Rainer Kiefmann

Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence

OBJECTIVE Survival after surgery and radiotherapy for the treatment of malignant gliomas is linked to the completeness of tumor removal. Therefore, methods that permit intraoperative identification of residual tumor tissue may be of benefit. In a preliminary investigation, we have studied the value of fluorescent porphyrins that accumulate in malignant tissue after administration of a precursor (5-aminolevulinic acid) for labeling of malignant gliomas in nine patients. METHODS Three hours before the induction of anesthesia, 10 mg 5-aminolevulinic acid/kg body weight was administered orally. Intraoperatively, red porphyrin fluorescence was observed with a 455-nm long-pass filter after excitation with violet-blue (375-440 nm) xenon light and was verified by analysis of fluorescence spectra. Fluorescing and nonfluorescing samples taken from the tumor perimeters were examined histologically or used to study the photobleaching of porphyrins by excitation light and white light from the operating microscope. Plasma and erythrocyte porphyrin levels were determined by fluorescence photometry. RESULTS Normal brain tissue revealed no porphyrin fluorescence, whereas tumor tissue was distinguished by bright red fluorescence. For a total of 89 tissue biopsies, sensitivity was 85% and specificity was 100% for the detection of malignant tissue. For seven of nine patients, visible porphyrin fluorescence led to further resection of the tumor. Under operating light conditions, fluorescence decayed to 36% in 25 minutes for violet-blue light and in 87 minutes for white light. Plasma and erythrocyte porphyrin contents increased slightly, without exceeding normal levels. CONCLUSION Our observations suggest that 5-aminolevulinic acid-induced porphyrin fluorescence may label malignant gliomas safely and accurately enough to enhance the completeness of tumor removal.

Dichloroacetate prevents restenosis in preclinical animal models of vessel injury.

Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.

Platelet-endothelial cell interaction in pulmonary microcirculation: the role of PARS

Accumulation of platelets might contribute to acute lung injury during systemic inflammation. The aim of the study was to elucidate the role of the poly (ADP-ribose) synthetase, a nucleotide-polymerizising enzyme, in mediation of platelet-endothelial cell interaction through regulation of adhesion molecules within the pulmonary microcirculation during endotoxemia. We used in vivo fluorescence microscopy to quantify kinetics of fluorescently labeled erythrocytes and platelets in rabbit pulmonary arterioles and venules. Six hours after onset of endotoxin infusion we observed a massive interaction of platelets with the microvascular endothelial cells, whereas under control conditions, no platelet sequestration was measured. An up-regulation of P- and E-selectin was detected in lung tissue following endotoxin infusion by immunohistochemistry and Western blot analysis. Blockade of endothelial P-selectin with fucoidin resulted in a reduction of the endotoxin-induced platelet-endothelial cell interaction. Inhibition of poly (ADP-ribose) synthetase by 3-aminobenzamide inhibited the endotoxin-induced expression of endothelial P- and E-selectin and the subsequent recruitment of platelets. In summary, we provide first in vivo evidence that platelets accumulate in pulmonary microcirculation following endotoxemia. Poly (ADP-ribose) synthetase seems to mediate this platelet-endothelial cell interaction via P- and E-selectin expressed on the surface of microvascular endothelium.

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.

Glycocalyx degradation causes microvascular perfusion failure in the ex vivo perfused mouse lung: hydroxyethyl starch 130/0.4 pretreatment attenuates this response.

The endothelial glycocalyx (GLX) is pivotal to vascular barrier function. We investigated the consequences of GLX degradation on pulmonary microvascular perfusion and, prompted by evidence that hydroxyethyl starch (HES) improves microcirculation, studied the effects of two HES preparations during GLX diminution. C57 BL/6 black mice lungs were explanted and perfused with 1-mL/min buffer solution containing autologous erythrocytes (red blood cells) at a hematocrit of 5%. Microvessel perfusion was quantified by video fluorescence microscopy at 0 and 90 min. To register interstitial edema, alveolar septal width was quantified. Pulmonary artery pressure (PAP), airway pressure, and left atrial pressure were recorded continuously. Lungs were randomly assigned to four groups (each n = 5): (i) control: no treatment, (ii) HEP1: heparinase I (1 mU/mL) was injected for GLX degradation, (iii) HES 130, and (iv) HES 200: one third of perfusion fluid was exchanged for 6% HES 130/0.4 or 10% HES 200/0.5 before GLX degradation. Analysis of variance on ranks and pairwise multiple comparisons were used for statistics, P < 0.05. Compared with control, GLX degradation effected perfusion failure in microvessels, increased PAP, and facilitated interstitial edema formation after a 90-min period of perfusion. In contrast to HES 200/0.5, pretreatment with HES 130/0.4 attenuated all of these consequences. Sequelae of GLX degradation in lung include perfusion failure in microvessels, interstitial edema formation, and increase in PAP. We assume that these effects are a consequence of vascular barrier dysfunction. Beneficial effects of HES 130/0.4 are presumably a result of its lower red blood cell bridging capacity compared with HES 200/0.5. ABBREVIATIONS ESL—endothelial surface layer FACS—fluorescence-activated cell sorting FITC—fluorescein isothiocyanate GLX—endothelial glycocalyx HEP1—heparinase I HPMECs—human pulmonary microvascular endothelial cells HES—hydroxyethyl starch HS—heparan sulfate PAP—pulmonary artery pressure MFI—mean fluorescence intensity RBC—red blood cell VRBC—red blood cell velocity

Tetrastarch sustains pulmonary microvascular perfusion and gas exchange during systemic inflammation

Objective: According to Ficks law of diffusion, gas exchange depends on the size and thickness of the blood perfused alveolocapillary membrane. Impairment of either one is tenuous. No data are available concerning the impact of hydroxyethyl starches and saline on pulmonary microperfusion and gas exchange during systemic inflammation. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: Thirty-two anesthetized rabbits assigned to four groups (n = 8). Interventions: Except for the control group, systemic inflammation was induced by lipopolysaccharide. Fluid resuscitation was performed with saline alone or in conjunction with tetrastarch or pentastarch. Pulmonary microcirculation was analyzed at 0 hr and 2 hrs using intravital microscopy. Thickness of the alveolocapillary membrane was measured using electron microscopy. Measurements and Main Results: Macrohemodynamics were stable in all groups. In pulmonary arterioles, lipopolysaccharide reduced the erythrocyte velocity and impeded the microvascular decrease of the hematocrit in the saline and pentastarch group. In contrast, infusion of tetrastarch normalized these perfusion parameters. In capillaries, lipopolysaccharide decreased the functional capillary segment density and the capillary perfusion index, which was prevented by both starches. However, compared with saline and pentastarch, treatment with tetrastarch prevented the lipopolysaccharide-induced reduction of the capillary erythrocyte flux and inversely reduced the erythrocyte capillary transit time. Thickening of alveolocapillary septae after lipopolysaccharide application was solely observed in the saline and pentastarch group. In contrast to pentastarch and saline, the application of tetrastarch prevented the lipopolysaccharide-induced increase of the alveoloarterial oxygen difference. Conclusions: Tetrastarch sustains pulmonary gas exchange during experimental systemic inflammation more effectively than saline and pentastarch by protecting the diffusion distance and the size of the microvascular gas exchange surface. Improved capillary perfusion resulting from tetrastarch therapy, which is typically applied to increase blood pressure, may according to the Ohms law locally decrease hydrostatic perfusion pressures in the pulmonary microvasculature during systemic inflammation.

IDH3 mediates apoptosis of alveolar epithelial cells type 2 due to mitochondrial Ca2|[plus]| uptake during hypocapnia

In adult respiratory distress syndrome (ARDS) pulmonary perfusion failure increases physiologic dead-space (VD/VT) correlating with mortality. High VD/VT results in alveolar hypocapnia, which has been demonstrated to cause edema formation, atelectasis, and surfactant depletion, evoked, at least in part, by apoptosis of alveolar epithelial cells (AEC). However, the mechanism underlying the hypocapnia-induced AEC apoptosis is unknown. Here, using fluorescent live-cell imaging of cultured AEC type 2 we could show that in terms of CO2 sensing the tricarboxylic acid cycle enzyme isocitrate dehydrogenase (IDH) 3 seems to be an important player because hypocapnia resulted independently from pH in an elevation of IDH3 activity and subsequently in an increase of NADH, the substrate of the respiratory chain. As a consequence, the mitochondrial transmembrane potential (ΔΨ) rose causing a Ca2+ shift from cytosol into mitochondria, whereas the IDH3 knockdown inhibited these responses. Furthermore, the hypocapnia-induced mitochondrial Ca2+ uptake resulted in reactive oxygen species (ROS) production, and both the mitochondrial Ca2+ uptake and ROS production induced apoptosis. Accordingly, we provide evidence that in AEC type 2 hypocapnia induces elevation of IDH3 activity leading to apoptosis. This finding might give new insight into the pathogenesis of ARDS and may help to develop novel strategies to reduce tissue injury in ARDS.

The Selective JAK1/3-Inhibitor R507 Mitigates Obliterative Airway Disease Both With Systemic Administration and Aerosol Inhalation.

Background The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models. Methods Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells. Results In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1&bgr;. Conclusions The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.