Blood flow in the rabbit tenuissimus muscle Influence of preparative procedures for intravital microscopic observation

LINDBOM, L., TUMA, R. F. & ARFORS, K.-E.: Blood flow in the rabbit tenuissimus muscle: Influence of preparative procedures for intravital microscopic observation. Acta Physiol Scand 1982, 114 :121–127. Received 21 April 1981. ISSN 0001–6772. Department of Experimental Medicine, Pharmacia AB, Uppsala, Sweden. The tenuissimus muscle in the rabbit and the cat is a suitable tissue for intravital microscopic investigation of skeletal muscle blood flow. In this study the influence of surgical procedures necessary for direct microscopic observation on the physiological state of the rabbit tenuissimus muscle was assessed by means of blood flow measurements. Mean resting blood flow was 2.8±0.8 (mean ± S.D.) ml. min^-1. 100 g^-1 in the left tenuissimus muscle when prepared for microscopic observation as determined by the radioactive microsphere method. This value was not significantly different from that in the intact unexposed muscle in the contralateral leg, 3.3 ±1.1 ml. min^-1. 100 g^-1. Exposure of the muscle to atmospheric oxygen tension resulted in a reduction of blood flow to 0.7±0.4 ml. min^-l. 100g^-l, suggesting that local metabolic control mechanisms were active. The normal range of vascular control seemed to be maintained, as demonstrated by an increase in blood flow to 64.2± 18.8 ml. min-¹. 100 g^-l during “maximal” vasodilation induced by topical application of PGE₁. The tenuissimus muscle showed a marked sensitivity to mechanical stimulation. Slight stretching of the muscle, similar to what may occur during surgical preparation, resulted in an increase in blood flow to 17.5±5.7 ml. min^-1. 100 g^-1. Flow values calculated from data obtained by direct microscopic measurements in the tenuissimus muscle agreed well with those obtained by the microsphere method.     

Cytokine flow cytometry differentiates the clinical status of multiple sclerosis (MS) patients

In this study we have examined intracellular cytokines in peripheral blood mononuclear cells (PBMC) of MS patients by flow cytometry (cytokine flow cytometry). MS progressive patients showed an increased number of cells producing interferon-gamma (IFN-gamma) after activation with phorbol 12-myristate 13-acetate and ionomycin, compared with patients with clinically inactive forms (P < 0001) and with healthy controls (P = 0001). These cells belonged to the CD4+ and CD8+ subsets in similar proportions. Clinically inactive patients showed a lower level of cells producing IL-2 than controls (P = 0.03) and active MS patients (P = 0.03). Most IL-2-producing cells were CD4+ lymphocytes, although a small part of the IL-2 was also produced by CD8+ cells. The percentage of cells producing simultaneously IL-2 and IFN-gamma was increased in active MS and they were mainly CD4+ lymphocytes. No differences in the production of IL-4 were observed between groups. However, we found an increased IL-10 production in clinically active MS patients (P = 0.03). Treatment with IFN-beta of active MS patients showed lower levels of cytokines when compared with untreated MS patients. This methodological approach could help in the follow up and therapeutic monitoring of MS patients.     

Positive selection is an early event in thymocyte differentiation: high TCR expression by cycling immature thymocytes precedes final maturation by several days

T cell antigen receptor expression by cycling and post-cycling thymocytes has been analysed by flow cytometry. Normal mice were pulsed with 5-bromo-2'-deoxyuridine (BrdUrd), a thymidine analogue detectable with a monoclonal antibody. Thymocytes were surface-stained with antibodies against several V beta gene products and against whole alpha beta receptors and detection of BrdUrd in the nuclei was performed after enzymatic generation of single-stranded DNA. A significant (10%) percentage of thymocytes expressing high levels of alpha beta TCR were found in the cycle: these cells were immature, as shown by the CD4+8+ phenotype and by high HSA expression. After division, most alpha beta high BrdUrd+ cells entered a resting state and their number remained constant for 3 days, decreasing in two steps thereafter. This post-mitotic evolution was not modified by injection of an anti-mitotic drug. After day 4, a majority of the studied subset acquired a single positive phenotype. Location of BrdUrd+ V beta 8.2 high cells studied on frozen sections was found cortical at early times and medullary after day 3. V beta 6 expression by cycling and post-cycling thymocytes was analysed in various mouse strains, and early high expression by cycling thymocytes was found to be restricted to MIs 1b strains. These results suggest that high alpha beta TCR expression by cycling immature thymocytes corresponds to positive selection, which must therefore be considered as an early event in intrathymic differentiation.     

Effects of indomethacin on regional blood flow in conscious rabbits—a microsphere study

In an attempt to reveal the importance of prostaglandins in the control of regional blood flow 20 mg/kg b.wt. indomethacin was given i.v. in conscious resting rabbits. Regional blood flow determinations were made before and 20 min after the injection using the labelled microsphere technique. The blood flow in the stomach wall was reduced by 0.75 ± 0.17 g·min^-1·g^-1 from a level of 1.64 ± 0.24 g·min^-1·g^-1. In jejunum the corresponding figures were 0.44 ± 0.12 and 1.26 ± 0.17 and in the brain 0.29 ± 0.10 and 1.24 ± 0.10. The blood flow in the liver via the hepatic artery increased by 0.20 ± 0.02 g·min^-1·g^-1 from a level of 0.13 ± 0.02 g·min^-1·g^-1. In the retina there was a reduction in blood flow by 2.75 ± 1.03 mg·min^-1 from a starting level of 15.1 ± 2.3 mg·min^-1. In a number of other tissues investigated there were no significant effects of the drug. The results suggest that under resting conditions prostaglandins play a role in the control of blood flow in the gastrointestinal tract, the brain and the retina—tissues which are likely to be rather active under such conditions.     

Intraaxonaler transport von Ethidium-Bromid-sensitiven RNS- und niedermolekularen 3H-Uridin-Verbindungen im tractus opticus von teleosteern

Zusammenfassung Am Sehtrakt von Carassius carassius und Scardinius erythrophthalmus wurde die axonale Ausbreitungsweise hoch- und niedermolekularer 3H-Uridin-Verbindungen untersucht. Dabei wurde nach intraocularer Injektion des Tracers für TCE-resistente Verbindungen eine intraaxonale Transportgeschwindigkeit von 2–4 mm/d bestimmt, für TCE-lösliche Verbindungen eine von ca. 30–50 mm/d. Durch Applikation des spezifisch mitochondrialen RNS-Synthese-Hemmers Ethidium-Bromid konnte die Einbaurate von 3H-Uridin in hochmolekulare RNS um 70–80% erniedrigt werden, was dafür spricht, daß die langsam im Axoplasma wandernden Mitochondrien einen Großteil der axonalen RNS synthetisieren.In der TCE-löslichen Fraktion konnten durch dünnschichtchromatographische Analyse noch nach 8d p. i. 3H-Uridin und 3H-UDPG nachgewiesen werden. Dieser Befund wird hinsichtlich eines transneuronalen Stoffübertritts von Uridin und der möglichen Bedeutung des UDPG-Transportes im Nervengewebe diskutiert.

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Intraaxonal transport of ethidium-bromide-sensitive RNA- and lowmolecular 3H-uridine-compounds in the optic tract of teleost

Summary In the optic system of teleosts (Carassius carassius and Scardinius erythrophthalmus) the axonal flow of high and low molecular 3H-uridine-compounds was investigated. After injection of the tracer into one eyeball and TCA-extraction of the samples a transport-rate of 2–4 mm/d was demonstrated. By the specific inhibitor of mitochondrial RNA-synthesis, Ethidium-Bromide, the amount of axonal radioactivity could be reduced to 20–30% of the control. This indicates the mitochondria as being the site of synthesis most of axonal RNA. Considering the TCA-soluble 3H-uridine-compounds, an intraaxonal flow also could be demonstrated, with a transport-rate of 30–50 mm/d, 16 times higher as the one of RNA. The analysis by thin layer chromatography indicated the existence of 3H-uridine and 3H-UDPG in the axonal fraction of TCA-soluble compounds still after an incorporation time of 8d. The possibility of a transneuronal convection of uridine and the function of UDPG-transport in the axon are discussed.

Frau Prof. Dr. H. Kersten (Erlangen) danke ich für die Überlassung einer Probe Ethidium-Bromid.     

平行平板流动腔的合理设计和使用

高度远小于横向和纵向几何尺寸的平行平板流动腔，是当前用以体外研究细胞力学行为，特别是细胞粘附特性的主要工具之一。本文从目前常用的平行平板流动腔内流体定常流动时的切应力表达式出发，指出该切应力表达式仅适用于小雷诺数条件，由此确定平行平板流动腔几何尺寸的合理选择；通过对小孔入流和出流的平行平板流动腔流场的分析，指出该切应力表达式只在远离孔口边界一定距离，即达到均匀流动后成立。     

优化原理与小动脉血流量─管径关系

作者假设血液为符合幂定律的非牛顿流体，对小动脉血管系统进行了详细的优化分析。导出了血流量与管径立方成正比的结论以及壁面切变率与管径无关的推论。此结果既包含了前人对于牛顿血流的研究成果，又能克服后者的不足，而与近期的生理测定一致。     

Antibody penetration of viable human cells. I. Increased penetration of human lymphocytes by anti-RNP IgG.

Antibody penetration of viable cells and interaction with intracellular antigens may have major consequences for immunopathological processes in connective tissue diseases. We have reported previously that antibody can penetrate viable human lymphocytes. To assess further the role of antinuclear antibodies in this process, peripheral blood lymphocytes (PBMC) were incubated with FITC-conjugated IgG fractions from sera containing anti-RNP (anti-RNP IgG), Ro(SS-A), La(SS-B) and dsDNA antibodies and control sera for 24 h. Using crystal violet to quench cell surface staining, intracellular fluorescence of viable lymphocytes was quantified on the flow cytometer. It was noted that anti-RNP IgG entered 46.4 +/- 7.2% of lymphocytes which was significantly higher than anti-Ro(SS-A) (29.9 +/- 4.1%, P less than 0.05), La(SS-B) (22.0 +/- 7.5%, P less than 0.01) IgG and control IgG (28.8 +/- 2.1%, P less than 0.05) and not statistically different from anti-dsDNA IgG (32.6 +/- 14.3%). Inhibition experiments showed that the increased number of cells penetrated by anti-RNP IgG was a specific process. Time-course studies showed that anti-RNP IgG entry into cells was different from pooled control IgG. With anti-RNP IgG, positive-staining lymphocytes gradually increased in number from 12 to 24 h incubation, whilst with pooled control IgG, the peak was reached within 5 min. Dual staining experiments suggested that whereas both anti-RNP IgG and pooled control IgG entered B and NK cells, anti-RNP IgG also entered T cells. Using IgG F(ab')2 and Fc fragments from either anti-RNP IgG or pooled control IgG to compete with their FITC-conjugated counterparts indicated that the entry of anti-RNP IgG into-viable cells appeared to involve both F(ab')2 and Fc fragments, and pooled control IgG depended exclusively on the Fc portion of IgG. Further investigation by incubating anti-RNP IgG with 35S-methionine-labelled monocyte-depleted PBMC (MD-PBMC) suggested that anti-RNP IgG might react with the corresponding antigens either on the cell surface or within the cytoplasm.     

IgG anti-D MAbs in tests by flow cytometry

Seventy-two IgG anti-D Mabs were studied by indirect immunoflorescence and flow cytometry (FCM) with R₁r, rr and various D variant phenotype red blood cells (RBCs) (D^IIIb, D^VI, D^VII, Rh33 and uncategorized variant ISBT number 48). The results were compared with those obtained by indirect antiglobulin test (IAT).     

Primary renal angiosarcoma: A case report and review of the literature

Primary renal angiosarcoma is very rare. To our knowledge, only 15 cases have been reported to date. A 77-year-old Japanese man with a unilateral kidney presented with massive hematuria followed by renal failure. A renal tumor was suspected and a left nephrectomy was performed. The histopathological diagnosis was angiosarcoma of the kidney. A hemorrhagic tumor measuring 10 × 5 cm and clotted blood was found in the modularly area. The atypical tumor cells had a sinusoidal and solid appearance, and showed Immunohistochemically positive reactions for some of the endothelial markers. The patient died about 21 months after the nephrectomy and the autopsy revealed massive metastases to the liver and retroperitoneum. One of the differential diagnoses of the case was anglomyolipoma, because the tumor cells were relatively bland in their histological appearance with entrapped fat cells in the pelvic area. Fifteen case reports with titles that included the term 'hemangiosarcoma/anglosarcoma', 'hemangioendothelloma/endothelloma' or 'vascular sarcoma' of the kidney were reviewed and compared to the present case.