Cross-Circulation for Extracorporeal Liver Support in a Swine Model.

Although machine perfusion has gained momentum as an organ preservation technique in liver transplantation, persistent organ shortages and high waitlist mortality highlight unmet needs for improved organ salvage strategies. Beyond preservation, extracorporeal organ support platforms can also aid the development and evaluation of novel therapeutics. Here, we report the use of veno-arterial-venous (V-AV) cross-circulation (XC) with a swine host to provide normothermic support to extracorporeal livers. Functional, biochemical, and morphological analyses of the extracorporeal livers and swine hosts were performed over 12 hours of support. Extracorporeal livers maintained synthetic function through alkaline bile production and metabolic activity through lactate clearance and oxygen consumption. Beyond initial reperfusion, no biochemical evidence of hepatocellular injury was observed. Histopathologic injury scoring showed improvements in sinusoidal dilatation and composite acute injury scores after 12 hours. Swine hosts remained hemodynamically stable throughout XC support. Altogether, these outcomes demonstrate the feasibility of using a novel V-AV XC technique to provide support for extracorporeal livers in a swine model. V-AV XC has potential applications as a translational research platform and clinical biotechnology for donor organ salvage.

First Successful Open-Heart Surgery Utilizing Cross-Circulation in 1954.

A pioneering surgeon at the University of Minnesota, Dr C. Walton Lillehei, is still considered the "father of open-heart surgery". Dr Lillehei and his surgical team performed the first open-heart operations utilizing cross-circulation, including the first successful ventricular septal defect closure on a 3-year-old boy. Before his death at age 67, this patient arranged to donate his body to the University of Minnesota's Anatomy Bequest program. We describe this patient's medical history, and present unique images of internal/external cardiac anatomies and implanted devices obtained via direct visualizations, computed tomography, and fluoroscopy post-mortem. Additionally, we present computational models and 3-dimensional printed models.

Ex Vivo Heart Perfusion for 72 Hours Using Plasma Cross Circulation.

Preservation of a donor heart for transplantation is limited to 6-8 hours. Based on our demonstration of 12 hour perfusion with plasma cross circulation, this study aimed to evaluate ex vivo heart perfusion (EVHP) for up to 72 hours using cross plasma circulation (XC-plasma) from a live, awake paracorporeal sheep (PCS). Six ovine hearts were perfused for 72 hours using plasma cross circulation at a rate of 1 L/min with a live, awake PCS. Controls were seven perfused hearts without cross circulation. Experiments were electively ended at 72 hours, and epinephrine (0.1 mg) was delivered to demonstrate hormonal responsiveness. All controls failed at 6-10 hours. All six hearts perfused for 72 hours maintained normal heart function, metabolism, and responsiveness to epinephrine. Blood gases, electrolytes, and lactate levels were normal and stable throughout the study. All hearts appeared suitable for transplantation. We have demonstrated successful normothermic EVHP for 72 hours.

Regeneration of severely damaged lungs using an interventional cross-circulation platform.

The number of available donor organs limits lung transplantation, the only lifesaving therapy for the increasing population of patients with end-stage lung disease. A prevalent etiology of injury that renders lungs unacceptable for transplantation is gastric aspiration, a deleterious insult to the pulmonary epithelium. Currently, severely damaged donor lungs cannot be salvaged with existing devices or methods. Here we report the regeneration of severely damaged lungs repaired to meet transplantation criteria by utilizing an interventional cross-circulation platform in a clinically relevant swine model of gastric aspiration injury. Enabled by cross-circulation with a living swine, prolonged extracorporeal support of damaged lungs results in significant improvements in lung function, cellular regeneration, and the development of diagnostic tools for non-invasive organ evaluation and repair. We therefore propose that the use of an interventional cross-circulation platform could enable recovery of otherwise unsalvageable lungs and thus expand the donor organ pool.

Exogenous biological renal support ameliorates renal pathology after ischemia reperfusion injury in elderly mice.

We established an exogenous biological renal support model through the generation of parabiotic mice. At 72 hours after ischemia reperfusion injury (IRI), the aged mice that received exogenous biological renal support showed significantly higher levels of renal cell proliferation and dedifferentiation, lower levels of renal tubular injury, improved renal function, and a lower mortality than those that did not receive exogenous biological renal support. Using the Quantibody Mouse Cytokine Antibody Array, we found that aged IRI mice that received exogenous biological renal support had an up-regulation of multiple inflammatory related cytokines compared to the group that did not receive exogenous biological renal support. We suggest that the exogenous biological renal support might promote renal tubular epithelial cell proliferation and dedifferentiation and improve the prognosis of aged IRI mice. Exogenous biological renal support may play an important role in the amelioration of renal IRI by regulating the expression of multiple cytokines.

Left ventricular mechanoenergetics in excised, cross-circulated rat hearts under hypo-, normo-, and hyperthermic conditions.

We investigated the effects of altering cardiac temperature on left ventricular (LV) myocardial mechanical work and energetics using the excised, cross-circulated rat heart model. We analyzed the LV end-systolic pressure-volume relationship (ESPVR) and linear relationship between myocardial oxygen consumption per beat (VO2) and systolic pressure-volume area (PVA; total mechanical energy per beat) in isovolumically contracting rat hearts during hypo- (32 °C), normo- (37 °C), and hyperthermia (42 °C) under a 300-beats per minute pacing. LV ESPVR shifted downward with increasing cardiac temperature. The VO2-PVA relationship was superimposable in these different thermal conditions; however, each data point of VO2-PVA shifted left-downward during increasing cardiac temperature on the superimposable VO2-PVA relationship line. VO2 for Ca2+ handling in excitation-contraction coupling decreased, which was associated with increasing cardiac temperature, during which sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity was suppressed, due to phospholamban phosphorylation inhibition, and instead, O2 consumption for basal metabolism was increased. The O2 cost of LV contractility for Ca2+ also increased with increasing cardiac temperature. Logistic time constants evaluating LV relaxation time were significantly shortened with increasing cardiac temperature related to the acceleration of the detachment in cross-bridge (CB) cycling, indicating increased myosin ATPase activity. The results suggested that increasing cardiac temperature induced a negative inotropic action related to SERCA activity suppression in Ca2+ handling and increased myosin ATPase activity in CB cycling. We concluded that thermal intervention could modulate cardiac inotropism by changing CB cycling, Ca2+ handling, and basal metabolism in rat hearts.

Methods for the Preparation of an Excised, Cross-Circulated Rat Heart.

The Emax-Pressure-Volume Area (PVA)-VO2 framework proposed by Dr. Suga for canine hearts has dramatically advanced the field of cardiac mechanical work and energetics, i.e., mechanoenergetics. He and his collaborators investigated mechanoenergetics in the left ventricle (LV) of excised, cross-circulated canine heart preparations. We instituted the excised cross-circulated rat whole heart preparations and found a curvilinear end-systolic pressure-volume relation (ESPVR) in the rat LV, in contrast to the linear ESPVR in canine, rabbit, and human LVs. Although Emax, the slope of the linear ESPVR, could be used as an index of LV contractility, it was not applicable for evaluating LV contractility in the rat LV. Thus, we proposed a new index of contractility, equivalent Emax (eEmax) in the rat LV. We also found a linear VO2-PVA relationship in the rat LV. Here, we introduce the methods for the preparation of excised, cross-circulated rat whole hearts and the eEmax-PVA-VO2 framework in the rat LV. Using this method, we can obtain accurate LV volume and myocardial O2 consumption in real time for estimating cardiac mechanoenergetics, which is very challenging in in vivo experiments.

Youthful systemic milieu alleviates renal ischemia-reperfusion injury in elderly mice.

The incidence of acute kidney injury (AKI) is high in elderly people, and is difficult to prevent and treat. One of its major causes is renal ischemia-reperfusion injury (IRI). A young systemic environment may prevent the senescence of old organs. However, it is unknown whether a young milieu may reduce renal IRI in the elderly. To examine this question, bilateral renal IRI was induced in old (24 months) mice three weeks after parabiosis model establishment. At 24 hours after IRI, compared to old wild-type mice, the old mice with IRI had significantly damaged renal histology, decreased renal function, increased oxidative stress, inflammation, and apoptosis. However, there was no increase in autophagy. Compared to old mice with IRI, old-old parabiosis mice with IRI did not show differences in renal histological damage, oxidative stress, inflammation, apoptosis, or autophagy, but did exhibit improved renal function. Compared to the old-old parabiosis mice with IRI, the old mice with IRI in the young (12 week)-old parabiosis showed less renal histological injury and better renal function. Renal oxidative stress, inflammation, and apoptosis were significantly decreased, and autophagy was significantly increased. Thus, a youthful systemic milieu may decrease oxidative stress, inflammation, and apoptosis, and increase autophagy in old mice with IRI. These effects ameliorated IRI injuries in old mice. Our study provides new ideas for effectively preventing and treating AKI in the elderly.

Normothermic Ex Vivo Heart Perfusion: Effects of Live Animal Blood and Plasma Cross Circulation.

Prolonged normothermic ex vivo heart perfusion could transform cardiac transplantation. To help identify perfusate components that might enable long-term perfusion, we evaluated the effects of cross-circulated whole blood and cross-circulated plasma from a live paracorporeal animal on donor porcine hearts preserved via normothermic ex vivo heart perfusion. Standard perfusion (SP; n = 40) utilized red blood cell/plasma perfusate and Langendorff technique for a goal of 12 hours. Cross-circulation groups used a similar circuit with the addition of cross-circulated venous whole blood (XC-blood; n = 6) or cross-circulated filtered plasma (XC-plasma; n = 7) between a live paracorporeal pig under anesthesia and the perfusate reservoir. Data included oxygen metabolism, vascular resistance, lactate production, left ventricular function, myocardial electrical impedance, and histopathologic injury score. All cross-circulation hearts were successfully perfused for 12 hours, compared with 22 of 40 SP hearts (55%; p = 0.002). Both cross-circulation groups demonstrated higher oxygen consumption and vascular resistance than standard hearts from hours 3-12. No significant differences were seen between XC-blood and XC-plasma hearts in any variable, including left ventricular dP/dT after 12 hours (1478 ± 700 mm Hg/s vs. 872 ± 500; p = 0.17). We conclude that cross circulation of whole blood or plasma from a live animal improves preservation of function of perfused hearts, and cross-circulated plasma performs similarly to cross-circulated whole blood.

A cross-circulated bicephalic model of head transplantation.

AIMS: A successful cephalosomatic anastomosis ("head transplant") requires, among others, the ability to control long-term immune rejection and avoidance of ischemic events during the head transference phase. We developed a bicephalic model of head transplantation to study these aspects. METHODS AND RESULTS: The thoracic aorta and superior vena cava of a donor rat were anastomosed with the carotid artery and extracorporeal veins of a recipient rat by vascular grafts. Before thoracotomy in the donor rat, the axillary artery and vein of the donor were connected to the carotid and the extracranial vein of the third rat through a silicone tube. The silicone tube was passed through a peristaltic pump to ensure donor brain tissue blood supply. There is no ischemia reperfusion injury in donor brain tissue analyzed by electroencephalogram. Postoperative donor has pain reflex and corneal reflex. CONCLUSIONS: Peristaltic pump application can guarantee the blood supply of donor brain tissue per unit time, while the application of temperature change device to the silicone tube can protect the brain tissue hypothermia, postoperative experimental data show that there is no brain tissue ischemia during the whole operation. The application of vascular grafting can also provide the possibility of long-term survival of the model.

Temperature-dependent inotropic and lusitropic indices based on half-logistic time constants for four segmental phases in isovolumic left ventricular pressure-time curve in excised, cross-circulated canine heart.

Varying temperature affects cardiac systolic and diastolic function and the left ventricular (LV) pressure-time curve (PTC) waveform that includes information about LV inotropism and lusitropism. Our proposed half-logistic (h-L) time constants obtained by fitting using h-L functions for four segmental phases (Phases I-IV) in the isovolumic LV PTC are more useful indices for estimating LV inotropism and lusitropism during contraction and relaxation periods than the mono-exponential (m-E) time constants at normal temperature. In this study, we investigated whether the superiority of the goodness of h-L fits remained even at hypothermia and hyperthermia. Phases I-IV in the isovolumic LV PTCs in eight excised, cross-circulated canine hearts at 33, 36, and 38 °C were analyzed using h-L and m-E functions and the least-squares method. The h-L and m-E time constants for Phases I-IV significantly shortened with increasing temperature. Curve fitting using h-L functions was significantly better than that using m-E functions for Phases I-IV at all temperatures. Therefore, the superiority of the goodness of h-L fit vs. m-E fit remained at all temperatures. As LV inotropic and lusitropic indices, temperature-dependent h-L time constants could be more useful than m-E time constants for Phases I-IV.

Preclinical Assessment of Young Blood Plasma for Alzheimer Disease.

IMPORTANCE: Alzheimer disease (AD) pathology starts long before clinical symptoms manifest, and there is no therapy to treat, delay, or prevent the disease. A shared blood circulation between 2 mice (aka parabiosis) or repeated injections of young blood plasma (plasma from 2- to 3-month-old mice) into old mice has revealed benefits of young plasma on synaptic function and behavior. However, to our knowledge, the potential benefit of young blood has not been tested in preclinical models of neurodegeneration or AD. OBJECTIVES: To determine whether young blood plasma ameliorates pathology and cognition in a mouse model for AD and could be a possible future treatment for the disease. DESIGN, SETTING, AND PARTICIPANTS: In this preclinical study, mice that harbor a human mutant APP gene, which causes familial AD, were aged to develop AD-like disease including accumulation of amyloid plaques, loss of synaptic and neuronal proteins, and behavioral deficits. The initial parabiosis studies were done in 2010, and the final studies were conducted in 2014. Alzheimer disease model mice were then treated either by surgically connecting them with a young healthy mouse, thus providing a shared blood circulation through parabiosis, or through repeated injections of plasma from young mice. MAIN OUTCOMES AND MEASURES: Neuropathological parameters and changes in hippocampal gene expression in response to the treatment were assessed. In addition, cognition was tested in AD model mice intravenously injected with young blood plasma. RESULTS: Aged mutant amyloid precursor protein mice with established disease showed a near complete restoration in levels of synaptic and neuronal proteins after exposure to young blood in parabiosis (synaptophysin P = .02; calbindin P = .02) or following intravenous plasma administration (synaptophysin P < .001; calbindin P = .14). Amyloid plaques were not affected, but the beneficial effects in neurons in the hippocampus were accompanied by a reversal of abnormal extracellular receptor kinase signaling (P = .05), a kinase implicated in AD. Moreover, young plasma administration was associated with improved working memory (P = .01) and associative memory (P = .02) in amyloid precursor protein mice. CONCLUSIONS AND RELEVANCE: Factors in young blood have the potential to ameliorate disease in a model of AD.

Search for an Endogenous Bombesin-Like Receptor 3 (BRS-3) Ligand Using Parabiotic Mice.

Bombesin-like receptor 3 (BRS-3) is an X-linked G protein-coupled receptor involved in the regulation of energy homeostasis. Brs3 null (Brs3-/y) mice become obese. To date, no high affinity endogenous ligand has been identified. In an effort to detect a circulating endogenous BRS-3 ligand, we generated parabiotic pairs of mice between Brs3-/y and wild type (WT) mice or between WT controls. Successful parabiosis was demonstrated by circulatory dye exchange. The Brs3-/y-WT and WT-WT pairs lost similar weight immediately after surgery. After 9 weeks on a high fat diet, the Brs3-/y-WT pairs weighed more than the WT-WT pairs. Within the Brs3-/y-WT pairs, the Brs3-/y mice had greater adiposity than the WT mice, but comparable lean and liver weights. Compared to WT mice in WT-WT pairs, Brs3-/y and WT mice in Brs3-/y-WT pairs each had greater lean mass, and the Brs3-/y mice also had greater adiposity. These results contrast to those reported for parabiotic pairs of leptin receptor null (Leprdb/db) and WT mice, where high leptin levels in the Leprdb/db mice cause the WT parabiotic partners to lose weight. Our data demonstrate that a circulating endogenous BRS-3 ligand, if present, is not sufficient to reduce adiposity in parabiotic partners of Brs3-/y mice.

Blood as the carrier of ischemic tolerance in rat brain.

This study provides clear evidence that the factor inducing tolerance to ischemia is transmitted via the circulating blood. By using the remote ischemia and the cross-circulation model, the tolerance to ischemia was transmitted from donor to recipient. For this study, the following experimental groups were designed: I, sham control group; II, group of tolerant hindlimb tourniquet-treated rats; III, positive control group; IV, control for cross-circulation influence; preconditioned animals: V, tolerant animals subjected to middle cerebral artery occlusion (MCAO); VI, tolerant animals cross-circulated with SHC, followed by MCAO; VII, SHC animals cross-circulated with tolerant animals and subsequently subjected to MCAO; VIII, tolerant animals cross-circulated with ischemic rats, followed by MCAO; IX, SHC animals cross-circulated with ischemic animals and subjected to MCAO; postconditioned animals: X, ischemic animals treated with a remote limb tourniquet; XI, ischemic animals cross-circulated with SHC control rats; and XII, ischemic animals cross-circulated with tolerant rats. Results confirmed that remote ischemia induced reduction of infarct volume in the preconditioned (V, 60%) as well as in the postconditioned group (X, 52%). Significant diminution was also observed in group XII (56.6%). In the preconditioned group, decreased infarct volume was detected in groups VI and VII (about 65%) and in group IX (about 50%). The greatest infarct reduction (84%) was induced by the presence of ischemic blood in a tolerant rat before ischemia induction. In summary, the factor inducing tolerance to ischemia is generated by remote ischemia and by ischemia itself; from the site of origin to the rest of the body, it is transported by the systemic blood circulation and can be transferred from animal to animal. The effect of conditioning with two different ischemic events (brain and hindlimb ischemia) led to a cumulative, stronger tolerance response.

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

RATIONALE: Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. OBJECTIVE: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. METHODS AND RESULTS: We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. CONCLUSIONS: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.