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PubMed1989

Biological and cellular responses to grass pollen in sensitive patients.

Using a noninvasive skin chamber technique, we studied the in vivo development of anaphylactic reactions in 8 pollen-sensitive patients suffering from seasonal allergic rhinitis/conjunctivitis/asthma and showing positive cutaneous reactions after intradermal allergen challenge. As agonists, histamine and pollen were introduced into the skin chambers and left in contact with superficial dermis during 6 h. The release of mediators (histamine and prostaglandin [PG] D2) and the modifications in protein diffusion occurring during the immediate (30 min) and the late (6 and 24 h) cutaneous reaction phases were quantitatively analyzed. 24 h after agonist introduction, the recruitment of inflammatory cells on the superficial dermis was studied by use of Rebuck's windows. Histamine release in pollen-containing skin chambers was immediate and persisted until the 24th h despite replacement of the agonists by control medium at the 6th h. An intense PGD2 release occurred as soon as the first 30 min in chambers containing either exogenous histamine or pollen and was maintained until the 24th h. Protein diffusion induced by histamine and pollen was similar to the control one at 30 min but was intensely enhanced at the 6th h. At the 24th h, pollen-induced protein diffusion was still intense whereas that induced by histamine was analogous to the control one. 24 h after pollen challenge, numerous eosinophils were recruited on the superficial dermis but almost none were observed after control medium or histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Eosinophils↗
PubMed1998

[Microcirculation research in experimental surgery].

The microcirculation is the organ that provides the direct link between blood and tissue, and thereby between the whole organism and the single cell. Modern microcirculation research in experimental surgery is characterized by the use of high-resolution video fluorescence microscopy and quantitative computer-assisted image analysis coupled with the techniques of molecular biology and transgenic or knockout gene technology. These advances should improve knowledge about surgically relevant physiologic and pathophysiologic phenomena at the interface between blood and tissues and help us to understand the initial molecular mechanisms leading to organ dysfunction following inflammation, ischemia-reperfusion, and transplantation.

Animals↗
PubMed1992

Chronic idiopathic urticaria: profiles of skin mast cell histamine release during active disease and remission.

Chronic idiopathic urticaria (CIU) is characterized by the increased releasability of histamine by mast cells in normal-appearing skin. In active CIU, this abnormality is consistently present. To determine if this finding subsides when the disease goes into remission phase, we analyzed the histamine secretion in patients with CIU in remission (CIUR) compared with that of patients with CIU and in normal control (NC) subjects, with the skin-chamber technique. The profiles of histamine release in sites challenged with compound 48/80 were significantly different in the groups with CIU and CIUR. Furthermore, patients with CIUR did not differ from NC subjects in terms of histamine releasability under compound 48/80 stimulation (p greater than 0.1). These data suggest that the state of excessive skin mast cell sensitivity is a reversible and transient phenomenon in CIU disease.

Analysis of Variance↗
PubMedDate not supplied

The Cytoscan Model E-II, a new reflectance microscope for intravital microscopy: comparison with the standard fluorescence method.

The Cytoscan Model E-II (Cytometrics Inc., Philadelphia, Pa., USA) is a newly developed instrument which functions as an intravital microscope and is small and easily portable. Through the use of orthogonal polarization spectral (OPS) imaging, the Cytoscan Model E-II delivers images of the microcirculation which are comparable to those achieved with intravital fluorescence videomicroscopy (IFM), but without the use of fluorescent dyes. The purpose of this study was to validate the Cytoscan Model E-II instrument against IFM. The experiments were carried out on striated muscle in the dorsal skinfold chamber of the awake Syrian hamster. The following parameters were measured in identical regions of interest in the same animal under baseline conditions and 0.5 and 2 h after a 4-hour period of pressure-induced ischemia: arteriolar diameter, venular diameter and venular red blood cell velocity. Bland-Altman plots showed good agreement between the two techniques for venular red blood cell velocity. As expected, arteriolar and venular diameters as measured by the Cytoscan were on average 5 microm smaller than the values from IFM, since the Cytoscan measures the red blood cell column width and IFM measures luminal diameter. Thus, OPS imaging can be used to make valid measurements of microvascular diameter and red blood cell velocity in tissues.

Animals↗
PubMedDate not supplied

[Morphological changes of the cell surface during phagocytosis of asbestos fibers].

The Sandersand Shubik transparent chamber inserted in the golden hamster cheek pouch has been found a simple and easy technique to study the in vivo phagocytosis of asbestos fibers. Use of this device in a scanning and transmission electron microscopy study provided a three-dimension view of some phases of the process, of the cell-fiber relationships as well as of the modifications which occured on the cell surfaces in contact with the fibers in the chambers. The advantages presented by this experimental model over the others are discussed as well as some recent data regarding the cytotoxic effect of the asbestos fibres.

Animals↗
PubMed1991

Simultaneous multiple site arteriolar vasomotion measurement using digital image analysis.

An automatic digital image processing technique for simultaneous vasomotion analysis in peripheral microcirculation at multiple sites and in real time is presented. The algorithm utilizes either fluorescent or bright field micro-images of the vasculature as input. The video images are digitized and analyzed on-line by an IBM RT PC, using digital filtering and edge detection. The sampling frequency is higher than 5 Hz when only one site is tracked and decreases as the number of sites is incremented. Performance of the algorithm was tested for a hamster cutaneous microcirculation model.

Algorithms↗
PubMedDate not supplied

Angiogenesis under normal and pathological conditions.

Angiogenesis, i.e. the generation of new blood capillaries, occurs in utero (during embryonal and fetal development) and in both physiological and pathological situations during extrauterine life. Several angiogenic factors have now been isolated, including angiogenin, acidic and basic fibroblast growth factors, and alpha and beta transforming growth factors. Their amino acid sequences have been determined and their genes cloned. Other factors await complete characterisation. An account is given of techniques used in the investigation of angiogenesis, both in vivo (transparent chambers; corneal micropockets; implantation on chick chorioallantoic membrane; employment of polymers for the sustained release of angiogenesis factors) and in vitro (cloning and long-term culture of capillary endothelial cells). The angiogenesis induced by solid tumours differs from other forms in that it is not self-limited and continues indefinitely until eradication of the tumour or death of the host. Anti-angiogenic factors have also been identified, particularly a new class of nonglucocorticoid steroids. Their employment in tumour therapy is a possibility, since neoplastic expansion is essentially dependent on angiogenesis.

Angiogenesis Inducing Agents↗
PubMed1984

Enhanced neutrophil migration in vivo HLA B27 positive subjects.

Chemotaxis of polymorphonuclear leucocytes in vivo was studied in patients with previous yersinia arthritis and in healthy subjects with or without HLA B27 by means of a skin chamber technique. Irrespective of previous arthritis the number of neutrophils in the chamber media was significantly higher in HLA B27 positive subjects than in those without HLA B27. The amounts of prostaglandins E2, F2 alpha, and 6-keto-F1 alpha in the chamber media correlated positively with the corresponding cell counts. The present results give credence to the view that the hyperreactive neutrophils and the vasodilatory prostaglandins produced by them can together trigger a vicious circle which results in increased inflammatory symptoms in patients with yersinia arthritis who have HLA B27 as compared with those who lack this antigen.

6-Ketoprostaglandin F1 alpha↗
PubMedDate not supplied

Sodium nitroprusside (SNP) hypotension: intracranial pressure (ICP) and hemodynamics in pial arteriole in the rat.

A detailed description is made of an acute closed cranial window method. The method is used for the study of cerebral pial microcirculation by intravital microscopy in the rat. Using these methods and techniques, the effects of systemic hypotension by SNP, i.v., on pial microvessel hemodynamics and on ICP were simultaneously measured and characterized under normophysiological conditions. The pH, PO2, PCO2 and temperature of the artificial cerebrospinal fluid (CSF) in the closed cranial window, intermittently measured, remained relatively constant, 30 to 60 min following the period of stabilization of the preparation. The infusion of SNP (6.2-35.0 micrograms/kg/min, 0.02% sol., i.v.) significantly decreased BP (52.1 +/- 13.4 mm Hg, mean +/- SD). From measurement of microvessels internal diameter (I. D.) and microhemodynamics, significant increases in pial arteriolar I.D. (from 35.4 +/- 10.1, microns, to 47.1 +/- 5.7, microns, mean and S.D., 33.0%) and estimated bulk flow (51.2%), occurred during the hypotension. The changes in hemodynamic parameter were predominantly in the arteriolar system. Only minimal changes in the venular diameter occurred during the SNP hypotension. The observed moderate (22.0%) increase in ICP during SNP hypotension in pentobarbital anesthetized rat correlates well with the microhemodynamic changes of the cerebral microcirculatory system.

Animals↗
PubMed1993

Dorsal skinfold chamber technique for intravital microscopy in nude mice.

For many years, observation chambers, implanted in various animal species and in man, have been used for intravital microscopy of the microcirculation in granulation tissue, preformed tissue, or of the microvascularization of tissue implants. We describe herein a skinfold chamber model for the intravital microscopic investigation of striated skin muscle in immunoincompetent, nude mice over a minimum period of 2 weeks. Using fluorescent markers for contrast enhancement of plasma (fluorescein isothiocyanate-dextran) and leukocytes (acridine orange), the presented model allows the quantitative analysis of 1) the microhemodynamic parameters microvessel diameter and red blood cell velocity in arterioles (16 to 50 mu diameter), capillaries (4 to 9 mu diameter), and post-capillary venules (19 to 60 mu diameter), 2) leukocyte/endothelium interaction in these vessel segments, 3) functional capillary density and intercapillary distance, and 4) endothelial cell integrity. These parameters can be assessed in the microcirculation of the striated muscle tissue under normal or pathological conditions, as well as in the microcirculation of transplanted xenogeneic (human) neoplastic and non-neoplastic tissue grafts.

Animals↗
PubMed1989

Pharmacokinetics of intravenous cefmetazole with emphasis on comparison between predicted theoretical levels in tissue and actual skin window fluid levels.

Cefmetazole is a cephamycin antibiotic which is resistant to hydrolysis by various beta-lactamases. This study evaluated the pharmacokinetics of cefmetazole, including its intravascular and interstitial fluid distribution, by using the skin window (SW) technique. A 2-g dose of cefmetazole was given intravenously over 30 min to each of 12 healthy adult male volunteers every 6 h for nine doses. Plasma levels were assayed at predetermined intervals after doses 1, 5, and 9. Interstitial fluid levels were determined by the SW technique. Antibiotic levels were assayed by the agar well bioassay technique. A concentration-versus-time plot indicates that cefmetazole is rapidly distributed, with mean peak levels in plasma equal to 126 micrograms/ml at the end of the half-hour infusion. The mean plasma half-life was 1.1 h. Plasma and tissue distribution constants permitted calculation of theoretical levels in tissue. Parallel elimination slopes for SW and theoretical tissue level showed that the SW model distribution kinetics are closely related. The area under the curve for the SW was 73.9 mg.h/liter. This was comparable to the theoretical level in tissue, which was 96 mg.h/liter. Furthermore, the area under the curve of theoretical tissue level/plasma was 0.6 and that of SW/plasma was 0.47. These results demonstrate that the SW technique yielded a result quite close to the theoretical tissue level. Ultrafiltration analysis indicated that as cefmetazole levels in plasma increased from 10 to 250 micrograms/ml, plasma protein binding of the antibiotic dropped from 85 to 65%. Finally, 60 to 70% of the drug was recovered from the urine as biologically active drug over 6 h postinfusion.

Adult↗
PubMed1990

An improved closed cranial window technique for investigation of blood-brain barrier function and cerebral vasomotor control in the rat.

A modification of the closed cranial window technique for small laboratory animals is presented. The method facilitates investigation of blood-brain barrier permeability in association with studies of the cerebrovascular response under normal and pathological conditions. Following trephination and preparation of the pia-arachnoid surface, the skull defect is closed again by sealing a cover glass onto a wall of dental cement surrounding the cranial window. The intracranial pressure can then be studied and experimentally manipulated. Care is taken to maintain normal blood-brain barrier function to a small barrier indicator (Na(+)-fluorescein, MW: 376). This is accomplished by opening the skull and dura mater under a column of paraffin oil to avoid exposure of brain tissue to atmospheric pressure. Reactivity of the cerebral surface vessels was assessed during hypercapnia at an arterial PaCO2 of 50.8 +/- 1.1 mm Hg. It was found that small arterioles of 20-50 microns phi had a significantly larger CO2-response than large arterioles of 50-100 microns phi. Pial venules did not respond at all. Superfusion of the cranial window preparation at a rate of 5 ml/h with buffered artificial CSF was tolerated for hours. No alterations of arteriolar or venular diameters nor opening of the blood-brain barrier to Na(+)-fluorescein was observed. These technical modifications enable us to employ a closed cranial window model in small laboratory animals for studies of cerebral microcirculation and blood-brain barrier function under normal as well as under pathological conditions related to brain damage.

Animals↗
PubMed1989

Microscopic observation of leukocyte kinesis in the vascular bed during hemodialysis using the rabbit ear chamber technique.

Leukocyte kinesis in the capillary vascular bed during hemodialysis (HD) was investigated to elucidate the mechanism of transient leukopenia. Leukocyte movement was observed microscopically during HD using the rabbit ear chamber (REC) technique, which permits visualization of the movement of blood corpuscles in capillaries. Blood was drawn from the femoral artery and returned into the auricular and/or carotid artery so that the blood passing through the hollow fiber artificial kidney (HFAK) flowed into capillaries in the REC. Leukocyte counts of blood samples taken from the afferent and efferent limbs of the HD circuit, the right jugular vein and the right atrium were determined consecutively during HD. The difference in the leukocyte count was observed between the afferent and efferent limbs for the first 15 minutes and thereafter between the efferent limb and the jugular vein. The "transpulmonary" difference in the leukocyte count was not noticed throughout HD. Between 15 and 90 minutes after the start of HD, scarcely any circulating leukocytes were found in capillaries in the REC and some leukocytes were attached to the endothelial surface. Thereafter circulating leukocytes were seen again and detachment of leukocytes from the endothelial surface was observed. No leukocyte aggregation or embolization of aggregating leukocytes was noticed. This evidence suggests that leukopenia may be attributed to the transient shift of leukocytes to the marginal pool of the vessel lumen and this process may not be specific for the pulmonary vasculature, but may occur in the first capillary bed into which the blood passing through the HFAK flows.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

In vivo chamber angiogenesis assay: an optimized Matrigel plug assay for fast assessment of anti-angiogenic activity.

Efficient in vitro and in vivo angiogenesis assays, to assess and compare anti-angiogenic activity are a prerequisite for the discovery and characterization of anti-angiogenic targets. Here we describe an optimized Matrigel plug assay based on subcutaneously implanted chambers and two fast and reproducible measuring techniques. Plexiglas ring/nylon net filter-chambers (0.2 ml) containing growth factor-reduced Matrigel and 300 ng basic fibroblast growth factor (bFGF) were subcutaneously implanted into the right flank of rats. Chamber angiogenesis was scored on day 5 and day 10 post-implantation by computer image analysis of the chamber, and by optical density reading at 415 nm of a PBS solution of the chamber content. bFGF significantly induced chamber angiogenesis and histological examination confirmed that numerous blood vessels were present in the bFGF-induced chambers. The anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. In contrast, the anti-inflammatory or immuno-suppressive compounds cyclosporin A (15 mg/kg/d p.o.), indomethacin (1 mg/kg/d p.o.), and prednisolone (5 mg/kg/d p.o.) showed no anti-angiogenic activity, indicating that the bFGF-induced angiogenesis was not driven by an inflammatory response or by a foreign body reaction. Finally, two candidate anti-angiogenic compounds were tested in the assay. Continuous low-dose therapy with cyclophosphamide (25 mg/kg/d p.o.) significantly inhibited bFGF-induced angiogenesis, whereas 1alpha,25-dihydroxyvitamin D3 (0.5 micro g/kg/d p.o.) showed no significant anti-angiogenic activity. In conclusion, this in vivo chamber angiogenesis assay is a useful new tool for drug evaluation as well as research into anti-angiogenesis.

Angiogenesis Inhibitors↗
PubMed1989

The pattern and kinetics in human skin of erythema and mediators during the acute and late-phase response (LPR).

To investigate the kinetics and pattern of allergenically induced mediator release in the human skin, we have studied 24 ragweed- and grass-allergic patients with a blister-chamber technique. Chambers sealed to the skin around a denuded area, formed by unroofing a blister, were challenged with 0.5 ml of either diluent or 10 or 100 times the concentration of allergen required for 4+ early intradermal reaction. Chamber fluids were removed hourly 1 to 8 hours after antigen challenge and examined for the presence of histamine, leukotriene C4 (LTC4), and prostaglandin D2 to compare inflammatory mediator levels with the clinical early response and late-phase response (LPR), as assessed by erythema around the chamber. An initial erythema developed rapidly and began to subside after 1 hour in all patients, but a late-phase local erythema and subcutaneous swelling around the chamber (i.e., greater than 2.5 cm, the outside diameter of the chamber) developed in 13/15 challenges only when the higher concentration of antigen was used. At both allergen concentrations, histamine levels peaked sharply at the first hour (20.6 +/- 2.3 ng/ml) and progressively declined during the next 4 hours by 75%, but remained above control levels for at least 7 hours. Despite high control values, LTC4 levels were significantly elevated (p less than 0.01) 4 to 6 hours after challenge. In visible reactions, maximal LPR around the chamber correlated with LTC4 levels obtained 6 and 7 hours after challenge (p less than 0.05). Prostaglandin D2 rose gradually in antigen-challenged chambers to a peak at 5 to 6 hours. Thus, early rises in histamine were temporally related to the immediate erythema, whereas the arachidonic acid metabolites from both cyclooxygenase and lipoxygenase pathways that appeared in the skin after allergen challenge followed kinetics that corresponded to the time course of cutaneous LPR.

Adolescent↗
PubMed2002

Orthogonal polarisation spectral imaging as a new tool for the assessment of antivascular tumour treatment in vivo: a validation study.

Tumour angiogenesis plays a key role in tumour growth, formation of metastasis, detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour angiogenesis is an indispensable prerequisite for developing novel treatment strategies such as anti-angiogenic and antivascular treatment options. Therefore, it was our aim to establish and validate a new and versatile imaging technique, that is orthogonal polarisation spectral imaging, allowing for non-invasive quantitative imaging of tumour angiogenesis in vivo. Experiments were performed in amelanotic melanoma A-MEL 3 implanted in a transparent dorsal skinfold chamber of the hamster. Starting at day 0 after tumour cell implantation, animals were treated daily with the anti-angiogenic compound SU5416 (25 mg kg x bw(-1)) or vehicle (control) only. Functional vessel density, diameter of microvessels and red blood cell velocity were visualised by both orthogonal polarisation spectral imaging and fluorescence microscopy and analysed using a digital image system. The morphological and functional properties of the tumour microvasculature could be clearly identified by orthogonal polarisation spectral imaging. Data for functional vessel density correlated excellently with data obtained by fluorescence microscopy (y=0.99x+0.48, r2=0.97, R(S)=0.98, precision: 8.22 cm(-1) and bias: -0.32 cm(-1)). Correlation parameters for diameter of microvessels and red blood cell velocity were similar (r2=0.97, R(S)=0.99 and r2=0.93, R(S)=0.94 for diameter of microvessels and red blood cell velocity, respectively). Treatment with SU5416 reduced tumour angiogenesis. At day 3 and 6 after tumour cell implantation, respectively, functional vessel density was 4.8+/-2.1 and 87.2+/-10.2 cm(-1) compared to values of control animals of 66.6+/-10.1 and 147.4+/-13.2 cm(-1), respectively. In addition to the inhibition of tumour angiogenesis, tumour growth and the development of metastasis was strongly reduced in SU5416 treated animals. This new approach enables non-invasive, repeated and quantitative assessment of tumour vascular network and the effects of antiangiogenic treatment on tumour vasculature in vivo. Thus, quantification of tumour angiogenesis can be used to more accurately classify and monitor tumour biologic characteristics, and to explore aggressiveness of tumours.

Angiogenesis Inhibitors↗