTRACT Purpose: The aim of this study was to evaluate the physical and chemical characteristics of coconut water and to analyze the use of coconut water solution for the conservation of human corneas. Methods: This was an experimental and controlled study performed at the Eye Bank of the General Hospital of Fortaleza. The coconut water-based solution was prepared at the Goat Seed Technology Laboratory of the Department of Veterinary Medicine of the State University of Ceará. Discarded corneas from the Eye Bank were divided into two groups for sequential experiments: G1, coconut water-based solution (experimental group), and G2, conservative treatment with OPTISOL GS® (control group). The osmolality of corneas in G1 was analyzed sequentially at 275, 300, 325, 345, 365, and 400 mOsm/L. The viability of the corneas was determined by specular microscopy and biomicroscopy on the first, third, and seventh days. Results: Corneas preserved in a solution of 365 and 345 mOsm/L had a transparency of 8 mm until the third day and had diffuse edema in the periphery, central folds, and partial epithelium loss until the seventh day. The 365-mOsm/L solution was associated with the worst results during follow-up. Corneas placed in Optisol-GS retained their original aspects. Conclusions: Coconut water-based preservative partially maintained corneal transparency and epithelial integrity, especially during the first three days of follow-up. The coconut water-based solutions used were not effective for use as preservatives in a human eye bank.(AU)

RESUMO Objetivos: As características físico-químicas e o baixo custo da água de coco foram fundamentais para o este estudo. Analisar o uso de solução a base de água de coco como meio de conservação de córneas humanas em banco de olhos. Métodos: Estudo experimental e controlado realizado no Banco de Olhos do Hospital Geral de Fortaleza. Utilizou-se solução à base de água de coco preparada no laboratório de Tecnologia de Sêmen de Caprinos do Departamento de Medicina Veterinária da Universidade Estadual do Ceará. Foram usadas córneas de descartes divididas em dois grupos: G1 (Conservante com água de coco) - grupo experimental e G2 (grupo Conservante com OPTISOL GS®) grupo controle, em experimentos sequenciais. A osmolaridade do G1 foi analisada sequencialmente com 275, 300, 325, 345, 365 e 400 mOsm/L. A viabilidade das córneas foram realizadas por microscopia especular e biomicroscopia nos 1º, 3º e 7º dias. Resultados: As córneas em solução de 365 e 345 mOsm/L apresentavam transparência nos 8mm centrais até o 3º dia, com edema em toda periferia, dobras centrais e edema 2+, com perda parcial do epitélio até 7º dia, sendo o de maior osmolaridade com melhor transparência durante o seguimento. Grupo com 275, 300 e 400 mOsm/L, córnea opaca, edema difuso, perda total do epitélio no 3º dia. As córneas em Optisol mantiveram seus aspectos. Conclusões: O conservante à base de água de coco manteve em parte a transparência corneana e a integridade epitelial, especialmente nos primeiros 3 dias de seguimento. A solução conservante com água de coco nas formulações utilizadas não se mostrou eficaz para o uso em banco de olhos humanos.(AU)

Development and validation of HPLC method for simultaneous quantification of alpha-tocopherol (free or encapsulated) and cholesterol in semen cryopreservation media.

The HPLC method was developed and validated for assaying alpha-tocopherol and cholesterol in cryopreservation media. Chromatographic separation was performed on an isocratic system, using a C-18 column. The mobile phase was composed of a mixture of methanol:acetonitrile:water 68:28:4 (v/v/v), using a flow rate of 1.5 mL/min and 20 µL injection volume, at a wavelength of 208 nm. The method was validated according to International Conference on Harmonization guidelines. The method proved to be specific, accurate, precise, and linear with correlation coefficients greater than 0.996 over a wide concentration range of both analytes. Vitamin E and cholesterol presented limits of detection of 0.002 mg/mL, 0.026 mg/mL and limits of quantitation of 0.006 mg/mL, 0.086 mg/mL, respectively. This method is simple and rapid, shows high precision and accuracy, and offers the advantage of simultaneous assaying of vitamin E and cholesterol (alone, in cyclodextrins complexes or in liposome loaded) on semen cryopreservation media.

In Vitro/Ex Vivo Models for the Study of Ischemia Reperfusion Injury during Kidney Perfusion.

Oxidative stress is a key element of ischemia-reperfusion injury, occurring during kidney preservation and transplantation. Current options for kidney graft preservation prior to transplantation are static cold storage (CS) and hypothermic machine perfusion (HMP), the latter demonstrating clear improvement of preservation quality, particularly for marginal donors, such as extended criteria donors (ECDs) and donation after circulatory death (DCDs). Nevertheless, complications still exist, fostering the need to improve kidney preservation. This review highlights the most promising avenues of in kidney perfusion improvement on two critical aspects: ex vivo and in vitro evaluation.

HTK-N: Modified Histidine-Tryptophan-Ketoglutarate Solution-A Promising New Tool in Solid Organ Preservation.

To ameliorate ischemia-induced graft injury, optimal organ preservation remains a critical hallmark event in solid organ transplantation. Although numerous preservation solutions are in use, they still have functional limitations. Here, we present a concise review of a modified Histidine-Tryptophan-Ketoglutarate (HTK) solution, named HTK-N. Its composition differs from standard HTK solution, carrying larger antioxidative capacity and providing inherent toxicity as well as improved tolerance to cold aiming to attenuate cold storage injury in organ transplantation. The amino acids glycine, alanine and arginine were supplemented, N-acetyl-histidine partially replaced histidine, and aspartate and lactobionate substituted chloride. Several in vitro studies confirmed the superiority of HTK-N in comparison to HTK, being tested in vivo in animal models for liver, kidney, pancreas, small bowel, heart and lung transplantation to adjust ingredients for required conditions, as well as to determine its innocuousness, applicability and potential advantages. HTK-N solution has proven to be advantageous especially in the preservation of liver and heart grafts in vivo and in vitro. Thus, ongoing clinical trials and further studies in large animal models and consequently in humans are inevitable to show its ability minimizing ischemia-induced graft injury in the sequel of organ transplantation.

The Role of Hormones and Trophic Factors as Components of Preservation Solutions in Protection of Renal Function before Transplantation: A Review of the Literature.

Transplantation is currently a routine method for treating end-stage organ failure. In recent years, there has been some progress in the development of an optimal composition of organ preservation solutions, improving the vital functions of the organ and allowing to extend its storage period until implantation into the recipient. Optimizations are mostly based on commercial solutions, routinely used to store grafts intended for transplantation. The paper reviews hormones with a potential nephroprotective effect, which were used to modify the composition of renal perfusion and preservation solutions. Their effectiveness as ingredients of preservation solutions was analysed based on a literature review. Hormones and trophic factors are innovative preservation solution supplements. They have a pleiotropic effect and affect normal renal function. The expression of receptors for melatonin, prolactin, thyrotropin, corticotropin, prostaglandin E1 and trophic factors was confirmed in the kidneys, which suggests that they are a promising therapeutic target for renal IR (ischemia-reperfusion) injury. They can have anti-inflammatory, antioxidant and anti-apoptotic effects, limiting IR injury.

The Effectiveness of Rinsing Organs With Selected Preservation Solutions Based on the Analysis of Their Physicochemical Parameters.

BACKGROUND: One of the key elements of successful transplantation is effectively rinsing off the blood and preserving organs under controlled hypothermia. The aim of the study was to analyze the physicochemical parameters of Biolasol (Biochefa, Sosnowiec, Poland) and histidine-tryptophan-ketoglutarate (HTK) (Custodiol) solutions, which are recommended for perfusion and preservation of abdominal parenchymal organs. METHODS: Biolasol and HTK solution were used for the study. The solutions were subjected to physicochemical analysis involving pH, density, osmolarity, viscosity, refractive index, zeta potential, hydrodynamic diameter, and rheological properties. Rheological parameters were associated with morphologic features of fluids. RESULTS: HTK and Biolasol are non-Newtonian systems with pseudoplastic properties and yield stress. The solutions begin to flow under shear stress greater than the ultimate stress. In addition, a nonlinear relationship of their viscosity as a function of velocity gradient (shear rate) was observed. CONCLUSIONS: The solutions reproduce blood properties, which leads to the conclusion about their effective filling of the vascular bed and high efficiency in organ rinsing.

A novel histidine-tryptophan-ketoglutarate formulation ameliorates intestinal injury in a cold storage and ex vivo warm oxygenated reperfusion model in rats.

AIM: The present study aims to evaluate protective effects of a novel histidine-tryptophan-ketoglutarate solution (HTK-N) and to investigate positive impacts of an additional luminal preservation route in cold storage-induced injury on rat small bowels. METHODS: Male Lewis rats were utilized as donors of small bowel grafts. Vascular or vascular plus luminal preservation were conducted with HTK or HTK-N and grafts were stored at 4°C for 8 h followed by ex vivo warm oxygenated reperfusion with Krebs-Henseleit buffer for 30 min. Afterwards, intestinal tissue and portal vein effluent samples were collected for evaluation of morphological alterations, mucosal permeability and graft vitality. RESULTS: The novel HTK-N decreased ultrastructural alterations but otherwise presented limited effect on protecting small bowel from ischemia-reperfusion injury in vascular route. However, the additional luminal preservation led to positive impacts on the integrity of intestinal mucosa and vitality of goblet cells. In addition, vascular plus luminal preservation route with HTK significantly protected the intestinal tissue from edema. CONCLUSION: HTK-N protected the intestinal mucosal structure and graft vitality as a luminal preservation solution. Additional luminal preservation route in cold storage was shown to be promising.

Original and generic preservation solutions in organ transplantation. A new paradigm?

Solid organ transplantation is a very complex process, in which the storage of the graft in a preservation solution is mandatory in order to extend ischemic times and contain further damage. The condition in which the organ is transplanted is critical for the outcome of the organ recipient. The recent emergence of generic versions of organ preservation solutions (solutions with the same composition and under the same legislation as the original versions, but with different brands) compelled us to study whether the standards are maintained when comparing the original and its generic counterpart. Along these lines, we discuss and comment on some aspects concerning this issue of general interest in the organ transplantation field.

Cost-Effectiveness of Antifungal Supplementation of Corneal Cold Storage Media.

PURPOSE: To evaluate the cost-effectiveness of supplementing hypothermic cold storage media (CSM) with antifungal therapy. DESIGN: Cost-effectiveness analysis (CEA). PARTICIPANT: Base case of a patient with Fuch's endothelial dystrophy undergoing a first eye keratoplasty. METHODS: Cost-effective analysis of the base case with corneal tissue stored in CSM or CSM supplemented with antifungal therapy over a 16-year time horizon. Multiple clinical scenarios were considered, including endothelial keratoplasty (EK) and penetrating keratoplasty (PK); amphotericin B, voriconazole, caspofungin, and combination therapy; and third-party payer and societal perspectives. The incidences were derived from PubMed literature searches and average wholesale prices of medications; all costs were discounted 3% per annum and adjusted for inflation to 2019 US dollars. MAIN OUTCOME MEASURES: Incremental cost-effectiveness ratios (ICERs). RESULTS: In the reference case, a corneal endothelial graft stored in amphotericin B-supplemented CSM was the most cost-effective approach from a third-party payer and societal perspective. Probability sensitivity analysis (PSA) of the societal model for the EK was robust, with 93.5% being below an arbitrary willingness-to-pay threshold (WTP) of $20 000 per fungal infection averted. Voriconazole, caspofungin, and combination antifungals were less cost-effective than amphotericin B. The main factors influencing the CEA were the incidences of postkeratoplasty fungal infections, potential increases in graft failures, and antifungal costs. For grafts intended for PKs, antifungal supplementation was less cost-effective than for EKs. CONCLUSIONS: Antifungal supplementation with amphotericin B for EK grafts was the most cost-effective approach of the studied antifungals; however, the CEA was sensitive to potential changes in graft failure rates, underlining the importance of long-term safety studies. For full-thickness corneal grafts, antifungal supplementation was less cost-effective.

Renal Protection Against Ischemia Reperfusion Injury: Hemoglobin-based Oxygen Carrier-201 Versus Blood as an Oxygen Carrier in Ex Vivo Subnormothermic Machine Perfusion.

BACKGROUND: The optimal method of oxygen delivery to donor kidneys during ex vivo machine perfusion has not been established. We have recently reported the beneficial effects of subnormothermic (22°C) blood perfusion in the preservation of porcine donation after circulatory death kidneys. Since using blood as a clinical perfusate has limitations, including matching availability and potential presence of pathogen, we sought to assess hemoglobin-based oxygen carrier (HBOC-201) in oxygen delivery to the kidney for renal protection. METHODS: Pig kidneys (n = 5) were procured after 30 minutes of warm in situ ischemia by cross-clamping the renal arteries. Organs were flushed with histidine tryptophan ketoglutarate solution and subjected to static cold storage or pulsatile perfusion with an RM3 pump at 22°C for 4 hours with HBOC-201 and blood. Thereafter, kidneys were reperfused with normothermic (37°C) oxygenated blood for 4 hours. Blood and urine were subjected to biochemical analysis. Total urine output, urinary protein, albumin/creatinine ratio, flow rate, resistance were measured. Acute tubular necrosis, apoptosis, urinary kidney damage markers, neutrophil gelatinase-associated lipocalin 1, and interleukin 6 were also assessed. RESULTS: HBOC-201 achieved tissues oxygen saturation equivalent to blood. Furthermore, upon reperfusion, HBOC-201 treated kidneys had similar renal blood flow and function compared with blood-treated kidneys. Histologically, HBOC-201 and blood-perfused kidneys had vastly reduced acute tubular necrosis scores and degrees of terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining versus kidneys treated with cold storage. Urinary damage markers and IL6 levels were similarly reduced by both blood and HBOC-201. CONCLUSIONS: HBOC-201 is an excellent alternative to blood as an oxygen-carrying molecule in an ex vivo subnormothermic machine perfusion platform in kidneys.

Original and generic preservation solutions in organ transplantation. A new paradigm?

Abstract Solid organ transplantation is a very complex process, in which the storage of the graft in a preservation solution is mandatory in order to extend ischemic times and contain further damage. The condition in which the organ is transplanted is critical for the outcome of the organ recipient. The recent emergence of generic versions of organ preservation solutions (solutions with the same composition and under the same legislation as the original versions, but with different brands) compelled us to study whether the standards are maintained when comparing the original and its generic counterpart. Along these lines, we discuss and comment on some aspects concerning this issue of general interest in the organ transplantation field.

Pharmacological Benefits and Risk of Using Hormones in Organ Perfusion and Preservation Solutions in the Aspect of Minimizing Hepatic Ischemia-Reperfusion Injury during Storage.

For several years, research has been carried out on the effectiveness of solutions for perfusion and preservation of organs, including the liver. There is a search for an optimal pharmacological composition of these solutions, allowing to preserve or improve vital functions of the organ for as long as possible until it is transplanted into a recipient. Hormones due to their properties, often resulting from their pleiotropic effects, may be a valuable component for optimizing the composition of liver perfusion and preservation solutions. The paper presents the current state of knowledge on liver perfusion and preservation solutions modified with hormones. It also shows the characteristics of the hormones evaluated, taking into account their physiological functions in the body.

Impact of human-derived hemoglobin based oxygen vesicles as a machine perfusion solution for liver donation after cardiac death in a pig model.

The recent clinical application of perfusion technology for the machine preservation of donation after cardiac death (DCD) grafts has some advantages. Oxygenation has been proposed for the preservation of DCD liver grafts. The aim of this study is to clarify whether the use of HbV-containing preservation solution during the subnormothermic machine perfusion (SNMP) of the liver graft improves the graft function of DCD porcine livers in an ex vivo reperfusion model. Pig livers were excised after 60 minutes of warm ischemic time and were preserved under one of three preservation conditions for 4 hours. The preservation conditions were as follows: 4°C cold storage (CS group; N = 5), Hypothermic machine preservation (HMP) with UW gluconate solution (HMP group; N = 5), SNMP (21°C) with UW gluconate solution (SNMP group; N = 5), SNMP (21°C) with HbVs (Hb; 1.8 mg/dl) perfusate (SNMP+HbV group; N = 5). Autologous blood perfusion was performed for 2 hours in an isolated liver reperfusion model (IRM). The oxygen consumption of the SNMP and SNMP+HbV group was higher than the HMP groups (p < 0.05). During the reperfusion, the AST level in the SNMP+HbV group was lower than that in the CS, HMP and SNMP groups. The changes in pH after reperfusion was significantly lower in SNMP+HbV group than CS and HMP groups. The ultrastructural findings indicated that the mitochondria of the SNMP+HbV group was well maintained in comparison to the CS, HMP and SNMP groups. The SNMP+HbVs preservation solution protected against metabolic acidosis and preserved the liver function after reperfusion injury in the DCD liver.

Y-27632 preserves epidermal integrity in a human skin organ-culture (hSOC) system by regulating AKT and ERK signaling pathways.

BACKGROUND: The human skin organ culture (hSOC) developed a century ago has been widely used to study various aspects of human skin development, differentiation, function, disease as well as skin appendages biology, however, maintaining the integrity of epidermal structure in long-term culture, has remained a challenge. OBJECTIVES: Here we tried to establish a culture system using supplemented William's E medium in the presence of a ROCK inhibitor Y-27632 to maintain epidermal architecture in the long-term hSOC and to investigate the underlying mechanisms. METHODS: Human breast skins, cut into 5 mm × 5 mm pieces, were cultured in supplemented William's E medium in the presence of 30µM Y-27632. The cultured skin tissues were collected at different time points for analysis of epidermal cell proliferation and differentiation by real time qRT-PCR and immunofluorescence (IF) staining. The keratinocyte suspension assay and in vivo treatment of Y-27632 on mouse were also carried out to study that the regulation of Y-27632 on keratinocyte proliferation and differentiation. RESULTS: We found Y-27632 not only enhanced both basal cell proliferation and expression of suprabasal cell differentiation markers, but also maintained the balance of keratinocyte proliferation and differentiation through activation of AKT pathways on one hand and inhibition of ERK pathways on the other hand. The AKT inhibitor MK-2206 blocked the epidermal preservation effect of Y-27632, while the MEK/ERK inhibitor U0126 enhanced the preservation of epidermal structure in the hSOC. CONCLUSIONS: Y-227632 can maintain skin epidermal integrity through regulation of AKT and ERK activity in the hSOC.

Improving the viability of tissue-resident stem cells using an organ-preservation solution.

Human clinical specimens are a valuable source of tissue-resident stem cells, but such cells need to be collected immediately after tissue collection. To extend the timescale for collection from fresh human samples, we developed a new extracellular fluid (ECF)-type preservation solution based on a high-sodium and low-potassium solution containing low-molecular-weight dextran and glucose, which is used for preservation of organs for transplantation. In this study, we compared the preservation of tissue-resident stem cells using our ECF solution with that using three other solutions: PBS, Dulbecco's modified Eagle's medium and Euro-Collins solution. These solutions represent a common buffer, a common culture medium and a benchmark organ-preservation solution, respectively. Lung tissues were removed from mice and preserved for 72 h under low-temperature conditions. Of the solutions tested, only preservation in the ECF-type solution could maintain the proliferation and differentiation capacity of mouse lung tissue-resident stem cells. In addition, the ECF solution could preserve the viability and proliferation of human alveolar epithelial progenitor cells when stored for more than 7 days at 4 °C. The mean viability of human alveolar type II cells at 2, 5, 8 and 14 days of low-temperature preservation was 90.9%, 84.8%, 85.7% and 66.3%, respectively, with no significant differences up to 8 days. Overall, our findings show that use of our ECF-type preservation solution may maintain the viability and function of tissue-resident stem cells. Use of this preservation solution may facilitate the investigation of currently unobtainable human tissue specimens for human stem cell biology.

Mimicking the cauda epididymal plasma-like osmolality in extender improves liquid preservation of ram semen at 3-5°C.

Cauda epididymis in mammals is known to store mature sperm largely in quiescent state for several weeks without significantly affecting fertility. Hence, the aim of this study was to evaluate the effects of mimicking cauda epididymal plasma (CEP)-like conditions in extender on liquid preservation of ram semen at 3-5°C. Four experiments were conducted in this study: (1) evaluation of physicochemical properties of ram CEP, (2) effect of hyperosmotic solution on sperm motility and functional membrane integrity (FMI), and the effects of (3) CEP-like hyperosmolality (390 vs. 360 mOsmol/kg) and (4) pH in extender (pH 6.5 vs. 6.8) on liquid preservation of ram semen. Sperm treatment with hyperosmotic solution (450 mOsmol/kg) resulted in a decline (P < 0.05) in mass motility (3.5 ± 0.2 vs. 4.3 ± 0.2) and FMI (30.4 ± 3.2 vs. 52.1 ± 2.1%) compared to that with isoosmotic solution (360 mOsmol/kg). Overall, sperm viability, acrosomal integrity, and progressive motility were similar (P > 0.05) while straight-line velocity (77.8 ± 3.1 vs. 71.3 ± 2.7µm/s), linearity (47.4 ± 0.4 vs. 39.5 ± 0.9%), straightness (79.7 ± 0.5 vs. 74.0 ± 0.5%) and beat cross frequency (28.6 ± 0.8 vs. 26.0 ± 0.5 Hz) were higher (P < 0.05) and FMI (65.7 ± 1.5 vs. 75.4 ± 1.1%) was lower (P < 0.05) following liquid-preservation in hyperosmotic extender compared to that in isoosmotic extender. Both total motility (83.3 ± 1.8 vs. 75.4 ± 1.5%) and progressive motility (51.7 ± 2.3 vs. 39.5 ± 1.9%) were higher (P < 0.05) at 48 h of storage in hyperosmotic extender compared to the control. Overall, the seminal attributes were similar (P > 0.05) between the two pH's of the extender. In conclusion, semen extender having CEP-like osmolality but not the pH was superior to extenders having conventional osmolality and pH for liquid preservation of ram semen.Abbreviations: AI: artificial insemination; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; CASA: computer-assisted semen analyzer; CEP: cauda epididymal plasma; ELON: elongation; EYTF: egg yolk-Tris-citrate-fructose; FMI: functional membrane integrity; GLM: general linear model; GPC: glycerophosphatidylcholine; HOS: hypoosmotic swelling; LIN: linearity; pHe: external pH; PROG: progressive motility; S.E.M.: standard error of the mean; SLTF: soya lecithin-Tris-fructose extender; SP: seminal plasma; STR: straightness; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight-line velocity; TM: total motility.

Compared efficacy of University of Wisconsin and histidine-tryptophan-ketoglutarate solutions in ex-situ liver resection and autotransplantation for end-stage hepatic alveolar echinococcosis patients.

BACKGROUND: The University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions are the two most frequently used liver graft preservation fluids. The present study aimed to compare their efficacy in end-stage hepatic alveolar echinococcosis patients who underwent ex-situ liver resection and autotransplantation (ELRA). METHODS: A total of 81 patients received ELRA from August 2010 to March 2018. They were allocated into UW (n = 48) and HTK groups (n = 33) based on the type of solutions used. Demographic and operational data were retrospectively analyzed. Primary outcomes included 90-day mortality, incidence of early graft loss, primary dysfunction, and postoperative complications. RESULTS: Demographic and operational characteristics were similarly distributed in the two groups. No statistically significant differences were observed with regard to 90-day mortality (12.77% vs. 12.12%) and early graft loss rate (8.51% vs. 9.09%) between the two groups. Patients in the UW and HTK groups showed a primary dysfunction rate of 27.66% and 27.27%, respectively. The UW group exhibited a higher incidence tendency of biliary complications, albeit with no statistical significance. CONCLUSIONS: This is the largest cohort study comparing the efficacy of the UW and HTK organ-preserving solutions in end-stage hepatic alveolar echinococcosis patients in ELRA settings. UW and HTK solutions presented similar efficacy and safety. A randomized clinical trial with larger scale is needed for further investigation in future clinical applications.