The effects of over expressing aquaporins on the cryopreservation of hepatocytes.

During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt(2)cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments - confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt(2)cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt(2)cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability.

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming.

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

An Air Particulate Pollutant Induces Neuroinflammation and Neurodegeneration in Human Brain Models.

Fine particulate matter (PM2.5), a major component among air pollutants, highlights as a global health concern. Several epidemiological studies show the correlation between chronical PM2.5 exposure and incidents of neurological disorders including Alzheimer's disease. However, the mechanisms have not been well understood, partly due to the lack of model systems that reflect the physiologically relevant innate immunity in human brains. Here, PM2.5-polluted human brain models (PMBs) are created in a 3D microfluidic platform reconstituting key aspects of human brain immunity under the PM2.5 exposure. PM2.5 penetration across a blood-brain barrier (BBB) model and accumulation in the brain tissue side of the model are first validated. Second, the PMB model shows that the BBB-penetrating PM2.5 initiates astrogliosis, resulting in slight neuronal loss and microglial infiltration. Third, it is demonstrated that the infiltrating microglia obtain M1 phenotype induced by interleukin-1ß and interferon-γ from neurons and reactive astrocytes under the PM2.5 exposure. Finally, it is observed that additional proinflammatory mediators and nitric oxide released from the M1 microglia exacerbate neuronal damages, such as synaptic impairment, phosphoric tau accumulation, and neuronal death. This study suggests that PM2.5 can be a potential environmental risk factor for dementia mediated by the detrimental neuroinflammation.

Inhibition of TXA synthesis with OKY-046 improves liver preservation by prolonged hypothermic machine perfusion in rats.

BACKGROUND AND AIM: We previously reported that hypothermic machine perfusion (HMP) for liver preservation is feasible, but hepatic microcirculatory dysfunction and significant liver damage remain major obstacles in its application when the preservation is extended to 24 h. The underlying injury mechanism is not well understood. The present study sought to investigate the role of thromboxane A(2) (TXA(2)) in the pathogenesis of liver injury after prolonged HMP. METHODS: Livers isolated from Sprague-Dawley rats were subjected to continuous machine perfusion with University of Wisconsin (UW) solution at a flow rate of 0.4 mL/min/g liver at 4 degrees C for 24 h. A specific TXA(2) synthase inhibitor, OKY-046 (OKY), was added to UW solution during the preservation period and to the Krebs-Henseleit buffer during reperfusion. The performance of the livers after preservation was evaluated using an isolated liver perfusion system with Krebs-Henseleit buffer at a flow rate of 15 mL/min at 37 degrees C for 30 min. RESULTS: Prolonged HMP induced a significant release of TXA(2) into the portal circulation as indicated by markedly increased levels of TXB(2) in the perfusate during reperfusion (at 30 min, 1447.4 +/- 163.6 pg/mL vs 50.91 +/- 6.7 pg/mL for control). Inhibition of TXA(2) synthesis with OKY significantly decreased releases of TXA(2) (69.8 +/- 13.4 pg/mL) concomitant with reduced lactate dehydrogenase (LDH) releases (at 30 min, HMP + OKY: 144.9 +/- 27.9 U/L; HMP: 369.3 +/- 68.5 U/L; simple cold storage or SCS: 884.4 +/- 80.3 U/L), decreased liver wet/dry weight ratio (HMP + OKY vs SCS and HMP: 3.6 +/- 0.3 vs 4.4 +/- 0.1 and 3.9 +/- 0.2, respectively) and increased hyaluronic acid uptake (at 30 min, HMP + OKY vs SCS, HMP: 33.1 +/- 2.9% vs 13.9 +/- 3.6%, 18.6 +/- 2.4%, respectively). Liver histology also showed significant improvement in tissue edema and hepatocellular necrosis with OKY compared with HMP without OKY. CONCLUSION: The results demonstrate that TXA(2) is involved in the development of hepatocellular injury induced by HMP, and inhibition of TXA(2) synthesis during preservation and reperfusion protects liver hepatocytes and sinusoidal endothelial cells from injuries caused by prolonged HMP.

Improved preservation of warm ischemic livers by hypothermic machine perfusion with supplemented University of Wisconsin solution.

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Pronlonged hypothermic machine perfusion preserves hepatocellular function but potentiates endothelial cell dysfunction in rat livers.

BACKGROUND: Although hypothermic machine perfusion (HMP) preservation has been shown to improve organ function and to expand the organ donor pool, problems still exist with the current HMP technology for liver preservation. The present study was conducted to investigate endothelial and hepatocellular functions following extended HMP (> r =24 hr) in rat liver model. METHODS: Following 24-hour hypothermic HMP with University of Wisconsin (UW) solution or 24-hour simple cold storage (SCS), livers were reperfused with Krebs-Henseleit buffer solution at 37 degree C for 30 minutes. Hepatocyte damage and function were assessed by measuring lactate dehydrogenase (LDH) activity, bile production, and indocyanine green (ICG) extraction. Sinusoidal endothelial cell (SEC) function and permeability were determined by hyaluronic acid (HA) uptake and multiple indicator dilution (MID) method, respectively. RESULTS: After 24-hour hypothermic preservation, HMP livers showed lower released LDH levels, higher bile flow rate, and greater hepatic ICG uptake compared with SCS livers. However, LDH levels became significantly higher in HMP than in SCS after 30 minutes of warm perfusion. The increased enzyme levels were accompanied by a significant increase in endothelial permeability to albumin and a decrease in hyaluronic acid uptake in HMP compared to SCS. Liver wet/dry weight ratio confirmed a greater edema in HMP livers than SCS livers. CONCLUSION: These results suggest that 24-hour hypothermic HMP may help preservation of hepatocyte function, but endothelial cell dysfunction during the cold preservation may play a key role in hepatocyte dysfunction and parenchymal cell death upon reperfusion.

Functional recovery of preserved livers following warm ischemia: improvement by machine perfusion preservation.

BACKGROUND: Hypothermic machine perfusion preservation has the potential to relieve the current donor shortage problem by reclaiming and preserving marginal donor organs including those from viable non-heart-beating donors. A number of problems exist with the current machine perfusion technology for preserving livers, and much research is needed to determine the clinical impact of this technology in preserving non-heart-beating donor livers. METHODS: This study was conducted to compare the poststorage function and microcirculation of simple cold stored and machine perfusion preserved livers that had experienced 30 min of warm ischemia followed by a 10 hr preservation period. In an isolated rat liver perfusion model, lactate dehydrogenase activity, indocyanine green secretion, and portal pressure values were determined at major time points. An intravital microscopy was conducted to assess microcirculation. RESULTS: The results showed an increase in flow homogeneity of machine perfused livers, which correlated with the reduction in portal pressure when compared with simple cold storage (5.4+/-0.4 vs. 8.7+/-0.6 mm Hg). A reduction in lactate dehydrogenase levels in the perfusate (333+/-22 vs.103+/-8 U/L) and an increase in bile production of the machine perfused livers (4.9+/-0.5 vs. 33.2+/-1.7 microg/min/g liver) and indocyanine green secretion (11.7+/-1.7 vs. 21.2+/-2.1 Abs/g bile) were observed at all time points (mean+/-SE of final point given). Intravital microscopic examination indicated that large regions of non flow, as indicated by the absence of fluorescein isothiocyanate-labeled albumin, were observed in the simple cold stored tissue, whereas machine perfused liver showed increase flow homogeneity. Values of bile production, indocyanine secretion, and cellular damages were comparable with controls. Histologic examination confirmed that simple cold stored tissue displayed increased vacuolization, and machine perfused tissue showed regions of normal hepatic structure. CONCLUSION: These results suggest that machine perfusion for 10 hr improves both poststorage function and microcirculation while reducing cellular damage of liver tissue that has experienced 30 min of warm ischemia, when compared with simple cold storage. Further studies need to be conducted, but this study suggests that machine perfusion preservation has the potential to reclaim and preserve liver tissues after warm ischemic insult.

Survival transplantation of preserved non-heart-beating donor rat livers: preservation by hypothermic machine perfusion.

BACKGROUND: Non-heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs. METHODS: This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival. RESULTS: Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07+/-1.42 and 2.02+/-0.69 U (mean+/-SE), respectively, compared with SCS, 15.54+/-0.81 and 3.41.3+/-0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17+/-0.49 mg/dL) were comparable to controls (0.91+/-0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure. CONCLUSIONS: These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Preservation methods for kidney and liver.

With the successful testing of the immunosuppressive effects of cyclosporine in transplant patients in 1978, the field of organ transplants began an exponential growth. With that, the field of organ preservation became increasingly important as the need to increase preservation time and improve graft function became paramount. However, for every patient that receives a transplanted organ, there are four more on the waiting list. In addition, a patient dies from the lack of a transplant almost every 1(1/2) hour. To alleviate this donor crisis, there is a need to expand the donor pool to marginal donor organs. The main reason these organs are underutilized is because the current method of static preservation, simple cold storage, is ineffective. This article will provide a general review of the methods of preservation including simple cold storage, hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation. In addition, the article will provide a review of how these dynamic preservation methods have improved the recovery and preservation of marginal donor organs including Donation after Cardiac Death and Fatty livers.

Noninvasive blood perfusion measurements of an isolated rat liver and an anesthetized rat kidney.

A simple, cost effective, and noninvasive blood perfusion system is tested in animal models. The system uses a small sensor to measure the heat transfer response to a thermal event (convective cooling) imposed on the tissue surface. Heat flux data are compared with a mathematical model of the tissue to estimate both blood perfusion and thermal contact resistance between the tissue and the probe. The perfusion system was evaluated for repeatability and sensitivity using isolated rat liver and exposed rat kidney tests. Perfusion in the isolated liver tests was varied by controlling the flow of the perfusate into the liver, and the perfusion in the exposed kidney tests was varied by temporarily occluding blood flow through the renal artery and vein. The perfusion estimated by the convective perfusion probe was in good agreement with that of the metered flow of the perfusate into the liver model. The liver tests indicated that the probe can be used to detect small changes in perfusion (0.005 ml/ml/s). The probe qualitatively tracked the changes in the perfusion in the kidney model due to occlusion of the renal artery and vein.

Hypothermic perfusion preservation: the future of organ preservation revisited?

Hypothermic perfusion preservation (HPP) was an integral step in the development of early clinical transplantation programmes, and considerable progress was made in understanding the basic principles underlying the technique. In subsequent years, the development of better preservation solutions for cold hypoxic storage, along with pragmatic choices made on grounds of costs and logistics, saw a fall in the application of HPP. More recently, the acute shortage of suitable organ donors and the inevitable pressure to use organs from sub-optimal (or expanded criteria) donors, has forced a re-evaluation of HPP, and the development of a new generation of HPP machines and associated perfusion solutions. This review sets out the historical development of HPP across the range of organs in which the method was originally investigated, describes the biological benefits and drawbacks associated with HPP, and sets out the most recent literature on the topic (including comments on the interest in use of higher temperatures in organ perfusion).

3,5-bis[4-(diethylamino)benzylidene]-1-methyl-4-piperidone and 3,5-bis[4-(diethylamino)cinnamylidene]-1-methyl-4-piperidone: prospective biophotonic materials.

The structures of 3,5-bis[4-(diethylamino)benzylidene]-1-methyl-4-piperidone, C(28)H(37)N(3)O, (I), and 3,5-bis[4-(diethylamino)cinnamylidene]-1-methyl-4-piperidone, C(32)H(41)N(3)O, (II), have been characterized. Because of conjugation between donor and acceptor parts, the central heterocycles (including the carbonyl group) in (I) and (II) are flattened and exhibit a 'sofa' conformation, with a deviation of the N atom from the planar fragment. The dihedral angles between the planar part of the heterocycle and the two almost flat fragments that include a phenyl ring and bridging atoms are 23.2 (1) and 11.2 (1) degrees in (I), and 11.8 (1) and 8.7 (2) degrees in (II). One- and two-photon absorption of light and the fluorescence of (I) and (II) have also been characterized.

Ex-vivo study of flow dynamics and endothelial cell structure during extended hypothermic machine perfusion preservation of livers.

Liver transplantation is often the only effective treatment for end stage liver diseases resulting from cirrhosis, hepatitis, progressive jaundice, and biliary atresia. Hypothermic machine perfusion (HMP) preservation may enhance donor pool by extending preservation time and reclaiming marginal donor livers including those from non-heart beating donors (NHBD), as demonstrated in the kidney. However, current HMP protocols have not been successful in improving extended preservation of livers and the major cause of preservation injury remains unknown. An intravital microscopy study was conducted to understand the flow dynamics of sinusoidal perfusion during 24h HMP with cold modified University of Wisconsin (UW) solution. Fluorescein isothiocynate (FITC) labeled albumin was utilized to visualize microvascular space and FITC labeled red blood cells (RBCs) were used to visualize flow dynamics during HMP. A heterogeneous flow pattern with regions of red cell stasis was observed after 24-h HMP. To examine the cause of red cell stasis, intravital and confocal microscopy studies of endothelial cells (ECs) structure labeled with DiI acetylated low-density lipoprotein (DiI acLDL) were conducted. These studies suggest that morphological changes in EC structures occurred during 24h HMP, which may cause obstruction to the sinusoidal flow. Histological findings confirm these results. As a result, heterogeneous flow pattern, red cell stasis, and edema occur, which may lead to the failure of these tissues following extended HMP.