The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase--a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.

A potential hurdle when applying mass cytometry to the field study setting is the streamlining of sample collection while at the same time protecting the integrity of important cell epitopes. Whole blood preservation kits applying fixation and/or permeabilization agents are increasingly used in clinical trials to preserve leukocytes needed for downstream analysis. We here present a structured overview of leukocyte surface marker detectability in samples processed with four commercially available whole blood preservation kits; 1) Proteomic Stabilizer, 2) Stable-Lyse V2 and Stable-Store V2, 3) Cytodelics and 4) Lyse/Fix Buffer, as well as in samples treated with buffers included in Mass-tag Cellular Barcoding kits. Isolated leukocytes were stained with a 28-marker panel (including 7 chemokine receptors) of metal-conjugated antibodies and analysed on a mass cytometer. Exploration of the data by manual gating and viSNE analysis showed that although many markers were similarly detected across all sample conditions, most of the chemokine receptors in our panel, particularly CXCR3, CCR4, CCR6 and CXCR5, were incorrectly detected in the preserved samples and thus incompatible with the fixation and permeabilization agents found in whole blood preservation kits and in buffers used prior to barcoding.

The immediate processing of whole blood specimen is required in circulating tumor cell-based liquid biopsy. Reliable blood specimen stabilization towards preserving circulating tumor cells can enable more extensive geographic sharing for precise rare-cell technology, but remains challenging due to the fragility and rarity of circulating tumor cells. Herein, we establish a zwitterionic magnetic microgel platform to stabilize whole blood specimen for long-term hypothermic preservation of model circulating tumor cells. We show in a cohort study of 20 cancer patients that blood samples can be preserved for up to 7 days without compromising circulating tumor cell viability and RNA integrity, thereby doubling the viable preservation duration. We demonstrate that the 7-day microgel-preserved blood specimen is able to reliably detect cancer-specific transcripts, similar to fresh blood specimens, while there are up/down expression regulation of 1243 genes in model circulating tumor cells that are preserved by commercial protectant. Mechanistically, we find that the zwitterionic microgel assembly counters the cold-induced excessive reactive oxygen species and platelet activation, as well as extracellular matrix loss-induced cell anoikis, to prevent circulating tumor cell loss in the whole blood sample. The present work could prove useful for the development of blood-based noninvasive diagnostics.

Red blood cell (RBC) storage solution is used for suspending and preserving RBCs for later use in in vitro immunohematology testing. Proper RBC preservation is crucial for obtaining accurate results in RBC phenotyping and pretransfusion antibody screening tests. Haemolysis or RBC antigen degradation during storage can result in inaccurate RBC phenotyping, thereby decreasing the sensitivity of pretransfusion antibody screening and identification assays. The conventional RBC storage solutions usually contain adenosine, adenine, and antibiotics. We designed an RBC storage solution and determined whether it could preserve RBC integrity for 70 days.

Besides long-term trans-differentiation into neural cells, benefits of stem cell therapy (SCT) in ischemic stroke may include secretion of protective factors, which partly reflects extracellular vesicle (EVs) released by stem cell. However, the mechanism(s) by which stem cells/EVs limit stroke injury have yet to be fully defined.

Organ preservation solutions are designed to minimize organ damage during transplantation. A novel preservation solution, WMO-II, was developed to have a low viscosity and to improve microvasculature perfusion for kidneys. In an autologous canine transplantation model, kidney function and recovery were evaluated after organs were flushed and cold-stored with WMO-II or HTK solution, a perfusate currently approved for clinical use. The average number of red blood cells remaining in a single glomerulus after flushing with WMO-II was significantly reduced when compared with HTK solution. Additionally, WMO-II reduced the number of apoptotic bodies in stored kidneys compared to HTK treated tissue after 48 h of cold storage by reducing expression of Caspase-9, BiP, Chop, and Caspase-12. WMO-II solution reduced serum creatinine levels and serum potassium in kidneys stored for 48 h when compared to HTK perfusion. WMO-II preserves kidney function as evidenced by the reduction in serum creatinine and potassium during graft transplantation.

A variety of neurological diseases are characterized by disturbances of the blood-brain barrier (BBB) and its transporters. We recently introduced fibroblast growth factor treated cortical organotypic slice cultures of mice as a model for in vitro studies of the blood-brain barrier and have now further characterized the maintenance and function of transport-proteins typically expressed in the endothelium of cerebral blood vessels. The glucose transporter GLUT-1 is present in blood vessels of slice cultures derived from postnatal day 4 to 21 mice after 3 days in vitro. The endothelial multidrug resistance P-glycoprotein (P-gp) which is involved in the control of pharmacological substance transport across the blood-brain barrier is also maintained in blood vessels, most prominently in slice cultures derived from postnatal day 14 and 21 mice. To assess P-gp function, we tested rhodamine 123 transport in presence or absence of the P-gp inhibitor verapamil. Rhodamine 123-fluorescence accumulated rapidly in the vascular lumen both in acute slices and in slices cultured for 3 days in vitro. Our results provide evidence that endothelial transporters and their functional properties can be maintained in organotypic cortical slices cultures, thus making it an attractive model system for the study of the blood-brain barrier.

Rationale: Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic strategy for the acute ischemic stroke (AIS). However, the poor targeted migration and low engraftment in ischemic lesions restrict their treatment efficacy. The ischemic brain lesions express a specific chemokine profile, while cultured MSCs lack the set of corresponding receptors. Thus, we hypothesize that overexpression of certain chemokine receptor might help in MSCs homing and improve therapeutic efficacy. Methods: Using the middle cerebral artery occlusion (MCAO) model of ischemic stroke, we identified that CCL2 is one of the most highly expressed chemokines in the ipsilateral hemisphere. Then, we genetically transduced the corresponding receptor, CCR2 to the MSCs and quantified the cell retention of MSCCCR2 compared to the MSCdtomato control. Results: MSCCCR2 exhibited significantly enhanced migration to the ischemic lesions and improved the neurological outcomes. Brain edema and blood-brain barrier (BBB) leakage levels were also found to be much lower in the MSCCCR2-treated rats than the MSCdtomato group. Moreover, this BBB protection led to reduced inflammation infiltration and reactive oxygen species (ROS) generation. Similar results were also confirmed using the in vitro BBB model. Furthermore, genome-wide RNA sequencing (RNA-seq) analysis revealed that peroxiredoxin4 (PRDX4) was highly expressed in MSCs, which mainly contributed to their antioxidant impacts on MCAO rats and oxygen-glucose deprivation (OGD)-treated endothelium. Conclusion: Taken together, this study suggests that overexpression of CCR2 on MSCs enhances their targeted migration to the ischemic hemisphere and improves the therapeutic outcomes, which is attributed to the PRDX4-mediated BBB preservation.

Synthetic cathinones, one of the largest groups of new psychoactive substances, represent a large analytical and interpretative challenge in forensic laboratories. Of these is the synthetic cathinones' instability in different biological samples, which may lead to drug concentration discrepancies when interpreting toxicological findings. In this study, the stability of a panel of synthetic cathinones and their dihydro-metabolites (n = 26) together with internal standard was monitored in human whole blood stored at various temperatures over 6 months. The influence of sodium fluoride as a preservative in blood collection tubes was also investigated.

Background: Asphyxia is the most common cause of brain damage in newborns. Substantial evidence indicates that leukocyte recruitment in the cerebral vasculature during asphyxia contributes to this damage. We tested the hypothesis that superoxide radical ( O 2 ⋅ _ ) promotes an acute post-asphyxial inflammatory response and blood-brain barrier (BBB) breakdown. We investigated the effects of removing O 2 ⋅ _ by superoxide dismutase (SOD) or C3, the cell-permeable SOD mimetic, in protecting against asphyxia-related leukocyte recruitment. We also tested the hypothesis that xanthine oxidase activity is one source of this radical. Methods: Anesthetized piglets were tracheostomized, ventilated, and equipped with closed cranial windows for the assessment of post-asphyxial rhodamine 6G-labeled leukocyte-endothelial adherence and microvascular permeability to sodium fluorescein in cortical venules. Asphyxia was induced by discontinuing ventilation. SOD and C3 were administered by cortical superfusion. The xanthine oxidase inhibitor oxypurinol was administered intravenously. Results: Leukocyte-venular adherence significantly increased during the initial 2 h of post-asphyxial reperfusion. BBB permeability was also elevated relative to non-asphyxial controls. Inhibition of O 2 ⋅ _ production by oxypurinol, or elimination of O 2 ⋅ _ by SOD or C3, significantly reduced rhodamine 6G-labeled leukocyte-endothelial adherence and improved BBB integrity, as measured by sodium fluorescein leak from cerebral microvessels. Conclusion: Using three different strategies to either prevent formation or enhance elimination of O 2 ⋅ _ during the post-asphyxial period, we saw both reduced leukocyte adherence and preserved BBB function with treatment. These findings suggest that agents which lower O 2 ⋅ _ in brain may be attractive new therapeutic interventions for the protection of the neonatal brain following asphyxia.

Cerebral edema is a common complication following moderate and severe traumatic brain injury (TBI), and a significant risk factor for development of neuronal death and deterioration of neurological outcome. To this date, medical approaches that effectively alleviate cerebral edema and neuronal death after TBI are not available. Glucagon-like peptide-1 (GLP-1) has anti-inflammatory properties on cerebral endothelium and exerts neuroprotective effects. Here, we investigated the effects of GLP-1 on secondary injury after moderate and severe TBI. Male Sprague Dawley rats were subjected either to TBI by Controlled Cortical Impact (CCI) or sham surgery. After surgery, vehicle or a GLP-1 analogue, Liraglutide, were administered subcutaneously twice daily for two days. Treatment with Liraglutide (200 μg/kg) significantly reduced cerebral edema in pericontusional regions and improved sensorimotor function 48 hours after CCI. The integrity of the blood-brain barrier was markedly preserved in Liraglutide treated animals, as determined by cerebral extravasation of Evans blue conjugated albumin. Furthermore, Liraglutide reduced cortical tissue loss, but did not affect tissue loss and delayed neuronal death in the thalamus on day 7 post injury. Together, our data suggest that the GLP-1 pathway might be a promising target in the therapy of cerebral edema and cortical neuronal injury after moderate and severe TBI.

The midbrain is an extensively studied brain region in schizophrenia, in view of its reported dopamine pathophysiology and neuroimmune changes associated with this disease. Besides the dopaminergic system, the midbrain contains other cell types that may be involved in schizophrenia pathophysiology. The neurovascular hypothesis of schizophrenia postulates that both the neurovasculature structure and the functioning of the blood-brain barrier (BBB) are compromised in schizophrenia. In the present study, potential alteration in the BBB of patients with schizophrenia was investigated by single-nucleus RNA sequencing of post-mortem midbrain tissue (15 schizophrenia cases and 14 matched controls). We did not identify changes in the relative abundance of the major BBB cell types, nor in the sub-populations, associated with schizophrenia. However, we identified 14 differentially expressed genes in the cells of the BBB in schizophrenia as compared to controls, including genes that have previously been related to schizophrenia, such as FOXP2 and PDE4D. These transcriptional changes were limited to the ependymal cells and pericytes, suggesting that the cells of the BBB are not broadly affected in schizophrenia.

The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at -6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at -4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood.

Resolvins are endogenous lipid mediators derived from omega-3 fatty acids. Resolvin E1 (RvE1), derived from eicosapentaenoic acid (EPA), modulates osteoclasts and immune cells in periodontal disease models. The direct role of RvE1 in bone remodeling is not well understood. The objective of this study was to determine the impact of RvE1 on bone remodeling under inflammatory conditions. Our working hypothesis is that RvE1 downregulates bone resorption through direct actions on both osteoblast and osteoclast function in inflammatory osteoclastogenesis. A tumor necrosis factor-α induced local calvarial osteolysis model with or without the systemic administration of RvE1 was used. To evaluate osteoclastogenesis and NFκB signaling pathway activity, murine bone tissue was evaluated by Micro CT (μCT) analysis, TRAP staining, and immunofluorescence analysis. Mechanistically, to evaluate the direct role of RvE1 impacting bone cells, primary calvarial mouse osteoblasts were stimulated with interleukin (IL)-6 (10 ng/ml) and IL-6 receptor (10 ng/ml) and simultaneously incubated with or without RvE1 (100 nM). Expression of receptor activator of NFκB ligand (RANKL) and osteoprotegerin (OPG) was measured by ELISA. RNA sequencing (RNA-Seq) and differential expression analysis was performed to determine signaling pathways impacted by RvE1. The systemic administration of RvE1 reduced calvarial bone resorption as determined by µCT. Histologic analysis of calvaria revealed that osteoclastogenesis was reduced as determined by number and size of osteoclasts in TRAP-stained sections (p < 0.05). Immunofluorescence staining of calvarial sections revealed that RvE1 reduced RANKL secretion by 25% (p < 0.05). Stimulation of osteoblasts with IL-6 increased RANKL production by 30% changing the RANKL/OPG to favor osteoclast activation and bone resorption. The ratio changes were reversed by 100 nM RvE1. RvE1 decreased the production of RANKL maintaining an RANKL/OPG more favorable for bone formation. RNA-Seq and transcriptomic pipeline analysis revealed that RvE1 significantly downregulates osteoclast differentiation mediated by differential regulation of NFκB and PI3K-AKT pathways. RvE1 reduces inflammatory bone resorption. This action is mediated, at least in part, by direct actions on bone cells promoting a favorable RANKL/OPG ratio. Mediators of resolution in innate immunity also directly regulate bone cell gene expression that is modulated by RvE1 through at least 14 specific genes in this mouse model.

Allogeneic blood vessels are regarded as one of the best natural substitutes for diseased blood vessels due to their good vascular compliance and histocompatibility. Since the supply and demand of allograft blood vessels do not always match in time and space, a good preservation scheme for isolated blood vessels is essential. The abdominal aortas of 110 male Sprague-Dawley (SD) rats were randomly divided into three groups, including cold storage group (4°C) (CSG), frozen storage group (FSG) and ambient storage group (25 ± 2°C) (ASG). Seven time points of preservation for 1, 3, 5, 7, 14, 30 and 90 days were set for detection. The changes in vascular physiological function were evaluated by MTT test and vasoconstriction ability detection, and the changes in vascular wall structure were evaluated by the tension tolerance test and pathological staining. The vascular function of CSG was better than FSG within first the 7 days, but the result was opposite since the 14th day. The vascular wall structure, collagen and elastic fibres of vessels, in CSG, showed oedema within 30 days, and continuous disintegration and rupture at 90 days. The vessel wall structure of FSG remained intact within 90 days. The tensile strength of the vessels in CSG was better than that in FSG within 5 days, and there was no statistical difference between the two groups between the 7th and 30th day, and then, the FSG was higher than CSG on the 90th day. Both cold storage and frozen storage could be applied as safe and effective preservation schemes for isolated rat artery within first 30 days. Cold storage is recommended when the storage time is <14 days, and then, frozen storage is better.

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.

We investigated the clinical impact of D3 lymph node dissection preserving left colic artery (LCA) compared to D3 without LCA preservation using data from JCOG0404. LCA preservation is expected to maintain adequate blood supply, which is effective in preventing anastomotic leakage, intestinal paralysis, and bowel obstruction. Whether D3 with LCA preservation (Group A) improves clinical outcomes following resection of sigmoid colon cancer compared to D3 without LCA preservation (Group B) is unclear.

Pyloric preservation (PP) can frequently be performed at the time of pancreatoduodenectomy (PD), although some reports have linked it to inferior outcomes such as delayed gastric emptying (DGE). We reviewed records in a single-surgeon practice to assess outcomes after PD with or without PP. There were 133 PDs with 67 PPPDs and 66 PDs. Differences between PPPD and PD groups included cancer frequency, tumor size, OR time, blood loss, and transfusion rate. However, postoperative morbidity rate and grade, NG tube duration, NGT reinsertion rate, DGE, and length of stay were similar. There was no difference among patients with pancreatic cancer. No detrimental outcomes are associated with pyloric preservation during PD. Greater intraoperative ease and superior survival in the PPPD group are due to confounding, tumor-related variables in this nonrandomized comparison. Nevertheless, we intend to continue the use of PP with our technique in patients who meet the stated criteria.

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, plays a crucial role in regulating inflammation and oxidative stress that are tightly related to stroke development and progression. Consequently, BRD4 blockade has attracted increasing interest for associated neurological diseases, including stroke. dBET1 is a novel and effective BRD4 degrader through the proteolysis-targeting chimera (PROTAC) strategy. We hypothesized that dBET1 protects against brain damage and neurological deficits in a transient focal ischemic stroke mouse model by reducing inflammation and oxidative stress and preserving the blood-brain barrier (BBB) integrity. Post-ischemic dBET1 treatment starting 4 h after stroke onset significantly ameliorated severe neurological deficits and reduced infarct volume 48 h after stroke. dBET1 markedly reduced inflammation and oxidative stress after stroke, indicated by multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2, CXCL1 and CXCL10, and oxidative damage markers 4-hydroxynonenal (4-HNE) and gp91phox and antioxidative proteins SOD2 and GPx1. Meanwhile, stroke-induced BBB disruption, increased MMP-9 levels, neutrophil infiltration, and increased ICAM-1 were significantly attenuated by dBET1 treatment. Post-ischemic dBET1 administration also attenuated ischemia-induced reactive gliosis in microglia and astrocytes. Overall, these findings demonstrate that BRD4 degradation by dBET1 improves acute stroke outcomes, which is associated with reduced neuroinflammation and oxidative stress and preservation of BBB integrity. This study identifies a novel role of BET proteins in the mechanisms resulting in ischemic brain damage, which can be leveraged to develop novel therapies.

Current cold storage organ preservation technique fails to preserve marginal donor grafts sufficiently. Evidence from large animal experiments suggests superiority of normothermic machine preservation of liver allografts. Long-term organ preservation using normothermic perfusion might not only allow organ viability assessment before transplantation, but also provide the means for further organ modifications under physiologic conditions. Previous research has shown that porcine livers can be transplanted successfully after normothermic preservation of 20 hours. In the present study we investigate whether similar methodology is capable of further extending the safe limit to 48 hours. In this study, livers from White Landrace pigs were preserved by normothermic, oxygenated sanguineous perfusion. After a 48-hour period of preservation, livers were transplanted into recipient pigs and followed for 5 days. Outcome parameters measured included markers of synthetic and metabolic liver function as well as hepatocellular injury and blood gas analysis during perfusion and follow-up. Histological assessment of morphological liver integrity was performed. All livers showed sustained bile production and metabolic activity throughout the preservation period. Low levels of hepatocellular damage were found. Following transplantation all liver grafts revealed excellent graft function and death-censored graft survival was 100%. Porcine livers were transplanted successfully following 48 hours normothermic machine preservation.