Isolated Perfusion Research Articles

Enhanced cytotoxicity of imidacloprid by biotransformation in isolated hepatocytes and perfused rat liver

Imidacloprid (IMD) is a neonicotinoid insecticide widely used in crops, pets, and on farm animals for pest control, which can cause hepatotoxicity in animals and humans. In a previous study using isolated rat liver mitochondria, we observed that IMD inhibited the activity of FoF1-ATP synthase. The aim of this study was to evaluate the effects of IMD on rat isolated hepatocytes and perfused rat liver, besides the influence of its biotransformation on the toxicological potential. For the latter goal, rats were pretreated with dexamethasone or phenobarbital, two classical cytochrome P-450 stimulators, before hepatocytes isolation or liver perfusion. IMD (150 and 200 μM) reduced state 3 mitochondrial respiration in digitonin-permeabilized cells that were energized with glutamate plus malate but did not dissipate the mitochondrial membrane potential. In intact (non-permeabilized) hepatocytes, the intracellular ATP concentration and cell viability were reduced when high IMD concentrations were used (1.5–3.0 mM), and only in cells isolated from dexamethasone-pretreated rats, revealing that IMD biotransformation increases its toxicity and that IMD itself affects isolated mitochondria or mitochondria in permeabilized hepatocytes in concentrations that do not affect mitochondrial function in intact hepatocytes. Coherently, in the prefused liver, IMD (150 and 250 μM) inhibited gluconeogenesis from alanine, but without affecting oxygen consumption and urea production, indicating that such effect was not of mitochondrial origin. The gluconeogenesis inhibition was incomplete and occurred only when the rats were pretreated with phenobarbital, signs that IMD biotransformation was involved in the observed effect. Our findings reveal that changes in hepatic energy metabolism may be acutely implicated in the hepatotoxicity of IMD only when animals and humans are exposed to high levels of this compound, and that IMD metabolites seem to be the main cause for its toxicity.

Technique of Complete Heart Isolation with Continuous Cardiac Perfusion During Cardiopulmonary Bypass: New Opportunities for Gene Therapy

Cardiopulmonary bypass (CPB) featuring complete heart isolation and continuous cardiac perfusion is a very promising approach for solving the problem of efficient gene delivery. In the technique presented here, separate pumps are used for the systemic and cardiac circuits. This system permits continuous isolated arrested heart perfusion through optimizing a number of delivery parameters including temperature, flow rate, driving pressure, ionic composition, and exposure time to the cardiac vessels. During complete cardiac isolation, the blood vector concentration trended from 11.51 ± 1.73 log genome copies (GCs)/cm3 to 9.84 ± 1.65 log GC/cm3 (p > .05). Despite restructuring a very high concentration to the heart, GCs were detectable in the systemic circuit. These values over time were near negligible by comparison but detectable 1.66 ± .26 during 20 minutes of recirculation and did not change (p > .05). After the completion of the recirculation interval and subsequent washing procedure, the initial systemic blood vector GC concentration slightly increased to 2.08 ± .38 log GCs/cm3 (p > .05). During the recirculation period, we supported flow via the cardiac circuit around 300 mL/min. In this technique of heart isolation with continuous cardiac perfusion, >99% of the vector remains in coronary circulation during recirculation period. The animal’s non recirculation blood, or that in the system, was routinely tested during and after recirculation to contain much less than 1% of the original dose obtained via logging concentration of therapeutic over time. All of the sheep in this group recovered from anesthesia and received critical postoperative care, including all organ function, in the first 24–36 hours. Twenty-one sheep (84%) survived to euthanasia at 12 weeks. Average CPB time was 107 ± 19.0 minutes and cross-clamp time was 49 ± 7.9 minutes. This technology readily provides multiple pass recirculation of genes through the heart with minimal side effects of collateral expression of other organs.

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart.

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap - previously cast to match the average size of the rat heart - is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 µL/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

Minimizing Systemic Leakage of Cisplatin during Percutaneous Isolated Pancreas Perfusion Chemotherapy: A Pilot Study.

To evaluate the feasibility of percutaneous isolated pancreas perfusion (PIPP) by using a pig model. All experiments were approved by the institutional Animal Experiment Ethics Committee. Fifteen pigs were assigned to five groups, and PIPP was performed. Angiographic and dye injection studies were performed to confirm the patency of the PIPP system (group 1). Blood that contained cisplatin (1.5 mg per kilogram of body weight) in an extracorporeal circuit was circulated through the pancreas at three infusion rates (40, 60, and 80 mL/min) to determine the optimal infusion rate in terms of safety and pharmacologic effectiveness (groups 2, 3, and 4, respectively). Chronological laboratory data and histologic findings were assessed in group 5, which received the optimal infusion rate. Maximum platinum concentration (Cmax) and area under the platinum concentration-time curve were compared by using the Kruskal-Wallis and Mann-Whitney U tests. Angiography and dye injection confirmed the patency of the PIPP system. Histopathologic examinations showed no abnormalities in the pancreas or other organs at a 40 mL/min infusion rate of cisplatin. However, edematous changes in the pancreas were observed at higher infusion rates. The pharmacologic effectiveness did not differ significantly among groups; therefore, the optimal infusion rate of 40 mL/min was selected. The median pancreatic-to-systemic exposure ratios were 71.8 for Cmax and 54.8 for the area under the curve. All laboratory data remained normal or returned to pretreatment levels within 1 week. PIPP at a 40 mL/min infusion rate appears to be safe and feasible for perfusion of the pancreas.

Inhibition of carotid sinus baroreflex in neonatal rats exposed to chronic intermittent hypobaric hypoxia.

Chronic intermittent hypobaric hypoxia (CIHH) facilitates carotid sinus baroreflex (CSB) in adult rats, but the effect of CIHH on CSB in young rats is not known. The purpose of present study was to investigate the effect of CIHH on CSB in the young rat treated with CIHH from neonatal age, and the role of nitric oxide (NO) and Ca²⁺ in the effect of CIHH. Neonatal male Sprague-Dawley rats were randomly divided into three groups: 42-day CIHH treatment group (CIHH42), 56-day CIHH treatment group (CIHH56), and an age-matched control group (control). CIHH neonatal rats with the maternal rats were exposed to a simulated high-altitude hypoxia in a hypobaric chamber mimicking 5,000-m altitude (O₂ at 11.1%) for 42 or 56 days, 6 h per day, respectively. Isolated carotid sinus perfusion technique was used to test CSB of the rats. After 42-day and 56-day CIHH exposure, the CSB of the rats was inhibited significantly, manifesting as decrease of peak slope (PS) and reflex decrease (RD), and increase of threshold pressure (TP), equilibrium pressure (EP) and saturation pressure (SP). This inhibitory effect was canceled by L-type calcium channel activator Bay K 8644, but not by nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The data showed that CIHH inhibited CSB in anesthetized young rats through blocking L-type calcium channels in carotid sinus baroreceptor.

The role of hyperthermic intraperitoneal chemotherapy (HIPEC) and isolated perfusion (ILP) interventions in sarcoma

Locally advanced sarcomas in the extremity and in the retroperitoneum/abdominal cavity (peritoneal sarcomatosis, PS) can be managed administering chemotherapy locally using isolated limb perfusion (ILP) and hyperthermic intraperitoneal chemotherapy (HIPEC), respectively. In this review, the authors discuss the pros and cons of the use of these locoregional therapies in locally advanced soft tissue sarcoma, with a view to establishing their role in the multidisciplinary approach to these difficult diseases.

A Novel Vascular Homing Peptide Strategy to Selectively Enhance Pulmonary Drug Efficacy in Pulmonary Arterial Hypertension

A major limitation in the pharmacological treatment of pulmonary arterial hypertension (PAH) is the lack of pulmonary vascular selectivity. Recent studies have identified a tissue-penetrating homing peptide, CARSKNKDC (CAR), which specifically homes to hypertensive pulmonary arteries but not to normal pulmonary vessels or other tissues. Some tissue-penetrating vascular homing peptides have a unique ability to facilitate transport of co-administered drugs into the targeted cells/tissues without requiring physical conjugation of the drug to the peptide (bystander effect). We tested the hypothesis that co-administered CAR would selectively enhance the pulmonary vascular effects of i.v. vasodilators in Sugen5416/hypoxia/normoxia-exposed PAH rats. Systemically administered CAR was predominantly detected in cells of remodeled pulmonary arteries. Intravenously co-administered CAR enhanced pulmonary, but not systemic, effects of the vasodilators, fasudil and imatinib, in PAH rats. CAR increased lung tissue imatinib concentration in isolated PAH lungs without increasing pulmonary vascular permeability. Sublingual CAR was also effective in selectively enhancing the pulmonary vasodilation by imatinib and sildenafil. Our results suggest a new paradigm in the treatment of PAH, using an i.v./sublingual tissue-penetrating homing peptide to selectively augment pulmonary vascular effects of nonselective drugs without the potentially problematic conjugation process. CAR may be particularly useful as an add-on therapy to selectively enhance the pulmonary vascular efficacy of any ongoing drug treatment in patients with PAH.

EFFECTS OF CHRONIC INTERMITTENT HYPOBARIC HYPOXIA ON CAROTID SINUS BAROREFLEX IN DEVELOPING RATS

ObjectivesThe purpose of present study was to investigate the effect of CIHH on CSB and the underlying mechanism in developing rat.MethodsNeonatal male Sprague-Dawley rats were randomly divided into four groups:...

Isolated Hepatic Perfusion with High-Dose Melphalan Results in Immediate Alterations in Tumor Gene Expression in Patients with Metastatic Ocular Melanoma

Patients with ocular melanoma liver metastases have a poor prognosis, treatment options are limited, and median survival is less than 1 year. In this study, we characterized the early molecular changes that occur in tumors immediately after vascular isolation perfusion with melphalan with hyperthermia in patients with hepatic metastases from ocular melanoma. Patients underwent treatment on a clinical trial using a 60-min hyperthermic isolated hepatic perfusion (IHP) with melphalan. Microarray analysis was performed in 28 tumor samples obtained intraoperatively of which 12 were pre- and 16 were post-IHP. Various statistical analyses were performed to identify differentially expressed genes and gene categories between the groups. Median survival of 17 treated patients was 11.9 months. Unsupervised hierarchical clustering of all tumors resulted in separation of pre and post-IHP samples into two distinct groups. Analysis of genes showed that the Ras GTPase activator, ecotropic viral integration site 5 (EVI5), and several other melanoma-associated genes were overexpressed in pre-IHP tumors. In post-IHP samples the overexpression of a DNA replication associated gene, replication factor C (RFC5), was significantly associated with shortened survival (P < 0.003). Other major gene ontology categories identified in the post-IHP tumor samples were DNA-directed RNA polymerase activity and chromatin remodeling, both important categories involved in DNA replication and repair. These results demonstrate that acute changes in gene expression patterns occur in tumors immediately after treatment with melphalan administered via hyperthermic IHP. Rapid activation of DNA synthesis and repair pathways may be a mechanism of acquired tumor resistance in patients with ocular melanoma.

Development of Isolated Hepatic Perfusion via the Operative and Percutaneous Techniques for Patients With Isolated and Unresectable Liver Metastases

The development of effective treatment strategies to provide durable control of isolated diffuse metastases to the liver is a major challenge in clinical oncology. The number of patients afflicted annually with isolated liver metastases is considerable; of the 156,000 patients diagnosed with colorectal cancer in 2009, it is estimated that up to 40,000 will develop liver metastases as the sole or dominant site of disease progression and of whom only 10% to 20% will have tumors amenable to resection. Patients with neuroendocrine cancers and ocular melanoma will frequently develop isolated and diffuse liver metastases as the dominant mode of tumor metastasis and, although less frequent, patients with other types of cancers such as cutaneous melanoma or breast cancer can occasionally develop isolated diffuse metastases to the liver.Isolated hepatic perfusion and percutaneous hepatic perfusion are under clinical evaluation for patients with diffuse isolated liver metastases from various solid organ cancers. Both share the advantages of intensifying treatment to the cancer-burdened organ of the body to improve efficacy and limit unnecessary systemic toxicity by selectively delivering high-dose therapeutic agents into the hepatic arterial system from which established metastases derive their predominant blood supply. In this article, we will review the history and early clinical development of isolated perfusion, the techniques of isolated hepatic perfusion and percutaneous hepatic perfusion, the current clinical results with isolation perfusion, and discuss the potential future clinical use of these approaches.

The treatment of limb soft tissue sarcoma with TNF-alfa and pegylated liposomal doxorubicin (PLD): Phase I-II study

10583 Background: Hyperthermic isolation perfusion (HILP) has been employed for STS as limb salvage procedure. Fifty-eight patients affected with unresectable STS have been treated with doxorubicin and 1 mg of TNF-alfa. The CR and PR were 34% and 48% respectively, with a limb salvage rate of 84%. Unfortunately, the limb toxicity was quite high, therefore we decided to employ PLD. Methods: Fourteen patients have been treated with escalating dose of liposomal doxorubicin, starting from 10 mg/L of limb volume up to 18 mg/L, at mean muscle temperature of 41.5C. During HILP blood samples were taken from both systemic and perfusional circulation at 10 minutes intervals. At the end of HILP a tumor and muscle biopsy was carried out in order to evaluate the doxorubicin concentration. A total of 18 STS patients have been treated with PLD. All the patients were monitored in terms of grade of limb toxicity (Wieberdink classification) and systemic toxicity (WHO classification). Eleven patients have been later treated ...

Direct evidence for rapid and selective induction of tumor neovascular permeability by tumor necrosis factor and a novel derivative, colloidal gold bound tumor necrosis factor

Tumor necrosis factor (TNF) causes regression of advanced cancers when used in isolation perfusion with melphalan; evidence suggests these effects are mediated via selective yet uncharacterized actions on tumor neovasculature. A novel derivative, colloidal gold bound TNF (cAu-TNF) has been shown to have similar antitumor effects as native TNF with less systemic toxicity in mice. These studies were done to determine their effects on tumor neovasculature, using in vivo video microscopy. Female C57BL/6 mice bearing 20 mm(2) MC38 or LLC tumors that are TNF sensitive and resistant tumors, respectively, had dorsal skinfold chambers implanted. The rate of interstitial accumulation of Texas red fluorescently labeled albumin in tumor and normal vasculature was measured after intravenous TNF, cAu-TNF or PBS. Changes in interstitial fluorescent intensity over time were quantified as a reflection of alterations in vascular permeability. MC38 bearing mice treated with TNF or cAu-TNF demonstrated a rapid, selective and significant increase in tracer accumulation in areas of neovasculature compared to those of normal vasculature. Experiments in LLC tumor bearing mice showed similar results. Monoclonal antibody against tissue factor partially abrogated the effects of TNF on MC38 neovasculature. These data provide direct evidence that TNF and cAu-TNF selectively and rapidly alter permeability in tumor neovasculature; a phenomenon that may be exploited to enhance selective delivery of chemotherapeutics to tumor.

Gene Therapy in In Vivo Isolated Perfusion Models

Locoregional administration of a genetic construct by means of in vivo, in situ isolated perfusion (IP) of a target organ or extremity is a method that may increase in vivo efficacy. Vascular isolation and perfusion minimizes systemic exposure and thereby reduces unwanted side effects. Isolated hepatic perfusion (IHP) is the most extensively studied IP model, especially in gene therapy protocols for inborn errors of metabolism. To achieve stable transduction most frequently retroviruses are used in IHP. IHP is combined with hepatectomy or vascular ligation of liver lobes to induce liver regeneration increasing transduction efficacy. When adenoviruses are used in IHP high transduction percentages of hepatocytes can be achieved without significant toxicity. In tumor models adenoviral IHP has been performed, but has not been very successful up till now. Isolated limb perfusion (ILP) is a promising treatment modality in pre-clinical cancer gene therapy studies. After ILP a homogeneous distribution of transduced cells was demonstrated especially at the viable rim of the tumor and around tumor associated vessels. Moreover complete tumor responses have been observed. Isolated pulmonary perfusion (IPP) results in selective expression in the perfused lung and the duration of expression is longer than after systemic administration. In rats a significant decrease of tumor nodules upon IPP can be achieved. Furthermore other less studied perfusion models are discussed: isolated kidney perfusion (IKP), isolated spleen perfusion (ISP) and isolated cardiac perfusion (ICP). IP is a methodology that delivers vectors highly selectively, with a long exposure time and high concentrations at the target side. This results in higher transduction rates and thereby may improve therapeutic effects.

Augmentation of endothelial cell monolayer permeability by hyperthermia but not tumor necrosis factor: Evidence for disruption of vascular integrity via VE-cadherin down-regulation

Following isolated limb perfusion (ILP) with hyperthermia (H T), TNF and melphalan, there is immediate tumor softening secondary to augmentation of capillary leak in the tumor neovasculature. TNF can induce vascular permeability but is always used with HT during ILP and the contribution of the latter on permeability is not known. This study characterizes the effects of HT and TNF on vascular permeability in vitro. Permeability across confluent human umbilical vein endothelial cells exposed to HT (40 degrees C) with or without 0.1-1000 ng/ml TNF was assessed by quantitating flux of albumin bound Evan's Blue dye from the upper to lower chamber. Immunofluorescent staining for VE-cadherin and F-actin was performed after human umbilical vein endothelial cells (hUVECs) were exposed to these conditions. HT induced a significant and reversible increase in permeability compared to untreated hUVECs (p<0.001) whereas barrier function was not altered by TNF. Untreated hUVECs had uniform cell surface staining for VE-cadherin, the primary endothelial intercellular adhesion molecule, with colocalization of F-actin cytoskeletal elements. HT resulted in a marked decrease in VE-cadherin staining and contraction of F-actin at sites of endothelial cell-cell separation. These data demonstrate that under conditions relevant to those used in ILP, HT but not TNF contributes to a rapid and reversible change in endothelial cell permeability in association with a down regulation of VE-cadherin. These data support the use of HT in isolation perfusion and demonstrate a novel mechanism for alterations in microvascular permeability by HT.

Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor–dependent mechanism: relationship between the procoagulant and permeability effects of TNF

Tumor necrosis factor (TNF) has marked effects on permeability and procoagulant activity on tumor-associated neovasculature when used in isolation perfusion, the latter effect primarily mediated via induction of cell surface expression of tissue factor (TF) on endothelial tissue. However, the cellular events that result in rapid alterations in endothelial cell (EC) permeability after intravascular TNF administration in isolation perfusion are not well characterized. We demonstrate that short exposure intervals to TNF induces TF expression on ECs but has no effect on permeability as assessed by flux of Evans blue-bound albumin across confluent EC monolayers using a 2-compartment model under basal culture conditions. However, a rapid and significant increase in EC permeability occurred with TNF in the presence of factor VIII-deficient plasma. Permeability was induced only with luminal versus abluminal TNF exposure and was blocked by antithrombin III, TF pathway inhibitor, or anti-TF antibody cotreatment. These data indicate that EC surface expression of TF and extrinsic clotting factors are critical in augmenting capillary leak following intravascular TNF administration. Alterations in permeability were associated with intercellular gap formation at sites of down-regulation of vascular endothelial (VE)-cadherin expression, the primary endothelial intercellular adhesion molecule, and intracellular contraction and alignment of F-actin cytoskeletal elements. Rapid induction of TF by TNF may be the primary EC response that results in alterations in permeability and procoagulant activity observed following intravascular TNF administration in isolation perfusion.

Isolation perfusion of the liver.

Thousands of patients die annually from unresectable metastatic or primary hepatic cancers confined to liver. Isolated hepatic perfusion (IHP) is a regional treatment strategy in which the vascular supply to the liver is isolated and perfused with a therapeutic regimen using an extracorporeal circuit consisting of a reservoir, heat exchanger, and oxygenator. Drug doses that would cause severe toxicities if delivered systemically can be confined to the liver by isolated hepatic perfusion, resulting in the ability to intensify treatment to the cancer-burdened region of the body. Agents and mechanisms commonly used in IHP include melphaIan, hyperthermia, and tumor necrosis factor. IHP appears to be efficacious for patients with advanced disease, as reflected by large tumor size, high number of lesions, or significant overall tumor burden in the liver. In addition, responses are observed for patients whose cancer is refractory to systemic and hepatic arterial infusion chemotherapy. Recent clinical trials have demonstrated that IHP has anti-tumor efficacy against primary hepatic neoplasms and metastases from various primary tumors, such as colorectal carcinoma, ocular melanoma, and neuroendocrine tumors. Current studies demonstrate that combining hepatic arterial infusion with floxuridine after IHP for patients with colorectal cancer metastases is associated with significant and durable response rates. Continued clinical evaluation is warranted for the use of IHP in the treatment of unresectable liver metastases.

Angiotensin-converting enzyme inhibitor preserves p21 and endothelial nitric oxide synthase expression in monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is associated with structural changes in the pulmonary vasculature characterized by the proliferation of cellular components of the vessels. ACE inhibitor (ACEI) may have beneficial effects in treating PAH, but its precise mechanism of action in the remodeling process is unclear. p21 is a cyclin-dependent kinase inhibitor that may have a protective role in this process by inhibiting cellular proliferation. Endothelial nitric oxide synthase (eNOS) has also been shown to be protective by its vasodilatory effect. Therefore, we investigated whether expression of p21 and eNOS was modulated by ACEI treatment in a rat model. Monocrotaline (MCT) was administered to 2 groups of Sprague-Dawley rats fed a high-cholesterol diet, ie, one group received MCT concomitantly with enalapril treatment (MCT(+)/ACEI(+) rats), and the other group did not receive enalapril (MCT(+)/ACEI(-) rats). After 5 weeks, MRI showed right ventricular hypertrophy in MCT(+)/ACEI(-) rats. MCT(+)/ACEI(+) rats showed a preserved right ventricular morphology. Isolated pulmonary perfusion studies showed that ACEI significantly upregulated NO production, as measured by nitrite levels. Addition of N-methyl-D-glucamine dithiocarbamate-Fe solution, an NO-trapping agent, reversed the basal vasodilatory effect of ACEI in the pulmonary vasculature. Immunoblot analysis showed decreased p21 and eNOS expression in the lung in MCT(+)/ACEI(-) rats, whereas their expression was preserved with enalapril treatment. ACEI suppresses the development of MCT-induced PAH in rats. The mechanism of action might involve the preservation of p21 and eNOS expression. Both p21 and endothelium-derived NO appear to have protective roles in the development of PAH.

Continuous leakage measurement during hyperthermic isolated limb perfusion.

Continuous measurement of perfusate leakage into the systemic circulation is of the utmost importance and can be performed with the help of radioactive tracers. The purpose of this study was to assess changes in the perfusion leakage rate between two periods: 1977-1990 and 1991-2000, and to determine the factors responsible for these changes. During the 1991-2000 period, 119 patients underwent HILP mainly for locally recurrent melanoma or locally advanced soft tissue sarcoma. HILP was performed with melphalan (33%) or in combination with TNFalpha (65%). There were 67 iliacal, 12 femoral, 25 popliteal, and 15 axillary perfusions performed. Leakage into the systemic circulation was monitored continuously with the help of 131I-albumin and a stationary scintillation detector placed above the heart. The median maximum leakage was 2.7% (range 0%-21%) which is significantly less than the previous period (1977-1990) where leakage of 8% (range 0%-30%) was reported (P < .05). A statistical difference in leakage was detected among perfusion locations where the iliac and femoral vessels showed more leakage than the axillary and popliteal vessels (P < .05). Furthermore, there appeared to be significantly less leakage when TNFalpha was used than when melphalan was the sole drug (P < .05). Nowadays leakage from isolated perfusions into the systemic circulation is further minimized compared with the days when melphalan was the sole drug used. Increased awareness about TNFalpha leakage, continuous external monitoring with 131I-albumin as the main isotope, flow rate regulation in the perfusion circuit, and regulation of the patient's systemic blood pressure have all been major contributors to this improvement.

Ventilatory responses to specific CNS hypoxia in sleeping dogs.

Our study was concerned with the effect of brain hypoxia on cardiorespiratory control in the sleeping dog. Eleven unanesthetized dogs were studied; seven were prepared for vascular isolation and extracorporeal perfusion of the carotid body to assess the effects of systemic [and, therefore, central nervous system (CNS)] hypoxia (arterial PO(2) = 52, 45, and 38 Torr) in the presence of a normocapnic, normoxic, and normohydric carotid body during non-rapid eye movement sleep. A lack of ventilatory response to systemic boluses of sodium cyanide during carotid body perfusion demonstrated isolation of the perfused carotid body and lack of other significant peripheral chemosensitivity. Four additional dogs were carotid body denervated and exposed to whole body hypoxia for comparison. In the sleeping dog with an intact and perfused carotid body exposed to specific CNS hypoxia, we found the following. 1) CNS hypoxia for 5-25 min resulted in modest but significant hyperventilation and hypocapnia (minute ventilation increased 29 +/- 7% at arterial PO(2) = 38 Torr); carotid body-denervated dogs showed no ventilatory response to hypoxia. 2) The hyperventilation was caused by increased breathing frequency. 3) The hyperventilatory response developed rapidly (<30 s). 4) Most dogs maintained hyperventilation for up to 25 min of hypoxic exposure. 5) There were no significant changes in blood pressure or heart rate. We conclude that specific CNS hypoxia, in the presence of an intact carotid body maintained normoxic and normocapnic, does not depress and usually stimulates breathing during non-rapid eye movement sleep. The rapidity of the response suggests a chemoreflex meditated by hypoxia-sensitive respiratory-related neurons in the CNS.

EFFECTS OF HYPERTHERMIA AND TUMOUR NECROSIS FACTOR ON INFLAMMATORY CYTOKINE SECRETION AND PROCOAGULANT ACTIVITY IN ENDOTHELIAL CELLS

The application of hyperthermia (HT) and tumour necrosis factor alpha (TNF) in isolation perfusion of the limb or liver results in regression of advanced cancers confined to these regions of the body in most patients and are thought to exert anti-tumour effects primarily on tumour neovasculature. However, the individual contribution of either treatment factor on endothelial cells (EC) are not known. In this study, we investigated the in vitro effects of moderate and severe HT on human umbilical vein EC (HUVEC) with and without TNF in clinically relevant doses. HUVEC were exposed to normothermia (37°C) or moderate (39°C) and severe (41°C) HT for 90 or 180min with or without TNF (1μg/ml). Cell viability, cytokine secretion (IL-6, IL-8, VEGF, ICAM-1, VCAM-1, RANTES, E-selectin, P-selectin, L-selectin, and PECAM-1), and induction of procoagulant activity as reflected in tissue factor (TF) production were assessed at the end of the treatment period and at several time points thereafter. Neither HT nor TNF exerted significant cytotoxic effects on EC at the doses and temperatures used. HT resulted in increased production of PECAM-1 with little or no additional effect when combined with TNF. TNF caused increased secretion of IL-6, IL-8, ICAM-1, and VCAM-1 with little or no additional effect from HT. Increased E-selectin and RANTES levels were observed with TNF and HT only at 24h after treatment. HT and TNF had mainly antagonistic effects on VEGF secretion with HT causing primarily decreased production and TNF causing increased VEGF secretion under all temperatures. Most notably, there was a rapid, prolonged and synergistic peak increase in procoagulant activity when TNF and HT were used in combination compared to TNF or HT treatment alone. These results indicate that TNF and HT exert primarily independent effects on inflammatory cytokine production in EC but synergistically increase procoagulant activity as reflected in TF production. These data provide a possible mechanism for the thrombotic effects in tumour neovasculature seen following isolation perfusion with these agents and provide a rationale for their combined use in this treatment setting.