MICROVASCULAR
RESEARCH
22,
232-237 (1981)
Abstracts of Papers Presented British Microcirculation
at the 1981 Joint Meeting of the Society and the Skin Club
April 8 and 9, St. Thomas’s Hospital Medical School, London, England
1. Hydraulic Conductivity of the Blood-Joint Barrier. A. D. KNIGHT AND J. R. LEVICK, Department of Physiology, St George’s Hospital Medical School, London SW17 ORE, England. The hypothesis has been advanced that the hydraulic conductivity of the lining of the joint cavity (synovial intima) increases as a function of intraarticular pressure (P,) in the rabbit knee (J. R. Levick, 1980, J. Physiol. 306, 445-461). The synovial intima, along with capillary endothelium, separates blood from the joint cavity. The hypothesis therefore predicts that the conductivity of the blood-joint barrier should depend upon P,. This prediction has been tested. The hydraulic conductivity of the blood-joint barrier equals the slope relating synovial capillary pressure (P,) to transsynovial flow (b,), and was determined by measurement of es, and P, in the isolated perfused rabbit hindquarter as described previously (Knight and Levick, 1980, J. Physiol. 307, 69P). & was found to be a linear function of P, at constant P,, hence endothelial conductivity is independent of pressure. Slope dQ,ldP, was determined at constant P,‘s of 4, 6, 8, 11, 12, 14, 18, 20, 25, and 30 cm H,O. The corresponding slopes, fitted by linear regression analysis, were respectively -0.06 (n = IO), -0.10 (n = 18), -0.14 (n = 24) -0.23 (n = 35), -0.39 (n = 58), -0.38 (n = 47) -0.43 (n = 142), -0.47 (n = 25), -0.49 (n = 67), and -0.61 (n = 58) pl.rnin.-’ mm Hg-‘; values of n represent number of capillary pressure-flow pairs at each P,. The conductivity d&/dP, displays a significant correlation with Pi (Spearman rank correlation coefficient = 0.99; P < 0.01). The hypothesis of a labile synovial conductivity is thus strongly supported by the results.
2. The Influence of Hormones for MicroJilament-Mediated
on the Microcirculation Vasoconstriction.
of a Single
Jejunal
Rat
Villus
With Evidence
M. KNOBLAUCH, C. VOGT, C. HOLLIGER, M. NEFF, AND J. M. METRY, Institute of Biomedical Engineering, University of Zurich and Department of Internal Medicine, Hospital of Mannedorf, Switzerland.
Applying an in vivo microscopy technique, we studied the effect of systemically administered glucagon, VIP, and vasopressin on the microcirculation of an intact single villus of the jejunum of the rat. Blood flow changes were calculated from measurements of RBC velocity and RBC column diameter. Glucagon increased villous flow to 150.1-13.7% of control, while superior mesenteric artery (SMA) flow, measured simultaneously, remained unchanged. VIP led to a dilatation of vessels but also to a reduction of velocity, leaving flow unchanged. Vasopressin caused a marked reduction of flow at the villus and SMA level, but a similar reduction of villous flow without altering SMA flow was achieved with topical application of vasopressin. The vasoconstrictor effect of topical vasopressin could be prevented by pretreatment of the villus with cytochalasin B, which is known to inhibit the contractility of microfilaments. In a search for contractile elements in the villous arcade vessels, we examined serial horizontal sections by electron microscopy. We failed to detect any muscular sphincters but found that the vessels were surrounded by pericytes and dendrites of tibroblasts, all containing abundant microfilaments. Microtilaments were also found in endothelial cells. These studies suggest that a jejunal villus has a microvascular bed which can be specifically influenced by hormones and that vasoconstriction may be mediated by microfilaments. 232 0026-2862/81/050232-~~2,~/0 Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
MICROCIRCULATORY
SOCIETY ABSTRACTS
233
and Reflexion Coefficients in the Isolated Perfused Rabbit Submandibular Gland. J. GAMBLE,* P. D. SPENCER,L. H. SMAJE, AND W. P. PENN, Department of Physiology, Charing Cross Hospital Medical School, London W6 8RF, and *Department of Physiology, Medical College of St Bartholomew’s Hospital, London EClM 684, England.
3. Filtration
Fenestrated capillaries are highly permeable to water and small solutes, but seem to be no more permeable than continuous capillaries to albumin and y-globulins (Koo et al., 1980, Microvasc. Res. 20, 256-257). The present experiments were designed to investigate the selectivity of the fenestrated capillaries of rabbit submandibular glands in vitro. Glands from four rabbits, anesthetised with urethane, were isolated, perfused in vitro with a Krebs-Ringer-bicarbonate solution containing 5 g% bovine serum albumin (KRB-A) and 0.1 mM adenosine, and the weight and perfusion pressures continuously monitored. Filtration coefficients (&) were determined using graded elevations of venous pressure, and reflexion coefficients to raffinose, inulin, and albumin were determined using the osmotic transient method of Vargas and Johnson (1964, J. Gem Physiol. 47, 667-677). An attempt was made to correct for outward diffusion of solute during the earliest stages of the transient using a simple computer model. Kf varied between 0.54 and 3.86 mVmin.100 g.mm Hg (mean = 2.07; SE = 0.4). Reflexion coefficients are shown in the table, where the predicted values are based on the equations of the fibre matrix theory (see Curry and Michel, 1980, Microvasc. Res. 20, 96-99). The reflexion coefficients of raffinose and inulin suggest that fenestrated capillaries exhibit a degree of selectivity consistent with the fiber matrix theory, but the rather low reflexion coefficient in this perfused preparation for albumin requires further investigation. Reflexion coefficient
Raffinose Inulin Albumin
n
Observed
Predicted
4 21 2
0.013 k 0.001 0.24 f 0.02 0.6
0.02 0.18 0.83
In&fences Involved in Angiogenesis. R. L. BARNHILL AND T. J. RYAN, University of Oxford and Department of Dermatology, The Slade Hospital, Oxford OX3 7JH, England.
4. Physical
The aim of the project was to study the physical influences of epithelial injury and proliferation on angiogenesis in the chorioallantoic membrane of the chicken embryo (CAM). Two stimuli-(l) silver nitrate cautery of the outer ectodermal epithelial layer of the CAM, and (2) continuous air exposure, which has been shown to result in proliferation and keratinization of the outer epithelium (Moscona, 1959, Develop. Bioi. 1, I-23)-were applied or initiated and the response was examined after 4 days. Each preparation was photographed and processed for histological examination at this time. All specimens demonstrated significant radial patterns of angiogenesis about the foci of injury or hyperplasia. On histological examination all specimens demonstrated buckling and contraction of the foci of epithelial hyperplasia injury. Specimens sectioned through the centre of a focus showed orientation of connective fibres and vessels inward towards the focus of epithelial hyperplasia. Although this phenomenon has never been reported in angiogenesis, previous work (Weiss, 1959, Harvey Let. Ser. 55, 13-42) has demonstrated the syneretic activity of foci of cellular proliferation which generate radial lines of tension, orientating connective tissue fibres and providing preferential pathways for cellular migration. Other mechanisms may account for or play a role in this process of epithelial contraction (Wessells, 1977, Tissue Interact. Develop. 84-102; Shelley et al., 1980, Brit. J. Dermatol. 102, 123-233) as well. Whether wound contraction involves a similar process, myofibroblast activity, or both, the result, i.e., the production of lines of tension, is the same. Thus while chemical factors are undoubtedly important in new-vessel growth, this study emphasises the often neglected role of physical influences on neovascularization. in Optical Properties of Skin During PlJVA Therapy. J. A. COTTERILL, J. DAWSON,* J. W. FEATHER,* AND K. RYATT, Departments of Dermatology and *Medical Physics, The General Infirmary at Leeds, Leeds LSl 3EX, England.
5. Changes
Reflectance spectrophotometry was used to derive indices of haemoglobin and melanin content of both psoriatic lesions and adjacent clinically normai skin in five patients under going photochemo-
234
MICROCIRCULATORY
SOCIETY
ABSTRACTS
therapy. The pretreatment haemoglobin index of a lesion was, on average, three times greater than that of clinically normal skin. Within 2 days of the start of treatment an increase in haemoglobin content of the uninvolved skin was observed while that of the lesion decreased. As treatment progressed the haemoglobin index of uninvolved skin at first increased, plateaued, and then decreased, whilst melanin pigmentation increased steadily. The rapid decrease in the haemoglobin index of lesions continued for the first 2 weeks of treatment after which the change in index followed the pattern of the adjacent unaffected areas of skin. This marked change in response of the lesion after 2 weeks may mean that continued ultraviolet radiation may be unnecessary beyond this time. This possibility is under active investigation. Melanisation appeared earlier in uninvolved skin and remained higher throughout the course of the treatment than that of the psoriatic lesion.
of Irradiation on Blood Flow in Split-Thickness Skin Grafts in the Pig. CAROLINE YOUNG AND J. W. HOPEWELL, Churchill Hospital Research Institute, University of Oxford, Oxford OX3 7LJ, England.
6. The Effects
The radiosensitivity of free skin grafts is generally considered to be greater than that of normal skin. Clinically, this may result in grafted areas being excluded from an irradiated field receiving a fully therapy dose. However, the scientific evidence on which this practice is based appears to be poor, so the present study was designed to compare change in normal and grafted pig skin following irradiation. Split thickness skin grafts (approximately 0.07 mm thick), were cut with an electrodermatome from the backs of 3-month-old pigs. The grafts were returned to their original beds when bleeding had been controlled. Revascularisation of grafts was monitored using an isotope clearance technique (Young and Hopewell, 1980, Microvasc. Res. 20, 182-194). Measurements were made at regular intervals from 2 to 21 days after surgery. Twenty-one days after surgery, areas of grafted and normal skin were irradiated with 1800 or 2340 rad 250-kVp X-rays. The radiation reactions were recorded weekly for 20 weeks using a skin scoring system and at 3, 6, 12, and 26 weeks after irradiation using isotope clearance. Revascularisation of the skin grafts became apparent 3 days after surgery. Initially isotope clearance in the grafts was slower than in normal skin but became faster from 10 to 21 days. Following radiation treatment, isotope clearance in the irradiated graft was also faster than in irradiated normal skin. The visible skin reaction in the graft was less than that in normal skin. These findings indicate that established skin grafts are less sensitive to radiation than normal skin, possibly as a result of the increased vascular density in the graft.
7. Reflectance
Spectrophotometry
and Bioassay
of Topical
Corticosteroid
Blanching
Activity.
RYAT~, J. DAWSON,* J. W. FEATHER,* AND J. A. COTTERILL, Departments of Dermatology *Medical Physics, The General Infirmary at Leeds, LSl 3EX, England.
K. S. and
Reflectance spectrophotometry was used to determine the degree of blanching produced by topical steroids. The intensities of reflected light from skin (fl and a white tile (1,) are measured at selected wavelengths and the logarithm of the invtrse of the ratio IdI, the LIR, is obtained which approximates to absorbance. The area under the LIR spectral curve between 510 and 610 nm in used to calculate an erythema index (Dawson et al., 1980, Physiol. Med. Biol. 25, 695-709) and from the ratio of post-treatment to pretreatment erythema indices, a blanching index is derived which was used to assess the blanching efficacy of several widely used steroid ointments and their bases. The following local steroids and bases were assessed and their blanching indices at 8 hr are shown in brackets. Dermovate ointment (0.49), Metosyn ointment (0.52), Betnovate ointment (0.65), Eumovate ointment (0.83), Metosyn ointment base (0.92), Efcortelin ointment (1.05), Betnovate 1:4 in ung. emulsificans 4 weeks old (0.90), 3 weeks old (0.91), 2 weeks old (0.82), 1 week old (0.80) and fresh Betnovate 1:4 in ung. emulsificans (0.65). (Data for the diluted form of Betnovate are from 6 subjects as opposed to 10 subjects for other steroids.) Steroid-induced blanching can be quantitated with a standard error of the mean of 5%, using reflectance spectrophotometry. The results show ranking of different steroids, and deterioration in blanching of the diluted form within a week. This technique is time consuming and requires elaborate equipment, but the results obtained are reproducible and smaller changes in the degree of blanching are detected than is possible by experienced observers using the naked eye. The results were in agreement with those of other workers using subjective assessment.
MICROCIRCULATORY
SOCIETY
ABSTRACTS
235
8. An Ultrastructural Study of Human Arterioles with Particular Reference to Their Innervation. CHRISTOPHERJ. JONES AND JANET E. KENDALL, Department of Zoology, University of Durham, Science Laboratories, South Road, Durham DHl 3LE, England. Our previously reported ultrastructural studies of human breast material (Jones and Kendall, 1980, Microvasc. Res. 20, 253) have now been extended to include 21 arterioles in specimens from seven patients and both normal and malignant areas have been examined. In all cases, no periarteriolar axons were observed, indicating that blood flow through the breast in the age group studied (34-63 years) is not controlled by autonomic nerves running along the vessel length. The possibility remains, however, that axons supply the vessels at discrete sites and we are currently searching for an adrenergic population using catecholamine fluorescence histochemistry. In contrast, studies of arterioles in skin and axillary lymph node have revealed a perivascular innervation. Staining techniques coupled with electron microscopy have indicated an adrenergic component and we are presently trying to determine whether any other populations are present. (The support of the British Heart Foundation is gratefully acknowledged.)
9. Prostaglandins, Leukotrienes, and Inj?ammation. Homerton Grove, London E9, England.
M. W. GREAVES, Institute
of Dermatology,
Prostaglandins appear to play an important role as mediators of inflammation since they fulfill the major criteria for proinflammatory mediators. The role of leukotrienes and other hydroxy fatty acid products is much less clear although their participation in cellular events seems probable. Steroid and nonsteroidal antiinflammatory drugs owe their activity at least in part to inhibition of biosynthesis of prostaglandins, although these two classes of drugs appear to act at different points in the pathways. It is becoming clear that the oxygenation of arachidonic acid leads to formation of a multiplicity of pharmacologically active fatty acids of which only a fraction have thus far been identified.
10. The Effects of Vasoactive Agents on Human Endothelial Cell Growth In Vitro. R. A. FRASER, W. II. ELMSLIE, AND J. G. SIMPSON,Department of Pathology, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, Scotland. Malignant tumours are dependent on a newly formed microcirculatory network for nutrition, growth, and blood spread. It has been suggested that neovascularisation is produced by tumour angiogenesis factor (TAF) secreted by the tumour itself. The mode of action of TAF has not yet been characterised. We have previously shown that TAF and certain vasoactive agents cause increased vascularity in the chick chorioallantoic membrane (CAM). To determine if this neovascularisation could be due to a direct effect on endothelium, the same vasoactive substances have now been used to supplement the growth medium of human endothelial cells obtained by collagenase digestion of umbilical veins; culture was established in T25 flasks using medium 199 supplemented with 20% foetal bovine serum. When almost confluent, the cells were subcultured into 35mm dishes at lo5 cells/dish using the same medium. After 24 hr to allow cell attachment, the medium was changed to one supplemented with ATP, ADP, SHT, adrenaline, or histamine at a concentration of lo-’ or 10m6 M. Five days later the total cell count was measured in a Coulter counter. Cultures supplemented with ADP, 5HT, or histamine had higher cell counts compared to control cultures, whereas those supplemented with ATP or adrenaline had lower counts; in very few instances, however., did the cell count reach the starting level. Those vasoactive agents which produced the higher endothelial cell counts were also those which had produced the greatest neovascularisation in the CAM. Since these agents are found in mast cells and platelets, it is possible that these cells not only play a role in maintaining normal vascular integrity but are also involved in the in vivo mediation of the effects of TAF.
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SOCIETY ABSTRACTS
11. Changes
in Blood Rheology following Venesection. T. CHALLONER, C. BRIGGS, M. W. RAMPLING, J. A. SIRS, AND D. J. THOMAS, Departments of Biophysics and Neurology, St Mary’s Hospital
Medical School, Paddington, London W2 IPG, England. There are several reports of the advantages of lowering blood viscosity by venesection. The variation of the component factors in blood viscosity following venesection have not been individually studied. Three patients with a previous history of vascular disease whose haematocrit was 48% or more were studied during routine treatment by venesection. Blood (250 ml) was withdrawn on the first and second day and subsequently each week until the haematocrit was reduced to 40%. Wholeblood viscosity at low shear rate (0.675 see-I), as measured by Contraves viscometer, fell on average 45%, and at high shear rate (230 see-I), using a Wells-Brookfield viscometer, from about 5 to 4 cP. Plasma fibrinogen was only significantly altered in one patient in whom it fell from 375 to 300 mg/ dl. No significant changes were noted in albumin and total globulin or platelet adhesion. Plasma viscosity at 37.3” and 230 set-’ changed from about 1.5 to 1.4 cP. Erythrocyte flexibility, measured by the centrifuge technique, progressively decreased to less than half its normal value. This is possibly due to progressive loss of iron, though no change in MCV and MCH occurred in the first few weeks. Only at the end of treatment were the patients becoming iron deficient as assessed by routine laboratory techniques. 12. Characterisation Radiometric
of the Molecular Heterogeneity Method. M. J. SEGHATCHIAN AND
of FVIII
L. J.
MACKIE,
by Electrophoresis
Using
a Simplljied
North London Blood Transfusion
Centre, Edgware, Middlesex, England. With the recent progress in uncovering antibodies which react separately and specifically against different parts of the FVIII molecule, immunoradiometric assay of FVIII has become the most productive technique in evaluation of FVIII protein. Since radiometric methods measure the concentration of protein having FVIII antigenic determinants, even though it may lack FVIII procoagulant activity, we have attempted to compare the selectivity of the currently used methods for the assay of FVIII-related activities using an agarose gel electrophoresis method (M. J. Seghatchian, 1980, Z’hromb. Res. 19, 757-766). The molecular distribution of several clinical concentrates (FVIII and FIX) as well as blood from patients with coagulation abnormalities investigated with and without preincubation with 1251-anti-VIII:C (I&i or FAB) and parallel assays were performed on the electrophoretically separated fractions. In FVIII concentrates w$h high VIIIR:Ag (Z-30 III/ml) two or more VIIIR:Ag peaks were seen. In the presence of IiG or FAB, only one main peak of radiolabelled VIII:C was noted, which corresponded with the larger molecular forms of VIIIR:Ag. FIX concentrates were also heterogeneous and consisted of an altered form of FVIII with a faster mobility and with both VIII:C functional and VIII:Cag neutralization activities. The method was also found suitable for identifying FVIII:C abnormality in patients with various coagulation disorders and since only small sample volumes are required it could be used on finger prick and foetal samples. Considerable differences existed between biological assays, immunological and radiometric methods. It is concluded that various forms of FVIII, which may represent a native subpopulation or proteolytic breakdown products, are not measured to the same extent by various methods. Based on this in vitro analysis it seems to be possible to predict which preparation can be used successfully for therapy as well as investigating the in vivo or in vitro proteolytic degradation of FVIII molecule. 13. The Trafuril Reaction. OX3 7JH, England.
T. S.
SONNEX
AND
T. J. RYAN, The Slade Hospital, Headington, Oxford
For several years Trafuril has been sold over the counter as an agent that will increase blood supply to the skin. During this time many researchers have tried to determine the mechanism by which the associated hyperaemia and oedema are produced (Thune et al., 1976, Acta Dermatol. (Stockholm), 56, 217-221), and this literature is reviewed. The problem is still unresolved although involvement of prostaglandins and fibrinolysis would explain many of the observations. We have investigated the action of Trafuril in normal subjects using a cell-mediated hypersensitivity system. When an allergen is applied under an occlusive chamber to the back of an individual who is allergic
MICROCIRCULATORY
SOCIETY
ABSTRACTS
237
to this substance, an area of erythema and oedema (? vesicles) is produced within 48-96 hr-a “positive patch test reaction.” This is a manifestation of cell-mediated immunity and although the inflammatory mediators remain unclarified reports have indicated the importance of prostaglandins and lymphokines. All patients attending the industrial dermatitis clinic at the Slade Hospital were tested to various substances in this manner after the application of Trafuril (thurfyl nicotinate ointment). The same procedure on the other half of the patient’s backs without Trafuril acted as a control. Three times as many positives and almost four times the intensity of reactions were obtained in the pretreated area. Using the same technique these reactions were reduced in number and intensity if the patients were ingesting prostaglandin synthetase inhibitors-aspirin or indomethacin. A similar increase in “positives” has been reported if the test area is preirradiated with UVB. Inflammation produced by UVB is believed to be mediated by prostaglandins (Sdndergaard and Greaves, 1970, J. Path/. 101, 93) and is inhibited by prostaglandin synthetase inhibitors (Snyder and Eaglestein, 1974, J. Invest. Dermatol. 62, 47). From the results of our work and by a direct comparison with the UVB findings it is inferred that Trafuril inflammation is mediated by an effect on prostaglandins and/or lymphokines.