Changes in interstitial pressure during and after blood volume expansion in rats

Summary Interstitial fluid pressure was measured via a chronically implanted capsule before, during and after acute isotonic, iso-oncotic blood volume expansion in normal or in 48-h dehydrated rats. At the same time, the patterns of body fluid distribution, of selected renal responses and of mean arterial and mean central venous pressure responses were studied. Dry tissue weight (DTW) was subsequently determined by freeze drying of the shaved carcass.Dehydration decreased plasma volume and interstitial fluid volume significantly below normal values. The initial intracapsular pressure in dehydrated animals (–3.7±0.6 mm Hg) was not significantly different from that in normal rats (–2.5±0.5), but dehydrated rats showed initially a very significantly lower effective interstitial compliance (0.0005 ml/mm Hg per gram DTW) than did the normal group (0.0704).In the course of the renal response to the volume load, effective interstitial complicance increased to 0.0350 in dehydrated rats but showed no change in normal rats. Neither group completely corrected its elevated blood volume; both returned their central venous pressures to pre-infusion levels; both decreased their interstitial fluid volumes below preinfusion levels and both decreased their intracapsular fluid pressures 1 mm Hg below the level prevailing in non-infused animals at that time.It is concluded that a reduction in interstitial, hydrostatic pressure can be a functionally important influence in the apparent control of central venous pressure following acute blood volume expansion.This study was supported by the Canadian Heart Foundation.     

Morphometric evaluation of volume shifts between intra- and extra-cellular space before and during global ischemia

Background: It is well known that all forms of cardiac arrest lead to global ischemia combined with alterations in cellular and interstitial volume. The aim of this study was to investigate the nature of these alterations with respect to different methods of cardiac arrest and establish the extent of their mutual influence at the onset as well as during the course of global ischemia. Methods: Three tested clinical methods were employed to induce cardiac arrest by a) aortic cross clamping, b) coronary perfusion with the cardioplegic solution St. Thomas, and c) coronary perfusion with the cardioplegic solution histidine-tryptophane-ketoglutarate (HTK). The arrested hearts were subjected to global ischemia at 25°C. The size of the myocytes, as well as the interstitial space of myocytes, was determined morphometrically. The contraction state of myocytes was evaluated according to a score. Results: We found that the degree of contraction, as well as nature of alterations in the cellular and interstitial volumes, depended both on the form of cardiac arrest and on the duration of ischemia. The following relationships were established. High contraction at the onset of ischemia leads to expulsion of fluid from the interstitium between bundles of myocytes into the tissue clefts increasing their size. The decrease in contraction during ischemia leads to narrower tissue clefts. Cellular swelling at the onset of and during ischemia is caused by volume shifts between intracellular and interstitial space. An increase in cellular volume during global ischemia and/or additional contraction reduce the interstitium within bundles of myocytes. Sufficient relaxation and/or interstitial edema enlarge the interstitium. Conclusions: Cellular and intersticial alterations seen at the onset and during the course of ischemia are dependent upon the method of cardiac arrest. Furthermore, a considerable mutual influence is exerted by the alterations in cellular and interstitial spaces. © 1995 Wiley-Liss, Inc.     

Compliance of the interstitial space in rats I. Studies on hindlimb skeletal muscle

Interstitial compliance, defined as the ratio between changes in interstitial fluid volume (ΔIFV) and interstitial fluid pressure (ΔIFP), was determined for rat skeletal muscle. IFV was measured as the extravascular distribution space for ⁵¹Cr-EDTA, while sharpened micropipettes connected to a servo-controlled counterpressure system were used to measure IFP. The experimental protocol was designed to bring about acute (2–4 h) and chronic (24–28h) tissue over- and dehydration. During dehydration, the average compliance was 0.056 ml/g dry weight · mmHg, corresponding to 1.40 ml/100 g wet tissue mmHg, and was not significantly different in acute and chronic experiments. In hydration (acute and chronic), compliance increased several-fold when IFV increased. Even at greatly increased IFV, IFP did not rise more than 1 to 1.5 mmHg above control level. Since control IFV amounts to 10 ml/100 g wet tissue, IFV will decrease by 14% when IFP falls by 1 mmHg from this control level. Provided unchanged interstitial protein mass the dehydration will cause interstitial fluid colloid osmotic pressure to increase by somewhat more than 1 mmHg—from a control level of 9 mmHg. Furthermore, since IFP was not increased by more than 1 to 1.5 mmHg during hydration, an increase in IFP plays a minor role in edema-prevention compared to dilution and/or washout of interstitial proteins.     

Interstitial exclusion of albumin in rabbit lung during development of pulmonary oedema

The modifications of the macromolecular sieving properties of the pulmonary extracellular tissue matrix were studied in adult anaesthetized rabbits ( n = 10) exposed to increased tissue hydration. Exclusion of albumin from the perivascular pulmonary interstitial space was determined by using the continuous infusion method coupled with direct sampling of interstitial fluid performed through the wick technique. The rabbits underwent an intravenous infusion of saline amounting to 10 ( n = 5) or 20 % ( n = 5) body weight. Extracellular albumin distribution volume was derived from the steady state tissue concentration of radioactive rabbit serum albumin (¹²⁵I-RSA). Pulmonary extracellular and intravascular fluid volumes ( V _x and V _v, respectively) were measured as distribution volumes of ⁵¹Cr-EDTA and ¹³¹I-RSA, respectively, and interstitial fluid tracer concentrations were determined in interstitial fluid collected through implanted wicks. At the highest degree of hydration, interstitial fluid volume ( V _i= V _x− V _v) and extravascular albumin distribution volume ( V _a,w) significantly increased by 38.5 and 240.2 %, respectively, compared to control. Albumin-excluded volume ( V _e,a= V _i− V _a,w) was 398.9 ± 17 μl (g dry tissue weight)⁻¹; the albumin-excluded volume fraction ( F _e,a= V _e,a/ V _i) was 0.23 ± 0.01, 33.2 % of the control value. Data indicate that, at variance with what is observed in tissues like skin and muscle, pulmonary F _e,a is highly sensitive to tissue fluid content.     

Impression technique for the assessment of oedema: comparison with a new tactile sensor that measures physical properties of tissue

To measure tissue oedema, the impression technique and a new tactile sensor technique are compared and evaluated in a silicone rubber model and in an in vivo rat testis model. The principles of the two techniques differ in that the impression technique evaluates interstitial fluid flow FT and peak force F(0) when tissue is compressed, whereas the tactile sensor evaluates the hardness/softness or change in resonance frequency Δf when a vibrating rod is attached to tissue. Both techniques can detect changes in silicone hardness/softness or in hormone-induced changes of testes, interstitial fluid. Although both F(0) and FT are significantly correlated to Δf in the experiments, it is concluded that F(0) is the most promising impression parameter to give valuable information about the hardness of living tissue as compared with Δf. The comparison indicates that the impression technique in the most easy, to interpret, non-invasive tool to assess tissue oedema so far developed.     

The interstitial distribution of macromolecules in rat tumours is influenced by the negatively charged matrix components

Knowledge of macromolecular distribution volumes is essential in understanding fluid transport within normal and pathological tissues. In this study in vivo we determined the distribution volumes of several macromolecules, including one monoclonal antibody, in tumours and tested whether charges associated with the tumour extracellular matrix influence their available volumes. Steady state levels of the monoclonal antibody trastuzumab (Herceptin) (pI = 9.2), IgG (pI = 7.6) as well as native (pI = 5.0) and cationized albumin (pI = 7.6) were established in rats bearing dimethylbenzanthracene (DMBA)-induced mammary tumours by continuous infusion using osmotic minipumps. After a 5–7 day infusion period, the rats were nephrectomized and the extracellular volume was determined with ⁵¹Cr-labelled EDTA. Plasma volumes were measured with ¹²⁵I-labelled human serum albumin or rat IgM in a separate series. Steady state concentrations of probes were determined in the interstitial fluid that was isolated by centrifugation from tumours or by post mortem wick implantation in the back skin. Calculations were made for interstitial fluid volume ( V _i), along with the available ( V _a/ V _i) and excluded ( V _e/ V _i) relative interstitial volume fractions. The V _e/ V _i for the positively charged trastuzumab in tumours averaged 0.29 ± 0.03 ( n = 16), a value which was significantly lower than the corresponding one for IgG of 0.36 ± 0.02 ( n = 16). Native albumin was excluded from 38% of the tumour interstitial fluid, whereas cationization of albumin reduced the excluded volume by ∼50%. Our experiments suggest that the tumour interstitium acts as a negatively charged matrix and is an important factor in determining the macromolecular distribution volume.     

Interstitial fluid flow in tendons or ligaments: A porous medium finite element simulation

The purpose of this study is to describe interstitial fluid flow in axisymmetric soft connective tissue (ligaments or tendons) when they are loaded in tension. Soft hydrated tissue was modelled as a porous medium (using Darcy’s Law), and the finite element method was used to solve the resulting equations governing fluid flow. A commercially available computer program (FiDAP) was used to create an axisymmetric model of a biomechanically tested rat ligament. The unknown variables at element nodes were pressure and velocity of the interstitial fluid (Newtonian and incompressible). The effect of variations in fluid viscosity and permeability of the solid matrix was parametrically explored. A transient loading state mimicking a rat ligament mechanical experiment was used in all simulations. The magnitude and distribution of pressure, stream lines, shear (stress) rate, vorticity and velocity showed regular patterns consistent with extension flow. Parametric changes of permeability and viscosity strongly affected fluid flow behaviour. When the radial permeability was 1000 times less than the axial permeability, shear rate and vorticity increased (approximately 5-fold). These effects (especially shear stress and pressure) suggested a strong interaction with the solid matrix. Computed levels of fluid flow suggested a possible load transduction mechanism for cells in the tissue.     

单侧输尿管梗阻肾间质纤维化模型大鼠与川芎嗪的干预

背景：肾间质纤维化与组织基质金属蛋白酶2和金属蛋白酶组织抑制因子2表达失衡有关。 目的：建立单侧输尿管梗阻肾纤维化模型,观察川芎嗪治疗后肾间质病理变化及肾组织组织基质金属蛋白酶2和金属蛋白酶组织抑制因子2的表达变化。 方法：24只雌性SD大鼠分为3组,除假手术组外,模型组及川芎嗪组均在无菌条件下行左侧输尿管结扎建立单侧输尿管梗阻模型,川芎嗪组于术前1 d开始灌胃给药,40 mg/(kg•d),1次/d,连续2周。术后14 d处死各组大鼠,留取梗阻侧肾组织行苏木精-伊红染色和Masson染色以观察肾组织病理改变,免疫组化和反转录-聚合酶链反应方法检测肾组织基质金属蛋白酶2、金属蛋白酶组织抑制因子2的表达水平。 结果与结论：川芎嗪组可明显减轻肾小管的扩张和萎缩,减轻肾间质纤维组织增生及炎性细胞浸润。与假手术组相比,模型组基质金属蛋白酶2、金属蛋白酶组织抑制因子2蛋白和mRNA基因表达均明显增加(P < 0.01);川芎嗪组上述物质表达较模型组明显减少(P < 0.01)。提示川芎嗪通过下调基质金属蛋白酶2、金属蛋白酶组织抑制因子2的表达,从而改善单侧输尿管梗阻大鼠的肾间质纤维化。     

The influence of muscle interstitial volume on K+-induced heart rate drive in rats

Summary During exercise heart rate is influenced by reflexogenic drives which are elicited by receptors situated in the interstitial space. Since the structure of interstitial tissue is complex (e.g. fixed negative charges of glycosaminoglycans), the situation in the immediate surrounding of the receptors might differ from the free fluid phases of blood or lymph in which the concentrations of stimulating substances are usually determined. Physiological variations of the interstitial structure may be due to changes in interstitial volume induced by exercise or the hydrostatic effects on body fluids. The objective of the present study was to investigate the effect of the interstitial volume on the relationship between heart rate and K⁺ stimuli applied through the muscle blood vessels.The calves of 12 male Wistar rats were artificially perfused and separated from the rest of the body with the sciatic nerve remaining intact. In these preparations the heart rate (HR) responses to low (4 mM) and high (8 mM) potassium concentrations were determined at different interstitial volumes. Expansion of the interstitial volume was obtained by reducing the colloid-osmotic pressure of the perfusate. The combination of intracellular oedema and mechanical limitation of total volume expansion (tapeing) was utilized to decrease the interstitial volume.When switching between the low and high potassium concentrations, significant heart rate responses could be observed only with reduced interstitial volume. It is suggested that the interstitial structure surrounding the muscular receptors modifies the relationship between heart rate response and the K⁺ stimuli determined in blood or lymph.This study was supported by a grant of the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen     

Oxidative damage and TGF-β differentially induce lung epithelial cell sonic hedgehog and tenascin-C expression: implications for the regulation of lung remodelling in idiopathic interstitial lung disease

Idiopathic interstitial lung diseases (iILDs) are characterized by inflammation, hyperplasia of Type-II alveolar epithelial cells (AECs) and lung remodelling often with progressive fibrosis. It remains unclear which signals initiate iILD and/or maintain the disease processes. Using real-time RT-PCR and immunohistochemistry on archival biopsies of three patterns of iILD (usual interstitial pneumonitis/UIP, non-specific interstitial pneumonitis/NSIP and cryptogenic organizing pneumonia/COP) we investigated whether hedgehog signalling (previously associated with lung damage and repair) was functional and whether the damage associated extracellular matrix protein tenascin-C was present in activated Type-II AECs in all three iILDs. Using tissue culture, protein and mRNA detection we also determined how two signals (oxidative damage and TGF-β) associated with iILD pathogenesis affected Sonic hedgehog (SHH) and tenascin-C production by a Type-II AEC cell line. We report that SHH pathway and tenascin-C mRNA and proteins were found in UIP, NSIP and COP. SHH signalling was most active at sites of immature organizing fibrous tissue (fibroblastic foci) in UIP. In vitro Type-II AECs constitutively secrete SHH but not tenascin-C. Oxidative injury stimulated SHH release whereas TGF-β inhibited it. TGF-β and oxidative damage both upregulated tenascin-C mRNA but only TGF-β induced synthesis and release of a distinct protein isoform. SHH signalling is active in Type-II AECs from three types of ILD and all three express tenascin-C.     

The interstitial fluid content in working muscle modifies the cardiovascular response to exercise

Summary The volume of interstitial fluid in the limbs varies considerably, due to hydrostatic effects. As signals from working muscle, responsible for much of the cardiovascular drive, are assumed to be transmitted in this compartment, blood pressure and heart rate could be affected by local or systemic variations in interstitial hydration. Using a special calf ergometer, eight male subjects performed rhythmic aerobic plantar flexions in a supine position with dependent calves for periods of 7 min. During exercise heart rate, blood pressure, oxygen uptake (VO₂) and blood lactate concentrations were measured in two different tests, one before and after interstitial calf dehydration through limb elevation for 25 min, compared to the other, a control with unaltered fluid volume in a maintained working position. Impedance plethysmography showed calf volume to be stabilized in the control position. Leg elevation by passive hip flexion to 90° resulted in a fast (vascular) volume decrease lasting <2 min, followed by a slow linear fluid loss from the interstitial compartment. Then, when returned to the control position, adjustment of vascular volume was completed within 2 min and exercise could be performed with dehydration remaining in the interstitium only. Cadiovascular response was identical at the start of both tests. However, exercising with dehydrated calves elicited a significantly larger increase in heart rate compared to the control, whereasVO₂ was identical. The blood pressure response was shown to be only slightly enhanced. Structural interstitial features varying with hydration, most likely chemical or mechanical ones, may have been responsible for this amplification of signals.     

Body fluid distribution in man in space and effect of lower body negative pressure treatment

Summary The lack of hydrostatic forces in space eventually produces a fluid deficit within the circulatory system. This deficit may alter the circulatory regulation patterns. The aim of the present study was to determine how much of this fluid deficit is attributable to interstitial fluid losses and to determine the effects of lower body negative pressure (LBNP) treatment on fluid distribution. The body fluid distribution of one subject was assessed before, during, and after weightlessness using two electrical impedance methods: (a) standard quadripole impedance for the segments of upper torso, lower torso, thigh, and calf and (b) an electrical impedance tomography technique (applied potential tomography) for a thigh cross-section. To assess the content of interstitial free fluid a thigh cuff overlying the electrodes for applied potential tomography was inflated to suprasystolic values to ascertain how much fluid can be squeezed out of blood vessels and tissue of skin and muscle. After the first thigh cuff maneuver (CUFF I) the subject performed a cardiovascular stress test with LBNP to mimic the gravity-induced blood shift to the lower part of the body. Then the compression maneuver was repeated (CUFF II). (a) This experimental sequence demonstrated a reduction in interstitial fluid in weightlessness of roughly 40% at the thigh. (b) The CUFF I and LBNP experiment demonstrated a reduced ability to cope with blood pooling in microgravity. (c) The CUFF II experiment suggests that LBNP in microgravity can refill the interstitial spaces and counteract the associated cardiovascular deterioration. The impedance measurements provided estimates of the contribution of different body sections to the observed body weight loss of more than 6 kg. The chest contributed nothing of significance, the lower torso more than 0.5 1, and both calves roughly 1.5 1. The thigh segments of both legs contributed between 1.51 and 2.01 with an interstitial free fluid reduction in muscle and skin by 40%.Abbreviations APT applied potential tomography - AU arbitrary units - LBNP lower-body negative pressure     

On the edema-preventing effect of the calf muscle pump

Summary During motionless standing an increased hydrostatic pressure leads to increased transcapillary fluid filtration into the interstitial space of the tissues of the lower extremities. The resulting changes in calf volume were measured using a mercury-in-silastic strain gauge. Following a change in body posture from lying to standing or sitting a two-stage change in calf volume was observed. A fast initial filling of the capacitance vessels was followed by a slow but continuous increase in calf volume during motionless standing and sitting with the legs dependent passively. The mean rates of this slow increase were about 0.17%·min⁻¹ during standing and 0.12%·min⁻¹ during sitting, respectively. During cycle ergometer exercise the plethysmographic recordings were highly influenced by movement artifacts. These artifacts, however, were removed from the recordings by low-pass filtering. As a result the slow volume changes, i.e. changes of the extravascular fluid were selected from the recorded signal. Contrary to the increases during standing and sitting the calf volumes of all 30 subjects decreased during cycle ergometer exercise. The mean decrease during 18 min of cycling (2–20 min) was −1.6% at 50 W work load and −1.9% at 100 W, respectively. This difference was statistically significant (p≤0.01). The factors which may counteract the development of an interstitial edema, even during quiet standing and sitting, are discussed in detail. During cycling, however, three factors are most likely to contribute to the observed reduction in calf volume: (1) The decrease in venous pressure, which in turn reduces the effective filtration pressure. (2) An increased lymph flow, which removes fluid and osmotically active colloid proteins from the interstitial space. (3) An increase in muscle tissue pressure, which counteracts the intravascular pressure during the muscle contraction thus playing an important role as an edema-preventing factor, which has not been considered to date.     

人牙周膜细胞在左旋聚乳酸/羟基磷灰石生物材料上的生长观察

目的观察电纺左旋聚乳酸/羟基磷灰石(PLLA/HA)生物材料(简称生物材料)的生物相容性,并以人牙周膜细胞作为种子细胞与生物材料复合培养,探索该生物材料用于人牙周组织再生的可能性。方法用电纺法制备PLLA/HA纤维生物材料;组织块法分离培养人牙周膜细胞,并用免疫组织化学方法鉴定;MTT法评价生物材料的生物相容性;将人牙周膜细胞与生物材料复合培养,用扫描电镜进行观察;将转染人腺病毒介导的绿色荧光蛋白的人牙周膜细胞与生物材料复合培养,用激光扫描共焦显微镜观察。结果成功分离了人牙周膜细胞,波形蛋白染色阳性确定该细胞来源于中胚层;MTT法检测人牙周膜细胞在生物材料上的增殖状况与正常培养基本一致;扫描电镜下可见细胞在生物材料上生长旺盛、充分伸展;激光扫描共焦显微镜下可见人牙周膜细胞沿生物材料呈纤丝生长,表现出一定的三维结构。结论电纺PLLA/HA生物材料具有三维空间网状结构和良好的生物相容性,与人牙周膜细胞复合培养有利于细胞的生长、贴附和增殖,并呈现一定的立体结构,为人牙周组织再生提供了实验依据。     

Asbestosis: demonstration of distinctive interstitial fibroelastosis: A pilot study

Asbestosis has long been defined as a diffuse interstitial “fibrotic” process, in similarity to other chronic interstitial pulmonary diseases. To address the hypothesis (which was based on morphological nuances) that the interstitial connective tissue response in asbestosis may be fibroelastotic rather than fibrotic, a comparative characterization of the connective response in cases of asbestosis and other forms of interstitial lung disease was performed. Archival open lung biopsies or autopsy specimens of pulmonary diseases featuring interstitial connective tissue abnormalities (15 of asbestosis, 21 of organizing pneumonia, 15 usual interstitial pneumonitis/idiopathic pulmonary fibrosis [IPF], 9 organizing diffuse alveolar damage, 9 “nonspecific” interstitial pneumonitis, 4 sarcoidosis, 3 each of desquamative interstitial pneumonia and chronic amiodarone toxicity, 2 cryptogenic organizing pneumonias, and 1 each of chronic hypersensitivity pneumonitis and chronic eosinophilic pneumonitis [85 total]) were stained histochemically with hematoxylin and eosin, Perl's method, Gomori's trichrome procedure, and the Verhoeff-van Gieson technique. Representative subsets of the cases (n = 20) were also studied immunohistologically using an antibody to elastin. Fibroelastosis in each of the samples was assessed for the degree of response and its location using a 3-tiered scale. The degree of fibroelastosis in the 15 cases of asbestosis was variable, with the pattern being peribronchial and perivascular in all instances; at least 2 asbestos bodies were identified in fibroelastotic foci in each of the 15 cases as highlighted with Perl's stain. Forty-seven cases of nonasbestotic lung disease (71%) showed interstitial fibrosis with a variable (usually modest) amount of admixed elastic tissue; when present, elastic fibers were distributed in a diffuse interstitial pattern, with or without perivascular accentuation. All cases of IPF also showed areas of fibroelastosis, but those foci were confined to regions of overt “honeycomb” change. No asbestos bodies were seen in any disease except asbestosis, and a predominantly peribronchial pattern of fibroelastosis was not identified in any nonasbestotic interstitial lung disease in this study. The authors conclude that the types and patterns of pulmonary connective tissue response in interstitial lung diseases may provide additional diagnostic clues to the presence of asbestosis.     

Immunolocalization of major interstitial collagen types in human lumbar intervertebral discs of various ages

 We used complete transverse sections through 65 samples of human lumbar intervertebral discs for immunolocalization of the major interstitial collagen types I, II, III, V, VI and IX. The samples were selected from 47 patients ranging in age from 0 (fetuses) to 86 years. The results were compared with the histological findings in disc tissue degeneration and/or reparative alterations as indicated by tear and cleft formation, chondrocyte proliferation, mucous degeneration, granular matrix changes and fibrocartilage fibrillation. We observed a typical pattern for each antibody and each anatomical structure, with, however, remarkable inter- and intraindividual variability, which could be monitored only by use of the complete transverse sections. Accordingly, collagen I was seen in the normal annulus fibrosus and in the degeneratively altered nucleus pulposus, but not within the end-plate, regardless of degenerative changes. Collagens II and IX were found in the normal nucleus pulposus, the inner annulus fibrosus and the end-plate. The collagen II (and IX) staining seemed to be enhanced in areas of minor degenerative lesions, but reduced in advanced lesions and in the degenerated end-plate. Collagens III and VI were significantly increased in areas of minor to advanced degeneration in all anatomical settings, while collagen V showed only minor changes in its staining pattern. In general, histological signs of tissue degeneration coincided with significant quantitative, but also with certain qualitative, changes in the composition of the collagenous disc matrix. These observations indicate the association of degenerative and/or reparative alterations of the intervertebral disc and changes in the collagenous matrix, but document the variability in the extent of the abnormalities observed. Received: 5 December 1996 / Accepted: 30 May 1997     

Validation of a new calibration method for human muscle microdialysis at rest and during exercise

Microdialysis presents the unique possibility to measure metabolite concentrations in human interstitial fluid. During exercise, the recovery of these metabolites should be precisely monitored since it is known to increase greatly with muscle blood flow. The loss of ethanol, perfused at low concentration, can be accurately measured and reflects the changes in dialysis conditions. We evaluated whether using the relationship determined in resting metabolic conditions between the loss of ethanol, as reference substance, and the recovery for lactate or glucose would allow us to calculate precisely the concentration of these substances and their variations during exercise. Using the new catheter calibration method (slope method), the error of estimation of lactate and glucose in vitro was limited to –0.6 (5.8)% and –0.7 (6.2)%, respectively. In resting human muscle, the slope method proved to be as accurate as an established calibration technique (no net flux method) to evaluate interstitial lactate concentration [1.82 (0.58) vs 1.83 (0.47) mM, respectively]. During dynamic knee-extension exercise or light neuromuscular electrical stimulation, the estimated interstitial lactate and glucose concentrations varied differently, but their time course changes remained consistent with their respective plasma values. We conclude that, after an initial calibration step, the slope method allows accurate measurement of interstitial muscle metabolites and it could be used to monitor rapid metabolic changes during exercise.     

Computationally efficient algorithms for convection-permeation-diffusion models for blood-tissue exchange

Analysis of data on tissue depositions obtained by positron tomographic or NMR imaging, or of multiple tracer outflow dilution curves, requires fitting data with models composed of aggregates of capillary-tissue units. These units account for heterogeneities of flows and multisolute exchanges between longitudinally distributed regions across capillary and cell barriers within an organ. Because the analytic solutions to the partial differential equations require convolution integration, solutions are obtained relatively efficiently by a fast numerical method. Our approach centers on the use of a sliding fluid element algorithm for capillary convection, with the time step set equal to the length step divided by the fluid velocity. Radial fluxes by permeation between plasma, interstitial fluid, and cells and axial diffusion exchanges within each time step are calculated analytically. The method enforces mass conservation unless there is regional consumption. Solution for a 2-barrier, 3-region model, accurate to within 0.5%, are 100 to 1000 times faster than the corresponding, purely analytic solution, and over 10,000 times for a 4-region model. Applications include multiple indicator dilution studies of kinetics of transcapillary exchange and positron emission tomographic studies of the mechanisms of substrate transport into cells of organsin vivo.     

Mechanisms of Interstitial Flow-Induced Remodeling of Fibroblast–Collagen Cultures

Interstitial fluid flow, critical for macromolecular transport, was recently shown to drive fibroblast differentiation and perpendicular cell and matrix alignment in 3D collagen cultures. Here we explore the mechanisms underlying this flow-induced cell and collagen alignment. Cell and matrix alignment was assessed from 3D confocal reflectance stacks using a Fast Fourier Transform method. We found that human dermal and lung fibroblasts align perpendicular to flow in the range of 5–13 μm/s (0.1–0.3 dyn/cm²) in collagen; however, neither cells nor matrix fibers align in fibrin cultures, which unlike collagen, is covalently cross-linked and generally degraded by cell fibrinolysis. We also found that even acellular collagen matrices align weakly upon exposure to flow. Matrix alignment begins within 12 h of flow onset and continues, along with cell alignment, over 48 h. Together, these data suggest that interstitial flow first induces collagen fiber alignment, providing contact guidance for the cells to orient along the aligned matrix; later, the aligned cells further remodel and align their surrounding matrix fibers. These findings help elucidate the effects of interstitial flow on cells in matrices and have relevance physiologically in tissue remodeling and in tissue engineering applications.     

Three‐dimensional imaging of the fibrous microstructure of Achilles tendon entheses in Mus musculus

The whole‐organ, three‐dimensional microstructure of murine Achilles tendon entheses was visualized with micro‐computed tomography (microCT). Contrast‐enhancement was achieved either by staining with phosphotungstic acid (PTA) or by a combination of cell‐maceration, demineralization and critical‐point drying with low tube voltages and propagation‐based phase‐contrast (fibrous structure scan). By PTA‐staining, X‐ray absorption of the enthesial soft tissues became sufficiently high to segment the tendon and measure cross‐sectional areas along its course. With the fibrous structure scans, three‐dimensional visualizations of the collagen fiber networks of complete entheses were obtained. The characteristic tissues of entheses were identified in the volume data. The tendon proper was marked as a segment manually. The fibers within the tendon were marked by thresholding. Tendon and fiber cross‐sectional areas were measured. The measurements were compared between individuals and protocols for contrast‐enhancement, using a spatial reference system within the three‐dimensional enthesis. The usefulness of the method for investigations of the fibrous structure of collagenous tissues is demonstrated.