Hypothermic machine perfusion of liver grafts for transplantation: technical development in human discard and miniature swine models.

INTRODUCTION: Cold storage (CS) is the standard preservation technique for liver transplantation (LTx). Hypothermic machine perfusion (HMP) is an alternative preservation technique that provides a continuous supply of substrates and removes waste products. HMP improves early graft function in kidney transplantation, especially for marginal organs: To our knowledge there have been no reports HMP in human LTx. The aim of this study was to develop a reproducible technique for liver HMP prior to initiating a clinical trial. METHODS: For the discard protocol, between May 2001 and March 2002, 10 nontransplantable human livers were obtained. We designed a model of atraumatic, centrifugal HMP of the portal vein (PV) and hepatic artery (HA) via donor vascular conduit. Livers were perfused at 3 degrees C to 5 degrees C with Vasosol solution for 5 to 10 hours using a modified Medtronic Portable Bypass System. Perfusion variables (temp, flow, pressure) where recorded every 30 minutes. During the study, we also validated our techniques in an animal model. For the animal protocol; six swine were used as liver donors and randomized to 12 hours of CS in UW (n = 3) or 12 hours of HMP using Vasosol solution (n = 3). LTx was performed in six swine. Animals survived until postoperative day 5. RESULTS: For the discard protocol, mean HMP time was 6.7 +/- 1.8 hours. Target flow was 0.7 mL/g liver/min. PV and HA pressure ranged from 3 to 5 and 12 to 18 mm Hg, respectively. All grafts were maintained at 3 degrees C to 5 degrees C during HMP. For the animal protocol, all recipients had good liver function and survived to postoperative day 5. AST and TBili were similar between CS and HMP. CONCLUSIONS: Our method of liver HMP appears to be a safe and reliable method to preserve livers. A clinical trial is now underway to evaluate this technique in human LTx.

Pushing the envelope in renal preservation; improved results with novel perfusate modifications for pulsatile machine perfusion of cadaver kidneys.

INTRODUCTION: Novel preservation techniques may diminish ischemia/reperfusion (I/R) injury. Our preservation laboratory has modified Belzer MPS for machine perfusion (MP) with prostaglandin E1 (PGE 1), nitroglycerin (NTG), and polyethylene glycol-superoxide dismutase (PEG-SOD) to attenuate I/R injury. We reviewed our recent experience using this novel formulation (NF) compared with standard perfusates. RESULTS: Between January 1998 and March 2000, 1060 consecutive kidneys were preserved in our laboratory. One hundred forty-eight kidneys (14%) were discarded. Fifty-eight percent of kidneys during this time period underwent MP (n = 532). En bloc kidney pairs were randomly assigned to pulsatile MP using Waters RM3 or MOX-100 perfusion systems using 1 of 3 perfusates; NF (NF; n = 119), Belzer MPS (MPS; n = 201), or Belzer II albumin gluconate (ALB; n = 212) Significant improvements in delayed graft function (DGF) rate were seen with NF versus other perfusates (8% vs 14% vs 19%, respectively; P =.03). At 6 months, graft survival was significantly improved with NF compared with MPS and ALB (96% vs 90% vs 87%, respectively; P =.03). NF also produced a significantly higher percentage of recipients with a serum creatinine level < or = 1.5 mg/dL. CONCLUSIONS: Novel modifications of standard MP perfusate improved outcomes after renal transplantation. Preservation-based interventions targeted to ameliorate I/R injury can improve outcomes and may allow expansion of the donor pool.

The nitric oxide connection: hyperbaric oxygen therapy, becaplermin, and diabetic ulcer management.

Clinical experience with adjunctive hyperbaric oxygen therapy in the treatment of diabetic ulcers has shown that wound hyperoxia increases wound granulation tissue formation and accelerates wound contraction and secondary closure. In addition to wound hyperoxia, increased wound nitric oxide production caused by hyperbaric oxygen therapy also appears to be important for successful diabetic wound repair. The results of a preliminary retrospective study suggest that nitric oxide production is reduced in the nonhealing diabetic wound, and that topical becaplermin therapy is effective only when wound nitric oxide production deficiency is corrected. In addition, the data suggest that below a critical level of endogenous nitric oxide production, diabetic ulcer repair may not be achieved. Under this hypothesis, diabetic patients with chronic, nonhealing ulcers that respond to becaplermin should have substantially increased endogenous nitric oxide production compared with those ulcers that do not respond to becaplermin. The results of a preliminary clinical study support the use of combined therapy using topical becaplermin and hyperbaric oxygen therapy as a means of successfully treating the chronic diabetic ulcer patient with deficient nitric oxide production and local wound hypoxia.

The influence of pulsatile preservation on renal transplantation in the 1990s.

BACKGROUND: Unlike simple cold storage (CS), pulsatile machine preservation (MP) of kidneys for transplantation permits pharmacologic manipulation of the perfusate and aids in the pretransplant assessment of the kidney graft. These characteristics of MP may have importance in the era of increasing use of extended criteria donor kidneys. The overall aim of this article is to critically assess practices at our preservation unit with respect to graft function. Specific aims are to (1) compare the influence of MP versus CS on graft function, (2) determine which pretransplant variables have significance in pretransplant assessment, and (3) determine whether pharmacologic manipulation during MP is advantageous. METHODS: There were 650 consecutive kidneys preserved in our laboratory between January 1, 1993 and March 1, 999, by either MP or CS. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer-MPS or Belzer II solution. Perfusion parameters and electrolytes were measured serially during pulsatile perfusion. All CS kidneys were stored in University of Wisconsin solution. All kidneys obtained from donors exhibiting extended criteria features underwent pretransplant frozen section biopsies. Transmission electron microscopy (EM) was performed on a subset of kidneys undergoing pharmacologic manipulation. Four agents were assessed prospectively for their ability to influence MP characteristics when added to perfusate: PGE1, trifluoperazine, verapamil, and papaverine. RESULTS: MP was associated with improved immediate, 1-, and 2-year graft function and reduced length of initial hospital stay when compared with CS grafts. Changes in the machine perfusion variables flow and resistance, and the [Ca++] in perfusate, were significantly associated with delayed graft function (DGF) after the transplant. Biopsy information was not predictive of DGF. The addition of PGE1 to perfusate improved MP characteristics, reduced the release of [Ca++] into perfusate, and ameliorated mitochondrial ischemic injury in transmission EM images. Early graft function was improved in the presence of PGE1+MP, compared with function in the presence of other pharmacologic agents or CS alone. CONCLUSIONS: MP is associated with improved early and long term renal function. Moreover, PGE1 augments MP in improving graft function. The combination of MP+PGE1 may be important in optimizing the ability to use extended donor criteria kidneys and, thereby, improve the overall efficiency of cadaveric renal transplantation.

Histological methods to determine blood flow distribution with fluorescent microspheres.

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lee's stain or Richardson's stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Wound healing management: enhancing patient outcomes and reducing costs.

Biomedical technology as applied to wound healing management allows specific evaluations of the oxygen-related pathophysiology of non-healing wounds. In many of these cases the use of transcutaneous oxygen mapping of the skin and hyperbaric oxygen (HBO) therapy as an adjunctive treatment for non-healing wounds speeds the healing process. While HBO treatment has remained a covered service for hospital-based care, only recently have treatment algorithms for its application in an outpatient setting been available. This technological advancement has also been a factor in the development of cost effective wound healing centers (WHC) in community hospitals. Better outcomes for many chronic wounds are achieved by combining a multidisciplinary team approach using advanced technologies. In this article the case of a soft-tissue radiation necrosis ulceration of the leg successfully treated with adjunctive HBO is presented. In this example, a reduction in patient charges of greater than 30% was achieved as compared to costs associated with traditional surgical/hospital management of the condition.

Precipitation of dialysis dementia by deferoxamine treatment of aluminum-related bone disease.

Five patients with chronic renal failure, complicated by bone aluminum toxicity, were treated with deferoxamine (DFO). This treatment appeared to precipitate dialysis dementia, which was fatal in three patients. In two patients, continuous treatment with lower doses of DFO was possible. The development of dialysis dementia in chronic renal failure patients with very high serum aluminum levels may be a complication of DFO treatment.

Mechanisms of cimetidine protection following thermal injury.

Experimental studies have shown that treatment with cimetidine within 30 minutes of severe thermal injury decreases resuscitative fluid volume requirements by 70 percent. In treated animals, marked hemodynamic improvement was shown as compared with the untreated animals. Administration of the histamine (H2)-receptor antagonist ranitidine was not effective in reducing resuscitative fluid volume, leading to the hypothesis that cimetidine acts via predominantly non-H2-receptor antagonist mechanisms. Evidence is presented for a multifactorial mechanism by which cimetidine offers protection from burn shock. The mechanisms under investigation include inhibition of the hepatic cytochrome P-450 enzymes, cimetidine/copper scavenging of oxygen free radicals, inhibition of thromboxane synthesis, and imidazole buffering capacity. Acting via one or all of these mechanisms, cimetidine may protect against the vascular permeability changes and circulatory collapse that can follow severe thermal injury.

The acridine orange fluorochrome microassay: a new technique for quantitation of neutrophil function in burned patients.

A new assay for determination of neutrophil bacterial killing and phagocytosis is presented. The acridine orange (AO) fluorochrome microassay is a simple, reliable technique for assessing polymorphonuclear (PMN) function. It requires small amounts of blood and provides a rapid and reproducible quantitation of neutrophil activity. Using this technique, bacterial killing and phagocytosis were assessed in a group of five severely burned patients admitted to the Medical College of Virginia Burn Unit. All patients studied demonstrated a significant decrease in bacterial killing at some point during their clinical course. The AO assay was found to be a reliable and effective means of quantitating PMN bacterial phagocytosis and killing in this group of burned patients.

Cimetidine therapy for burn shock: a quantitative assessment.

Optimal cardiac output (CO) resuscitation for severely burned guinea pigs is obtained with intravenous volumes of lactated Ringer's (LR) calculated at 4 cc/kg/%burn/24 hr. When one half this volume of LR is given (2 cc/kg/%burn/24 hr) CO is significantly (p less than 0.05) reduced at 2, 4, and 8 hours after injury. When early postburn cimetidine therapy (0.5 hours after injury) is added to only 1 cc/kg/%burn/24 hr LR, CO is significantly elevated for the same time periods and is not significantly different from CO values of LR at 4 cc/kg/%burn/24 hr for the first 24 hours after injury. However, postburn cimetidine therapy delayed until 1 hour after burn injury did not improve CO compared to treatment with LR at 2 cc/kg/%burn/24 hr. These observations suggest that early postburn cimetidine therapy administered within 1/2-hour of severe scald injury will result in significant CO improvement while simultaneously reducing resuscitative fluid volume requirements by as much as 70% for the first 24 hours after injury.

Mechanisms of burn shock protection after severe scald injury by cold-water treatment.

Following a 3-second subxiphoid immersion of shaved, unresuscitated guinea pigs in 100 degrees C water, cardiac output (CO), mean systemic blood pressure (BP), total peripheral resistance (TPR), hematocrit (HCT), serum histamine (SH), and serum lactate (SL) were measured in untreated and cold-water treated animals up to 24 hours after injury. Animals receiving cold-water treatment (CWT) were immersed in 15 degrees C water for 15 minutes immediately after scald injury. CWT significantly (p less than 0.05) reduced SH and SL for up to 8 and 24 hours, respectively, after injury compared to untreated injured animals. HCT of CWT animals remained significantly lower than that of untreated animals for the first 8 hours after injury. However, CWT-animal HCT was not significantly different from control-animal HCT for this same period. CWT-animal BP was significantly greater than untreated-animal BP for the first 8 hours after injury. CWT- and untreated-animal TPR progressively rose after injury; however, at 24 hours after injury CWT-animal TPR was significantly reduced to 87% +/- 18.0 of preinjury values while untreated-animal TPR was maximally elevated to 170% +/- 10.0 of preinjury values. At 4 hours after injury CO of both untreated and SWT animals was significantly depressed at 56% +/- 3.0 and 50% +/- 10.0 of preinjury values, respectively. However, by 24 hours after injury untreated-animal CO remained depressed at 52% +/- 3.0 while CWT-animal CO was significantly improved at 92% +/- 19.0 of preinjury values. These studies document a beneficial hemodynamic response of severely burned animals after CWT and further verify the phenomenon of cold inhibition of burn wound tissue histamine release after severe scald injury. A correlation between decreased serum histamine and lactate levels and improved cardiovascular function following severe scald injury and CWT is also suggested.

Histamine-mediated delayed permeability response after scald burn inhibited by cimetidine or cold-water treatment.

Scald injury to one ear of the hairless mouse induced significant (P < .05) delayed edema formation in remote, uninjured skin. This remote edema formation was completely inhibited by immediate cold-water treatment of the scalded ear. Cold-water treatment significantly reduced histamine loss from the scalded ear, and the edema-inhibiting effect of the treatment could be mimicked by treating the animal prior to injury with the H2-histamine receptor antagonist cimetidine or a drug that causes histamine depletion. These observations suggest (i) that a histamine-mediated, delayed permeability response occurs after thermal injury that causes remote edema formation and (ii) that one mechanism of remote edema inhibition by cold-water treatment is the prevention of histamine release from thermally injured tissues.

In vivo microcirculation of a scald burn and the progression of postburn dermal ischemia.

The homozygous hairless mouse ear provides a reproducible model for the study of the microcirculatory changes of the burn wound during and following a scald burn injury. This model has allowed us to correlate the dynamic changes of the microcirculation to progressive zones of injury, which show an approximate tenfold increase in the area of complete capillary occlusion during the first 48 hours after injury. Platelet thromboembolism appears to be the major factor causing this progression of postburn dermal ischemia. Edema (increased skin water content) was greatest in the burned ear at 6 hours after the burn (20 percent greater than control values); edema of the unburned, contralateral ear was significant at 2 hours after the burn (9 percent greater than control values).