从血液流变学到微循环与再生医学(经典英文文献批注)

Clinical Hemorheology and Microcirculation 45 (2010) 79–99 DOI 10.3233/CH-2010-1312 IOS Press79From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 20091F. JungCenter for Biomaterial Development and Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Institute for Polymer Research, GKSS Research Center GmbH, Teltow, Germany该文是Jung F在2009年获得欧洲微循环学会Fahraeus Award时对自己过去研究工作的总结。

其中列举的都是他的代表性研究。

1. Introduction It is a great honor for me to receive the Fåhraeus Award and I would like to thank the European Society very much for this. The medal represents an important milestone in my life and to have achieved it fills me with immense joy. I was especially pleased that my laudation was held by Mike Rampling – whom I had the pleasure of presenting the Fåhraeus medal in Dresden – and that this year it was his turn to present it to me. Mike is a very good friend of mine and an outstanding personality. I have always enjoyed the articles he has published and the lectures he has given over the past years. To be honored with the Fåhraeus medal as a non-physician was only possible due to my close collaborative research relationship with many clinicians within a vast network. Therefore, my most sincere thanks go to all with whom I had the pleasure of working during the past 25 years. As has been the tradition for many years, I would now like to present some of the most important of my work projects. My first work in the field of hemorheology was to investigate under which conditions the HagenPoiseuille Law is valid. This question can be answered straightforwardly by continuum physics. General mass and impulse balance equations form coupled systems of partial differential equations that are not directly solvable with respect to the associated exit, transient and boundary conditions. ∂vi =0 ∂xi ρ∗ ∂ ij ∂vi ∂p − = ρ ∗ fi + ∂t ∂xi ∂xi1 Excerpts from the Fåhraeus Lecture 2009 in Pontresina (Switzerland), at the 15th European Conference for Clinical Hemorheology and Microcirculation.1386-0291/10/$27.50 © 2010 – IOS Press and the authors. All rights reserved80F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 2009However, the estimates for the velocity field (vi ) and the stress tensor ij can be selected in such a way that an analytical solution, known to us as the Hagen-Poiseuille equation, is possible: V= π∗ρ∗g ∗ R4 8∗ηAltogether, the following 10 conditions need to be satisfied: 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) the fluid is incompressible, the density of the fluid is constant, linear relation between shear stress and shear rate (Newton’s law), the viscosity is a material constant (it depends solely on temperature), the velocity field is one dimensional, it varies only in direction of the radius, the fluid flow is stationary (no time dependence), the vessel is an ideal cylinder, the radius of the vessel is constant (no peristaltic), no fluid exchange through the vessel wall, the shear rate at the wall is zero.It is clear that since most of the 10 conditions above are not fulfilled, the Hagen-Poiseuille equation can only offer an approximation of the blood flow in a vessel [22]. In 1981, I joined a research group at the Institute for Physiology at the RWTH Aachen, which, at that time, was focused on the development of rheological measurement technology. In a first study, a plasma viscometer was developed and evaluated, then the measurement accuracy, precision, and variability were tested [25, 26]. Also at that time, a consistent quality control feature was introduced [28], which has meanwhile become the standard in rheological methodology [3]. – precision in series: 1.14% – inter-individual variability: 3.2% – intra-individual variability: 1.59 ± 0.35%. A cross-sectional population based study followed in which the reference range and the relationship to age, body weight and sex was determined [28]: Reference range: 1.24 mPas (1.14–1.34 mPas) The results revealed that neither smoking nor sex or age had any effect on the plasma viscosity of apparently healthy subjects, whereas there was a clear relationship to the body weight [29]. However, all smokers suffering from bronchitis had been excluded from the study because such subjects cannot be regarded as healthy. Indeed, their plasma viscosity showed a marked increase. Studies conducted by Harkness suggested [20] that proteins affect the plasma viscosity in various ways. Thus, a further demographics study [36] with 2821 subjects investigated the influence of different proteins (albumin, fibrinogen, 2-macrogobulin, immunoglobulins) and also of different plasma lipids (cholesterol, triglycerides, HDL). Figure 1 shows the correlation between plasma viscosity and fibrinogen concentration (Fig. 1a) and HDL concentration (Fig. 1b). Fibrinogen has the strongest influence on plasma viscosity (r = 0.62). Interestingly, the HDL-cholesterol concentration is reversely correlated, meaning the plasma viscosity decreases as the HDL concentration increases.F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 200981a10 9 8y = 7.87x - 6.3 r = 0.62 n = 2676bHDL cholesterol (mmol/l)2.5 2 1.5 1 0.5 0y = -0.83x + 2.34 r = 0.15 n = 2686Filxinogen (g/l)7 6 5 4 3 2 1 1.2 1.3 1.4 1.5 1.6 1.7 1.81.21.31.41.51.61.71.8Plasma viscosity (mPas)Plasma viscosity (mPas)Fig. 1. (a) Correlation between plasma viscosity and fibrinogen concentration and (b) HDL-cholesterol concentration, (modified according to [36])."Beta-weights"0.6r = 0.69 R = 0.480.40.20.0-0.2 Fi b TG IGM Chol a2M HD Lmolecul/molecul complexFig. 2. Influence of different plasma proteins and plasma lipids on the plasma viscosity (according to [36]).The influence of various molecules on plasma viscosity can best be demonstrated by means of beta weights (see Fig. 2). The subsequent clinical trial [47] showed not only that the plasma viscosity in patients with peripheral arterial occlusive disease was significantly higher than in healthy study subjects, but also that it continuously increased with disease stage (see Fig. 3). The results of this study led to a discussion as to whether altered blood rheology influences the development of atherosclerosis. This led to another study, initiated in 1986 in Aachen, to elucidate whether changes in blood fluidity foster the development and progress of vascular disease [46]. The Aachen study was an epidemiological prospective cohort study with annual control examinations. The aim of the study was to prove whether the initially reduced blood fluidity in subjects without arterial82F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 20091.61.41.21.0healthy subjects n=283 POAD I n=33 POAD II n=184 POAD IV n=72Fig. 3. Plasma viscosity in patients with peripheral arterial occlusive disease.occlusive disease is a risk factor for the later occurrence of arterial circulation disorders. Based on the data of a prestudy, 2821 voluntary subjects of both sexes (1709 m; 1112 f) between 45 and 65 years of age were included. After the entry examination, two incidence groups of 456 participants each – without malignoma, chronic inflammations, and arterial or venous disease – were categorized according to their blood fluidity. The members of both groups were comparable with respect to cardiovascular risk factors such as hypercholesterolemia, hypertriglyceridemia, decreased HDL cholesterol, elevated blood pressure, diabetes mellitus, age, and sex. They only differed in blood fluidity: Group I had reduced blood fluidity (at least 1 rheological variable above the mean value + standard deviation limit in healthy subjects) and Group II had normal blood fluidity. Two years after the start of the study, 30 subjects in Group I had already developed an arterial occlusive disease but only 14 in Group II (see Fig. 4). This indicates that the relative risk (RR) of developing an arterial disease within 2 years is more than twice as high (RR: 2.14 CI: 1.14–4.01) for subjects between 45 and 65 years with intact vasculature but reduced blood fluidity compared to subjects with intact vasculature and normal blood fluidity [47]. Therefore, the Aachen study clearly showed that reduced blood fluidity is an important independent risk factor for arterial occlusive disease.AOD/Non-AOD [-]0.08 reduced 0.060.04 incidence group II: normal blood 0.02 p=0.0199 0.00 0 1 2Time [years]Fig. 4. Results of the Aachen study.F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 200983In addition, it was found that increased plasma viscosity – independent of classical risk factors – was a predictive factor for the later incidence of peripheral arterial occlusive disease. This finding was confirmed by two other epidemiological studies on rheological parameters – the Caerphilly-Speedwell and MONICA studies. Whether this is indicative of some causal relationship or only reflects an early prodromal stage of high plasma viscosity (thus, probably, a good early diagnosis method) cannot be concluded from the studies conducted so far. Ultimately, it still remains to be seen whether early treatment of impaired blood fluidity has an influence on the incidence of arterial occlusive disease. Such interventional studies, however, are not available to date. Blood is a highly concentrated suspension, whose components can reduce its fluidity when they interact with each other [10]. The extent of cell-cell interactions depends on the number and properties of the cells and plasma, and also on the shear forces present. During the 1970s, research was focused on the influence of microrheological variables on whole blood viscosity. The fundamental principles of this field were developed by the research groups Schmid-Schönbein [70], Barras [2], Meiselman [58, 63], Forconi [16–18, 72] and, in particular, Chien [10, 11]. Blood properties govern perfusion to a greater extent in the microvasculature than in the macrovasculature. During circulation, blood separates with a higher concentration of red blood cells in the center region of blood vessels compared to the marginal regions [56]. Moreover, in the capillary system – arterioles, capillaries, venules – blood cells are redistributed so that the actual capillary hematocrit is only 15% [32], whereas it may be double this value in thoroughfare channels. Hence the blood viscosity in the capillary system approaches the viscosity of plasma [2] and the plasma viscosity determines the blood flow velocity in the capillaries. A prerequisite is, however, that the erythrocyte aggregates in the arterioles are sheared – shear rates being the highest here – so that the single erythrocytes, whose diameters at rest are about 8.5 mm and which are thus considerably larger than the capillary diameters of 3 to 9 mm, can enter into the capillaries. Thus, in order to investigate this hypothesis, microcirculation investigation methods had to be developed or available methods needed to be adapted for use in a clinical situation and, most importantly, they had to be evaluated. The technologies conjunctival capillary videomicroscopy [23, 49, 79] and videofluorescence angiography of retinal blood circulation were developed in collaboration with Sebastian Wolf [4, 24, 77, 78, 80]; periungual capillary videomicroscopy [27, 31, 37], and oxygen partial pressure measurements in skeletal muscle using macroelectrodes were evaluated in collaboration with Holger Kiesewetter [43, 54, 55]. In Fig. 5, the left-hand image shows a perimacular capillary network [78] of a healthy study subject and, in contrast, the right-hand image shows the altered capillary network of a patient with diabetic retinopathy. In the latter, the foveal avascular zone is distinctly larger, capillary demise clearly having occurred, and arteriolar dropouts are also identifiable. Dynamic analyses have demonstrated that erythrocyte velocity in these capillaries of healthy subjects was about three times higher than in nail-fold capillaries [31, 77] – which could be ascribed to much lower hemodynamic resistance, the capillaries being considerably shorter here than in the skin. Conjunctival capillary videomicroscopy is particularly suitable for measuring the influence of mediators on the vessel diameter, but is less suitable for dynamic analysis. As is typical of the mucosa, the number of anastomoses found is very high so that flow reversals and stagnation or shuttle flow may be frequent. This makes it difficult to assess or statistically analyze blood circulation at rest. Figure 6 depicts an example. Here the influence of a vasoconstrictor and a vasodilator on vessel diameter of a conjunctival arteriole was studied. The results show that these arterioles are capable of changing their diameters by up to 300% and thus can regulate blood flow very effectively.84F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 2009abFig. 5. (a) Perimacular capillaries in healthy subjects and (b) in patients with diabetic retinopathy.constrictednormal statedilatedFig. 6. Conjunctival arteriole in the normal, constricted and dilated state.As an example the evaluation of periungual video capillary microscopy is described below. Skin capillaries can be observed under a reflected-light microscope [9, 31, 73]. The capillaries of the nail fold run parallel to the surface of the skin and are thus particularly suitable for dynamic analyses. The system setup comprised a reflected-light microscope with coarse and fine vertical adjustment, an objective with a long focal distance (Neofluar 6.3/0.20), an Optovar 1.0–2.0 (for quick magnification changes), a cold-light source with a green filter (in the spectral absorption range of hemoglobin (480 nm) for enhanced contrast between red blood cells and tissue), and a heat filter (to prevent heating of the finger during the measurement). The digital image data are recorded on video tape for off-line analysis (Fig. 8). The overall system is an optical transfer chain consisting of various components so that the total transfer function is derived by calculating the transfer function of the individual components. The spatial resolution of an optical system is characterized by the modulation transfer function, that is, modulation of an input signal as a function of the spatial frequency in the image plane. In the present case, the vertical resolution limit is 0.784 m [33], which is considerably smaller than the image structures to be measured so that they can thus be located with sufficient accuracy. Under these conditions, it is sufficient to describe the geometric transfer behavior. Because the transfer behavior is space-dependent, it is relatively easy蹄、甲、爪周围的F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 200985to investigate how the given distances are displayed in horizontal, vertical, and diagonal directions. The results show that the measuring procedure is correct with regard to measuring accuracy. For assessments of capillary erythrocyte velocities (usually in the vertical direction), the measurement error with respect to length measurements of 100 m is approx. 1.5% [33]. Detailed information on the transfer function and spatial resolution can be found in earlier publications [33, 35]. Accuracy, precision, and power of discrimination are not independent of each other (see Fig. 7): 1) The lower the precision => the broader the reference ranges. 2) The broader the reference ranges => the lower the discrimination. Thus, in order to achieve a high degree of discrimination between normal and impaired capillary flow, and to minimize false positive and also false negative results, it was necessary to fine-tune the individual components of the microscope and the video transfer chain with respect to each other in such a way that the highest possible resolution was obtained.Frequency distribution Plasma viscosity [mPas]reference rangepatientsplasma viscosity [mPas]false negativ false negativFig. 7. Discrimination between healthy and diseased subjects.表面的丆表浅的superficialmicroscopdigitalramification of the digital手指的乮数字的乯分支、衍生Fig. 8. System setup with sketch of the imaging area and the focused capillaries (modified according to [37]).86F. Jung / From hemorheology to microcirculation and regenerative medicine: Fåhraeus Lecture 2009When evaluating a measurement method, it is important to differentiate between the technical errors of measurement acquisition and biological errors, which are dependent on the biological structure of the studied area, as well as influencing variables. At the same time, the technical error should not exceed 30% of the biological error [35]. When evaluating the clinical significance of the measurement parameters, it is important to take account of the physiological fluctuations of such a parameter during repeated measurements, particularly in the case of assessments of long-term therapies. It is therefore important to determine the fluctuation range of a parameter throughout a day, a week, and a year. Since the result of a measurement essentially depends on how an investigation is performed, the measuring process is now explained first. 以下为甲皱微循环的测量过程及注意事项両 The hand is positioned at heart level under the objective on an adjustable X, Y microscope stage that has been preheated to 28◦ C. The pulse rate and blood pressure are then measured (in the contralateral arm to avoid capillary flow reactions to blood pressure measurement). During the measurement, the subject is seated on a chair with his or her arm resting relaxed on the microscope table. The measurements are always carried out on the fourth finger of the right or left hand. For the duration of the investigation the finger is held by a clamp at the front third dorsal portion of the nail bed so that perfusion in the nailfold is not affected [13]. Application of a drop of immersion oil causes the epidermis to become transparent and the capillaries in the nail cuticle can be brought into focus. In order to assess the concordance of capillary perfusion, the whole nail fold margin is first scanned at a low magnification (1 : 285). When perfusion has become concordant and continuous, the images obtained from one capillary area are recorded continuously on video tape for three minutes at a magnification of 1 : 570 for later analysis. The erythrocyte velocity is measured interactively via the frame-by-frame technique [14] or via an image processing system [48]. The velocity is measured approx. 100 m from the capillary vertex in a microscopic field of ±50 m. Repeated velocity measurements are performed to compensate for rhythmic fluctuations in the erythrocyte velocity, which have a frequency of 6 to 10 per minute [14] and are caused by vasomotion, i.e. oscillation of the vascular caliber of precapillary arterioles. Ten measurements are made per minute per capillary (thus, in total 30 velocity measurements are made during the 3 min recording time). The time-averaged erythrocyte velocities are calculated from these values. Given a normal capillary density (9 per millimeter epidermis margin [41]) about 4 to 5 capillaries can be seen on the monitor at a magnification of 1 : 570, the magnification at which the velocity measurements are performed. To ensure that 弯曲、曲度 all the capillaries are in good focus, an area with the smallest curvature at the center part of the nail fold is selected where preferably all capillaries are in the same depth of focus of the microscope objective and are sharply imaged. If this is the case, time-averaging is carried out on 4 capillaries as mentioned above. If 4 capillaries cannot be assessed (e.g. when capillary density is low, imaging quality is poor, or neighboring capillaries are not in the same depth of focus, etc.), a second area is imaged for another 3 min. The following specified mean velocities of erythrocytes are calculated over time (10 measurements/min over 3 min) and spatial averages (over 4 capillaries). The following Table 1 shows that, with unimpaired microcirculation in a finger, the same mean values for erythrocyte velocity can be obtained for two different capillary areas and that secondly, the thus calculated mean erythrocyte velocity is found 需要搞清他测的是动脉支还是静脉支的RBC流速 even in measurements on different fingers.根据后面的内容看丆应该是动脉支的RBC流速。