A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy.

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumour's course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.

Cryopreservation with dimethyl sulfoxide sustains partially the biological function of osteochondral tissue.

The clinical routine use of bone allograft transplants dates back to the discovery that grafts devitalized by freezing bear a reduced antigenicity. Graft failures, caused by a host versus graft reaction, however, remain a clinical problem. Previous investigations on pancreatic islet allografts revealed improved survival and biological function when fast cryopreservation (-70 degrees C/min) was performed in the presence of dimethyl sulfoxide (DMSO). The aim of this study was to determine the effect of fast freezing using DMSO on the biological function of osteochondral tissues. Organ culture was performed with neonatal femora of mice, untreated, rapidly frozen (-70 degrees C/min) with DMSO, or frozen without DMSO. After the culture, tissue morphology, cellular proliferation, osteoblast function, osteoclasts, and the presence of antigen-presenting cells were investigated. In untreated control femora histology appeared normal and proliferating and collagen-synthesizing osteoblasts, osteoclasts, and B-cells and macrophages were present. In frozen femora (with and without DMSO) a disintegration of the periosteum and the epiphyseal growth plate were observed and no active osteoblasts could be detected. Osteoclasts were partially detached from the bone surface. Cell proliferation was fully blocked in femora frozen in the absence of DMSO, while freezing in the presence of DMSO preserved cell proliferation in the medullary canal. The proliferating cells do not express epitopes present on the cells of the B-cell or macrophage lineages. Although the biological function of osteoblasts and osteoclasts was lost upon freezing of osteochondral tissue, DMSO included in freezing protocols preserves some residual cell viability which may be of importance for early graft revascularization as has been previously demonstrated by our group.

Development of transplanted fetal bones: differences between isografts and allografts in mice.

Allogeneic bone from bone banks frequently is used when large skeletal defects have to be bridged in orthopaedic surgery. Beside immunologic rejection of the graft, the loss in osteogenic potential caused by bone banking procedures may be a major reason for limited clinical success. Similar problems as described for bone have occurred with cartilage and osteochondral transplants. Improving the properties of allogenic bone so that its biologic activity becomes comparable to autologous bone could be substantially beneficial for the outcome of allograft transplantation. To dissect the steps involved in the integration of a fetal osteochondral graft as it matures to bone, the current study compared the development and biologic function of metatarsals from 18-day-old fetal mice freshly transplanted in three different immunologic settings. Morphologic assessment of (1) isografts and (2) allografts in nonsensitized hosts 12 days after transplantation revealed that the grafts bear an intrinsic potential to develop after transplantation. In allografts in nonsensitized hosts, however, a slight alteration in biologic activity as compared with isografts could be detected already in this early phase after transplantation by in situ hybridization for messenger ribonucleic acids encoding extracellular matrix proteins. (3) In contrast to isografts and allografts in nonsensitized hosts, morphologic features and biologic function of allografts transplanted to presensitized hosts were altered severely.

Dorsal skinfold chamber preparation in mice : studying angiogenesis by intravital microscopy.

Since 1924, when the first transparent chamber model in animals was introduced by Sandison (1), many other chamber models have been described in the literature for studying angiogenesis and microcirculation in a wide variety of neoplastic and nonneoplastic tissues by means of intravital microscopy (for reviews see 2-4). Because angiogenesis is an active and dynamic process, one of the major strengths of chamber models is the possibility of monitoring angiogenesis in vivo continuously up to several weeks with high spatial and temporal resolution. In addition, after the termination of experiments, tissue samples can be excised easily and further examined by various in vitro methods (e.g., histology, immuno-histochemistry, and molecular biology).

The effect of pedicle artery vasospasm on microhemodynamics in anatomically perfused and extended skin flap tissue.

The aim of this study was to evaluate quantitatively the influence of pedicle artery vasospasm on the microcirculation in skin flaps, particularly in the jeopardized extended portions. For this purpose, the hamster island skin flap model was used, which allowed for simultaneous assessment of hemodynamics in both the pedicle artery and the microvasculature of the flap by intravital microscopy. Vasospasm was induced by applying a V3 microvascular clamp for 30 seconds. Clamping resulted in a severe vasospasm, with the artery exhibiting a diameter of 7% +/- 2% (mean +/- standard error) of its original diameter (n = 10; p < 0.01), and with a reduction of total blood flow to the flap of 11% +/- 2% (p < 0.01). Diameter and blood flow recovered gradually to baseline levels after 25 and 15 minutes respectively. During recovery from severe pedicle artery vasospasm (moderate to mild vasospasm), the arterioles in the anatomically perfused flap tissue (n = 38) showed reactive vasodilation (p < 0.01), which was absent in the extended tissue (n = 49; p < 0.01 vs. anatomic). At a pedicle artery vasospasm of 50% of the original diameter, blood flow was restored to normal levels in the anatomically perfused arterioles, but remained below baseline in the extended part (partly p < 0.05 vs. baseline and anatomic). The findings suggest that the development of ischemic necrosis in extended flap portions may be promoted by prolonged, moderate vasospasm, which is well tolerated in the anatomically perfused tissue because of its high capacity for implementing compensatory local regulatory mechanisms.

Adenosine deaminase inhibition attenuates reperfusion low flow and improves graft survival after rat liver transplantation.

BACKGROUND: Low flow or no flow is a prefinal step after reperfusion of hepatic allografts. Adenosine is an intrinsic key regulator of physiological and pathological hepatic blood flow. METHODS: In a model of rat liver transplantation, the effect of donor pretreatment with adenosine deaminase inhibitors (0, 0.1, 1, 10 micromol erythro-9-[2-hydroxy-3-nonyl]adenine) was studied on hepatic interstitial adenosine concentrations, microcirculatory flow, leukocyte adhesion, and graft survival by means of microdialysis sampling, intravital video microscopy, and laser Doppler flowmetry. RESULTS: Donor pretreatment with 1 micromol erythro-9-[2-hydroxy-3-nonyl]adenine increased interstitial adenosine concentrations 5- to 10-fold, for more than 24 hr of cold storage. In LDF studies, mean donor blood flow was increased from 420 +/- 42 perfusion units (PU) to 832 +/- 52 PU and from 475 +/- 79 to 720 +/- 81 PU after reperfusion, and in intravital video microscopy studies from 247 +/- 24 to 281 +/- 39 pl/sec. There was no difference in the number of leukocytes sticking, but a significantly lower percentage of leukocytes rolling (26.1 +/- 1.9 vs. 36.5 +/- 7.5%) along the endothelial wall in the treatment group. Transplant survival after 44 hr cold storage in UW solution was 8/10 in the treatment group and 1/13 in the control group. CONCLUSIONS: Donor pretreatment with erythro-9-[2-hydroxy-3-nonyl]adenine increases survival of critically injured liver grafts. Donor or recipient treatment rather than addition of protectants to cold storage solutions are successful strategies to overcome preservation injury and possibly adverse donor factors.

Role of L-selectin in leukocyte sequestration in lung capillaries in a rabbit model of endotoxemia.

After a variety of pathophysiologic stimuli, neutrophils accumulate in lung capillaries and contribute to the pathogenesis of acute lung injury. Lung neutrophil sequestration has previously been attributed to mechanical retention of stiffened neutrophils, but L-selectin-mediated leukocyte/endothelial interaction may be an essential step. We investigated the effect of the anti-L-selectin antibody HuDreg 200 on leukocyte sequestration and microhemodynamics in alveolar capillaries in a model of acute endotoxemia. We used in vivo fluorescence microscopy to analyze kinetics of fluorescently labeled red and white blood cells in alveolar capillary networks of the rabbit lung. Investigations were performed over 2 h after an intravenous infusion of 0.2 ml/kg body weight (bw) NaCl, 2 mg/kg bw HuDreg 200, 20 microg/kg bw lipopolysaccharide (LPS) of Escherichia coli 0111:B4, or the combination of HuDreg 200 and LPS, respectively. Infusion of LPS induced leukocyte sequestration in alveolar capillaries, which was accompanied by a reduction of alveolar capillary perfusion and functional capillary density. These effects could be completely blocked by pretreatment of animals with HuDreg 200. We conclude that L-selectin-mediated leukocyte/endothelial interaction is a necessary prerequisite for leukocyte sequestration in alveolar capillaries in this model. Impaired alveolar capillary perfusion appeared to result directly from capillary leukocyte sequestration.

Quantitative assessment of angiogenesis and osteogenesis after transplantation of bone: comparison of isograft and allograft bone in mice.

We performed a vital microscopic study in mice bearing dorsal skinfold chambers to characterize microvascular perfusion and leukocyte/endothelium interaction and their effects on elongation and mineralization of neonatal isograft and allograft bone. Isograft (C57/BL to C57/BL) and allograft bone (C57/ BL to BALB/C) revascularized simultaneously. However, vascular perfusion and density were lower in allograft bone than in isograft bone. Leukocyte/endothelium interaction was the same in isograft and allograft bones. Revascularization was not detected in allograft bone transplanted to presensitized recipients. Moreover, in preexisting vessels at the transplantation site, leukocyte/endothelium interaction was altered in allograft bone of presensitized recipients, despite a normal systemic leukocyte count. Femoral growth resulting from thickening of both epiphyses did not differ between experimental groups, however, mineralization occurred in isograft bone only. Isograft bone was histologically intact, allograft bone hypovital and allograft bone in presensitized recipients necrotic 12 days after implantation. Our findings suggest that graft incorporation or rejection is mediated by the microvasculature and that presensitizing of recipients accelerates rejection of allograft bone.

The connective-tissue envelope in revascularisation of patellar tendon grafts.

Free patellar tendon grafts used for the intra-articular replacement of ruptured anterior cruciate ligaments (ACL) lack perfusion at the time of implantation. The central core of the graft undergoes a process of ischaemic necrosis which may result in failure. Early reperfusion of the graft may diminish the extent of this process. We assessed the role of peritendinous connective tissue in the revascularisation of the patellar tendon graft from the day of implantation up to 24 days in a murine model using intravital microscopy. The peritendinous connective-tissue envelope of the graft was either completely removed, partially removed or not stripped before implantation into dorsal skinfold chambers of recipient mice. Initial revascularisation of the grafts with preserved peritendinous connective tissues began after two days. The process was delayed by five to six times in completely stripped patellar tendons (p < 0.05). Only grafts with preserved connective tissues showed high viability whereas those which were completely stripped appeared to be subvital. The presence of peritendinous connective tissues accelerates the revascularisation of free patellar tendon grafts.

Targeting HER-2/neu for active-specific immunotherapy in a mouse model of spontaneous breast cancer.

The identification of tumor-associated antigens has led to increased interest in vaccination strategies to treat and/or prevent cancer. This study examined the feasibility of active-specific immunotherapy against the breast-tumor antigen HER-2/neu using a HER-2/neu transgenic (rNeu-TG) mouse model. rNeu-TG mice develop spontaneous breast tumors after pregnancy, indicating that they fail to mount an effective immune response against rNeu. Allogeneic fibroblasts expressing HER-2/neu were used as a cell-based vaccine. Vaccination induced a rNeu-specific anti-tumor immune response that prevented tumor formation of transplanted breast-tumor cells, and also protected mice from spontaneous tumor formation. Both T-cell-mediated and humoral immune responses were detectable in vaccinated mice. Vaccination also protected tumor-bearing mice from a challenge with cell suspensions isolated from spontaneous tumors, indicating that rNeu-TG mice are not tolerant to rNeu, even after spontaneous tumor formation. However, established spontaneous tumors themselves were never affected. This observation correlated with T-cell infiltrations in the injected but not in the established spontaneous tumor. Thus, allogeneic fibroblasts are efficient vaccine vectors to prime a specific immune response against an over-expressed tumor antigen. Moreover, our results suggest striking differences in the immunological requirements for the rejection of an established vs. a transplanted tumor.

Leukocyte sequestration in pulmonary microvessels and lung injury following systemic complement activation in rabbits.

Inflammatory reactions are associated with sequestration of leukocytes in the lung. Complement activation leads to accumulation of leukocytes in alveolar septa and alveoli, to lung edema and hemorrhage. Although in organs other than the lung leukocytes interact with the vascular endothelium only in postcapillary venules, alveolar capillaries are considered to be the site of leukocyte sequestration in the lung. However, pulmonary venules and arterioles have not been investigated systematically after complement activation so far. A closed thoracic window was implanted in anesthetized rabbits; leukocytes and red blood cells were stained, and the movement of these cells was measured in superficial pulmonary arterioles, venules and alveolar capillaries using fluorescence video microscopy before and 30 and 60 min after infusion of cobra venom factor (CVF). Erythrocyte velocity and macrohemodynamic conditions did not change after CVF infusion and were not different from the sham-treated controls. The number of sticking leukocytes increased significantly compared to baseline and control: by 150% in arterioles and in venules and by 740% in alveolar capillaries within 60 min after CVF infusion. The width of alveolar septa in vivo was significantly enlarged after CVF infusion, indicating interstitial pulmonary edema. At the end of the experiments, myeloperoxidase activity was higher in the CVF group, showing leukocyte sequestration in the whole organ. It is concluded that complement activation by CVF induces leukocyte sequestration in lung arterioles, venules and alveolar capillaries and leads to mild lung injury.

Dosage of inhaled nitric oxide: a simple method for experimental studies.

Few studies on treatment with inhaled nitric oxide (NOi) have been carried out in small laboratory animals yet, since commercially available dosing devices are not appropriate in this setting for technical or financial reasons. The aim of our study was to establish and validate a simple, cost-effective system for the application of NOi in small animals. The system mixes NOi with constant-flow inspiratory gas. A gas blender allows for a mixture of nitrogen, oxygen, and NO dissolved in nitrogen. A formula using the desired inspiratory oxygen fraction and the desired concentration of NOi as independent variables derives a somewhat higher inspiratory oxygen fraction, which is preset using an oximeter. Then the flow of NO in nitrogen is started, lowering the inspiratory oxygen fraction to the initially desired value, thereby adding NOi in the desired concentration. The method was validated by 153 adjustments, covering a variety of oxygen fractions and concentrations of NOi. NOi was measured by chemiluminescence as reference method. A close correlation (R = 0.994) was found, and the regression line was close to the line of identity with y = -0.0994 + 1.048x. No systematic errors could be identified. We conclude that the method described may serve as a simple, cost-effective way to administer NOi to small animals.

Primary tumor size-dependent inhibition of angiogenesis at a secondary site: an intravital microscopic study in mice.

Some primary tumors are capable of suppressing the growth of their metastases by presumably generating antiangiogenic factors such as angiostatin. We hypothesized that the amount of inhibitor(s) released by a tumor increases with tumor growth. We tested this hypothesis by evaluating the relationship between the size of a primary tumor and its ability to inhibit angiogenesis at a secondary site. Furthermore, we characterized the effects of the primary tumor on physiological properties of newly formed vessels at the secondary site. Angiogenesis and physiological properties were measured using intravital microscopy of angiogenic vessels in the gels containing basic fibroblast growth factor placed into cranial windows of immunodeficient mice bearing human prostatic carcinoma (PC-3) in their flank. The PC-3 tumor inhibited angiogenesis in the gels, and surgical resection of tumor reversed this inhibition. The inhibition of angiogenesis 20 days after gel implantation (range, 0-83%) correlated positively (r = 0.625; P < 0.008) with the tumor size on the day of gel implantation (range, 19-980 mm3). The primary tumor also suppressed leukocyte-adhesion in angiogenic vessels, thus helping them evade the immune recognition. These results provide an additional rationale for combining antiangiogenic treatment with local therapies.

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor.

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone-ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone-ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

Noninvasive measurement of regional cerebral blood flow by near-infrared spectroscopy and indocyanine green.

Clinicians lack a practical method for measuring CBF rapidly, repeatedly, and noninvasively at the bedside. A new noninvasive technique for estimation of cerebral hemodynamics by use of near-infrared spectroscopy (NIRS) and an intravenously infused tracer dye is proposed. Kinetics of the infrared tracer indocyanine green were monitored on the intact skull in pigs. According to an algorithm derived from fluorescein flowmetry, a relative blood flow index (BFI) was calculated. Data obtained were compared with cerebral and galeal blood flow values assessed by radioactive microspheres under baseline conditions and during hemorrhagic shock and resuscitation. Blood flow index correlated significantly (rs = 0.814, P < 0.001) with cortical blood flow but not with galeal blood flow (rs = 0.258). However, limits of agreement between BFI and CBF are rather wide (+/- 38.2 +/- 6.4 mL 100 g-1 min-1) and require further studies. Data presented demonstrate that detection of tracer kinetics in the cerebrovasculature by NIRS may serve as valuable tool for the noninvasive estimation of regional CBF. Indocyanine green dilution curves monitored noninvasively on the intact skull by NIRS reflect dye passage through the cerebral, not extracerebral, circulation.

[Effect of humanised anti-L- selectin antibody HuDreg 200 on leukocyte kinetics in pulmonary microcirculation in endotoxinemia]. / Effekt des humanisierten anti-L-Selektin Antikörpers HuDreg 200 auf die Leukozytenkinetik in der pulmonalen Mikrozirkulation bei Endotoxinämie.

In the pulmonary circulation, endotoxemia induces leukocyte/endothelium interaction in pulmonary arterioles and venules. Thus, leukocytes may contribute to the pathogenesis of acute lung injury. In order to investigate, whether this interaction can be inhibited by early blockade of the adhesion cascade, we studied the effect of the humanized anti-L-selectin antibody HuDreg 200 on leukocyte kinetics in pulmonary arterioles and venules following i.v.-infusion of endotoxin. In endotoxemia HuDreg 200 reduces sticking of leukocytes in both pulmonary arterioles and venules. Thus, we were able to demonstrate that early blockade of the adhesion cascade effectively prevents leukocyte accumulation in pulmonary microvessels in endotoxemia. Therefore, HuDreg 200 may exhibit protective effects on the manifestation of leukocyte-mediated acute lung injury.

[The effect of mechanical ventilation, thoracotomy, and one-lung respiration on intrapulmonary perfusion distribution. An animal experimental study]. / Einfluss von Beatmung, Thorakotomie und Ein-Lungen-ventilation auf die intrapulmonale Perfusionsverteilung. Eine tierexperimentelle Studie.

The physiological pattern of regional pulmonary blood flow is mainly determined by the relationship of pulmonary arterial, venous, and alveolar pressures. Changes in alveolar pressure and pulmonary geometry may therefore be expected to influence regional perfusion, which is a key determinant of pulmonary gas exchange. Unilateral thoracotomy is usually performed with the patient in the lateral decubitus position. The present study examined the influence of mechanical factors on regional pulmonary blood flow distribution in rabbits in the lateral decubitus position during normoxia and unilateral hypoxia. METHODS. Anaesthetised white New Zealand rabbits (n = 8) weighing 2200-3900 g (mean = 2860 g) received central venous injections of radioactive microspheres while in the left lateral decubitus position during spontaneous breathing (SB) and during mechanical ventilation (two-lung ventilation, 2LV), under closed (2LVC) and open chest (2LVT) conditions, as well as during unilateral hypoxia of the nondependent lung induced by nitrogen inflation (1LVN) or atelectasis (1LVA). The method used for one-lung ventilation (1LV) has been previously described in detail. Arterial, central venous, and pulmonary arterial pressures were recorded continuously. Lungs were excised, dried in the inflated state, and cut into 16 sagittal slices, which were further divided into lobar components, the lower lobes into center and periphery. The radioactivity of each specimen was measured in a gamma-counter; perfusion of the individual tissue specimens was quantified using the software program MIC III. The Friedman test followed by paired comparisons according to Conover was used for statistical analysis of differences between the experimental phases. Perfusion of central and peripheral parts of isogravitational slices was compared by use of the Wilcoxon matched pairs test. Values are given as means +/- SE; the level of significance was P < 0.05 unless otherwise indicated. RESULTS AND DISCUSSION. Haemodynamic parameters did not differ significantly between the experimental phases (Table 1). Compared to 2LV, a significant increase in venous admixture (P < 0.05) and a corresponding decrease in PaO2 (P < 0.01) were observed during 1LV. This effect was significantly more pronounced during 1LVA as compared to 1LVN (P < 0.01). Since inspiratory pressure was kept constant throughout the experiments, moderate respiratory acidosis developed during both phases of 1LV. Regional perfusion (Qr) of the nondependent lung was slightly reduced during 2LVC compared to SB and 2LVT. One-lung ventilation induced a significant decrease in perfusion of the hypoxic lung (P < 0.001 1LVN, 1LVA vs. SB,2LVC,2LVT). In accordance with the data obtained from blood gas analysis and oximetry, this effect was more pronounced during N2 insufflation than during atelectasis (P < 0.01 1LVN vs. 1LVA). Among the factors that may account for this effect, PaCO2 did not differ significantly between both phases of 1LV. During N2 insufflation PO2 at the hypoxia-sensitive site is lower than during atelectasis, where it equals mixed-versus PO2 (PvO2). The difference in local PO2 is unlikely, however, to have caused the changes in regional perfusion between 1LVN and 1LVA, since PvO2 was as low as 40 mmHg during 1LVA and the pulmonary vascular response to hypoxia has been found to reach its maximum in this PO2 range [2, 11]. Enhanced redistribution of regional perfusion during 1LVN as compared to 1LVA is therefore most likely attributed to differences in alveolar pressure and pulmonary geometry. Apart from a radial perfusion gradient in the right lower lobe during 2LVC and 2LVT, no isogravitational Qr gradients were observed. CONCLUSION. We conclude that controlled mechanical ventilation in the lateral decubitus position causes only minor changes in vertical blood flow distribution.

Effects of isoflurane on regional pulmonary blood flow during one-lung ventilation.

Isoflurane has been reported to inhibit hypoxic pulmonary vasoconstriction. However, the effects of one-lung ventilation and isoflurane on regional pulmonary blood flow (Qr) have not been investigated in detail. Therefore, using radionuclide labelled microspheres we measured Qr in rabbits (n = 8) in the left lateral decubitus position during two- and one-lung ventilation under i.v. baseline anaesthesia and during additional administration of 1.5% isoflurane. Macrohaemodynamic variables were recorded continuously. Isoflurane increased non-dependent lung blood flow during two-lung ventilation. One-lung ventilation caused a homogeneous decrease in Qr throughout the hypoxic lung, irrespective of isoflurane administration (P < 0.001). However, isoflurane significantly augmented Qr of the hypoxic lung during one-lung ventilation (P < 0.05). During all phases, Qr of the upper lobe was higher compared with that in the lower lobe in isogravitational slices of both lungs; a ventrodorsal perfusion gradient was found in the left upper lobe. We conclude that 1.5% isoflurane increased perfusion of the non-dependent lung, inhibited hypoxic pulmonary vasoconstriction-induced redistribution of pulmonary blood flow and did not influence isogravitational perfusion gradients.

Isoflurane inhibits hypoxic pulmonary vasoconstriction. An in vivo fluorescence microscopic study in rabbits.

BACKGROUND: Contradictory results have been reported in previous studies investigating the effect of isoflurane on hypoxic pulmonary vasoconstriction by indirect approaches. The current study measured the effects of one-lung ventilation (1LV) and isoflurane 1.5% by direct visual observation of the pulmonary microcirculation. METHODS: Ten New Zealand White rabbits were anesthetized with intravenous thiopental, alpha-chloralose, and piritramid. Arterial, central venous, pulmonary arterial, left atrial, and airway pressures and cardiac output were recorded continuously. 1LV was facilitated by a bronchial blocker in the right main bronchus. A transparent window was implanted into the right thoracic wall for videofluorescence microscopy of the subpleural pulmonary microcirculation. After intravenous injection of fluorescein isothiocyanate-labeled red blood cells, vessel diameters, red blood cell flux, red blood cell velocity, and dynamic microhematocrit were measured in pulmonary arterioles and venules during two-lung ventilation and 1LV during baseline anesthesia and with supplementary isoflurane 1.5%. RESULTS: During intravenous anesthesia, 1LV caused significant reduction of vessel diameters and red cell flux and velocity and an increase in microvascular hematocrit in pulmonary arterioles and venules. The decreases in arteriolar diameters and red blood cell flux and velocity induced by 1LV were significantly attenuated by isoflurane as compared with those measured during baseline anesthesia (P = 0.010, P = 0.029 and P = 0.047). Accordingly, 1LV-induced reduction of venular red cell flux (P = 0.023) and velocity (P = 0.036) were less pronounced during isoflurane. Isoflurane caused a significant decrease in arterial pressure. Venous admixture increased and arterial oxygen tension decreased significantly during 1LV; the changes were more pronounced during 1LV with isoflurane 1.5% than during 1LV with baseline anesthesia. CONCLUSIONS: 1LV leads to a marked reduction of microvascular diameters and blood flow in the hypoxic lung. Isoflurane 1.5% inhibits hypoxic pulmonary vasoconstriction in pulmonary arterioles and increases regional blood flow in the hypoxic lung.