- Email: [email protected]
A decrease in effective diameter of rat mesenteric venules due to leukocyte margination after a bolus injection of pentoxifylline—digital image analysis of an intravital microscopic observation
Microvascular Research 67 (2004) 237 – 244 www.elsevier.com/locate/ymvre
A decrease in effective diameter of rat mesenteric venules due to leukocyte margination after a bolus injection of pentoxifylline—digital image $ analysis of an intravital microscopic observation M.A. Hussain, a,* S.N. Merchant, b L.S. Mombasawala, c and R.R. Puniyani a a School of Biomedical Engineering, IIT Bombay, Powai, Mumbai 400-076, India Advanced Centre for Research in Electronics, IIT Bombay, Powai, Mumbai 400-076, India c Regional Sophisticated Instrumentation Centre, IIT Bombay, Powai, Mumbai 400-076, India b
Received 29 July 2003 Available online 11 March 2004
Abstract The ability of leukocytes to adhere to endothelial cells (EC) and then to migrate out of the blood stream into tissues enable them to perform their surveillance functions. Adhesion of leukocytes to EC is, however, only possible if the cells have marginated as a result of rheological interaction with other blood cells in flow. Using Pentoxifylline (PTX), a rheologically active drug, to manipulate this interaction, we have imaged and quantified this margination phenomenon in vivo. A system has been developing to perform this imaging via an intravital microscope connected to an image processing system. Albino rats were anesthetized and cannulated for intravenous bolus injection (0.5 ml) of PTX (1.25 mg/ml) through the femoral vein. A longitudinal incision exposed the mesentery, part of which was observed under microscope to visualize microcirculation. The image of interest was then stored on computer hard drive. Individual leukocyte velocities were determined before and after PTX infusion. The leukocytes, marginating and sticking after PTX infusion either remained attached, constituting the peripheral marginating leukocyte pool in the postcapillary venules, or detached with different step velocities. The reduction in effective venular diameters as a result of leukocyte margination was estimated to be 32 – 44%. These results demonstrate the biological importance of hemodynamic displacement leading to docking, adhesion, rolling and migration processes of leukocytes in blood. D 2004 Elsevier Inc. All rights reserved. Keywords: Intravital microscopy; Digital image processing; Microcirculation; Leukocyte; Pentoxifylline
Introduction Leukocytes are seen to adhere to the vascular endothelium of postcapillary venules under a variety of conditions. The molecular mechanisms involved in leukocyte adherence to and subsequent rolling on the endothelial cell (EC) lining of microvessel walls have been extensively studied (Bahra et al., 2001; Dominguez-Jimenez et al., 2002; Gerszten et al., 1998; Mohan et al., 1999; Ogawa et al., 1997; Pearson and Lipowsky, 2000; Stein et al., 1999; Warnock et al., 1998; Wojciak-Stothard et al., 1999). Biophysical aspects of $
Leukocyte margination event observed in rat venular microcirculation. * Corresponding author. Department of Surgery, Biomedical Engineering Institute, H 151, Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, PA 17033. Fax: +1-717-531-4464. E-mail address: [email protected] (M.A. Hussain). 0026-2862/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2004.01.005
the EC – leukocyte interaction leading to hemorheological manifestation in vivo are no less important, and although in vitro investigations exist, there is little in vivo data. Model studies have shown (Bagge et al., 1986) that adhering cells may cause a significant increase in flow resistance by reducing the effective diameter of the vessel, and this is supported by direct measurements in the cat microvessels (Lipowski et al., 1980). This aspect of leukocyte adhesion biology, although of great significance, is only observed after margination from the axial stream primarily because of rheological interaction with other blood cells in flow. An interesting study on this influence of blood rheological properties on the adhesion of leukocytes in vitro has recently been published (Abbitt and Nash, 2003). From a hemodynamic perspective, a leukocyte tends to remain in axial flow in the blood vessel, as it has greater mass than a red blood cell (RBC). However, when RBC
238
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
aggregates are formed and exceed the mass of a leukocyte, the latter becomes marginated. In essence, when as few as five RBCs unite, their mass is much higher than that of an individual leukocyte. Hence, any physical or chemical factor leading to RBC aggregate formation in vivo would automatically tend to displace leukocytes away from the axial stream (Schmid Scho¨nbein, 1996). One such chemical factor widely used clinically is 1-[5-oxohexyl]-3,7-dimethylxanthine, a derivative of theobromine and commercially known as Pentoxifylline (PTX), shown in vitro to induce RBC aggregation (Singh and Kumaravel, 1996). Clinical responses to long-term oral administration of PTX are thought to result primarily from improved erythrocyte flexibility and reduced blood viscosity (Weitz et al., 1996). Newer uses of PTX at the molecular level have been investigated, demonstrating that PTX has inhibitory effects ex vivo on various inflammatory mechanisms, including the complement cascade, neutrophil adherence and cytokine production (Krakauer, 2000; Krakauer and Stiles, 1999; Sliwa et al., 2002; William, 1995). Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF) promote adhesion and migration of leukocytes on endothelial cells (Springer, 1995). As a response to inflammation, the cytokines cause expression of E- and P-selectin, which support adhesion and rolling of leukocytes. Cytokines also upregulate endothelial intercellular adhesion molecule-1 (ICAM-1), which binds integrins on leukocytes and facilitates migration (Springer, 1995). Studies have shown that PTX has a definitive effect on the interaction of different types of leukocytes and endothelial cells at various stages of adhesion and migration (Bahra, 2001; Dominguez-Jimenez, 2002). At the cellular level, however, the effects of PTX on in vivo microhemodynamic behavior of leukocytes and RBCs remain obscure. In this study, we have used PTX to manipulate and an intravital microscopy to visualize leukocyte adhesion events in the microvascular blood flow in vivo. Intravital microscopy provides unique opportunities for visualizing cellular events in microvascular flow at the micrometer scale. Intravital cellular microscopy when combined with digital image processing allows easy handling of the resulting image data. Digital image processing is a rapidly evolving field with growing applications in science and engineering, and generally refers to the processing of a two-dimensional picture by computer. In a broader context, it implies digital processing of any two-dimensional data. An image is first digitized and stored as a matrix of binary digits in computer memory. The digitized image can then be processed and/or displayed on a high-resolution color monitor. Personal Computers (PCs) can be used to communicate and control the digitization, storage, processing, and display operations via a computer network. Digital image processing can be classified broadly into four areas: image enhancement, restoration, coding and understanding. In this work, tools from the first three areas have been extensively utilized. We specifically describe the quantitative measurement of a decrease in effective (flowing) diameter of the microvessel
due to leukocyte margination and subsequent docking along the venular wall. Our results show attaching and detaching events of leukocytes to the venular walls and quantify the step velocities of dislodged cells after detachment. In our analysis and discussion, we interpret the attachment duration of leukocytes to the endothelial cell lining of vascular wall in terms of ligand-receptor activation. Individual leukocyte velocity before and after PTX administration in the microvessel is reported using a semiautomatic method with the help of the digital image processing system.
Methods Animal preparation Albino rats (n = 8) were anesthetized by intraperitoneal injection of Pentothal Sodium (0.1 mg/kg body weight). Femoral veins were cannulated for a 0.5-ml intravenous bolus injection of 1.25 mg/ml PTX (Hoechst), this dose being selected from a published report (Quezado et al., 1999) and also from unstable effects on animals used in screening experiments, performed with higher dose to establish experimental protocol. A longitudinal incision was made to open the belly and a part of mesentery was placed under the microscope (Meiji, Japan) to visualize microcirculation via 40 objective. The mesentery was continuously flushed with normal saline to keep it wet. All experiments were performed in accordance with institutional guidelines. Intravital microscopic setup A CCD camera (Pulnix, USA) was attached to the microscope (Meiji, Japan) and connected with the power supply and image grabber card (Dazzle, Multimedia). The image grabber card, installed in a Personal Computer, was also connected to a VCR to record the experiment on videocassette (Fig. 1). Data acquisition Using the software supplied with the image grabber card, the image data of interest were digitized and stored on the computer hard disk while conducting experiments. A simultaneous recording was also made on videocassette through out the experimental duration to allow offline replay and further digitization of any data that might have been omitted during online digitization. Image analysis Image analysis was carried out using Soft Imaging System—analySIS software. The leukocyte velocities and microvessel diameters were calculated as described below.
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
239
their flow was no longer smooth. Leukocytes were now observed to flow in steps, such that an individual leukocyte appeared to form a contact at one location on the venular endothelial wall, detached to step up and attached again to another location in the same microvessel under focus. Such individual leukocytes were tracked by replaying digital video frame by frame and observed to stick to the venular wall before detachment with a jerk. The velocity of each leukocyte that detached after sticking was measured using the same semiautomatic method as described above and was termed the step velocity of an individual leukocyte. Duration of attachment of leukocyte to the microvessel wall
Fig. 1. Block diagram showing the scheme of image grabbing, editing and analysis systems.
The duration of attachment for a given leukocyte to the venular wall was determined by replaying the digital movie frame by frame. The ratio of the number of frames elapsed before an adhering leukocyte detached from the venular wall with a jerk, to the frame rate of the image grabbing system, determined the attachment duration of the adhering leukocyte.
Velocity determination of leukocytes Vessel diameter measurement The velocities of flowing leukocytes were determined before and after PTX administration using a semiautomatic method with the help of Soft Imaging System, analySIS. Two frames, namely, frame-1 and frame-n, were snapped by replaying the digital movie of the mesenteric microcirculation stored on the hard disk frame by frame in such a way that the movement of the leukocyte in question was visually tracked in each frame. The location of the leukocyte in the first frame (i.e., frame-1) and nth frame (frame-n) was determined by assigning a common reference point in both frames. This reference point was chosen to be present in both frames and was a static structure in the matrix of interstitial spaces in the microvascular bed. The distance blood cell moved was calculated by drawing a line between the cell and reference point in fame-1. The length of this line and the angle between this line and a vertical line were measured. This length and angle relative to the reference were then used to relocate the initial blood cell position in frame-n. The distance between the relocated postion-1 and the position-2 of the moving blood cell in the frame-n may then be determined. Representative images depicting this method for a leukocyte velocity calculation are shown in Fig. 2. The time was calculated by the number of frames elapsed (i.e., n frames) divided by the frame rate at which the image was grabbed (25 frames/s). Hence, with the distance traveled and time elapsed, leukocyte velocities were determined. The experiments were focused at measuring individual leukocyte velocities near the microvessel wall and not the flux of leukocytes flowing through the microvessel. Before PTX administration, leukocytes were occasionally found flowing smoothly along the microvessel wall and their velocities were determined as above. After PTX infusion, more leukocytes started appearing on the venular wall but
Two diameters were calculated: one, the ‘actual’ diameters of the microvessel including the marginating pool of leukocytes, and, two, the ‘reduced’ diameter of the flowing red cell column excluding the marginating pool of leukocytes. They were calculated by taking the average of five diameters measured at points within a length equal to 2.5 times the microvessel radius. The percentage of change in diameter was calculated using the following equation. Percentage diameter change ¼ ðda dr Þ=da 100% where da is actual diameter measured; dr is the reduced diameter.
Fig. 2. Representative image depicting the method for leukocyte velocity calculation. From a common reference points in two given images, a vertical line is drawn. In the given frame-1, another line is drawn from the reference point connecting it to the leukocyte and the angle that this line makes with vertical line is noted. In the frame-n now at this measured angle, a line of the same length is drawn to relocate the leukocyte position in this frame corresponding to the frame-1. A broken line with two-sided arrow shown in frame-n gives the distance traveled by the leukocyte during the time lapse from frame-1 to frame-n. The velocity was calculated from this time and distance data.
240
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
Statistical analysis Means and standard deviations are reported. The level of significance was set to P < 0.001 at 95% of confidence.
Table 1 Leukocyte velocity measured in postcapillary venules and capillaries before infusing pentoxifylline. Sixty leukocytes were analyzed from digitized movie of 16 venules and capillaries, roughly seven velocity measurements per two microvessels per rat Diameter ranges
Results Before administration of PTX, leukocytes were often observed passing through capillaries but occasionally appeared to crawl along venular walls. The flow of leukocytes observed in venules was smooth with a uniform velocity, although this was not always the case in capillaries. Rather, leukocytes would often obstruct the flow and even plug the capillaries momentarily. We measured the velocities of 60 smoothly flowing leukocytes from digitized movies of 16 venules and capillaries, with roughly seven velocity measurements per two microvessels per rat. The results of leukocyte velocity measurements in venules and capillaries are shown in Table 1. We did not find any significant correlation between leukocyte velocities and
Venules (n = 30) 15 – 25 Am Capillaries (n = 30) 3 – 8 Am
Leukocyte velocity (Am/s) 50.3 F 3.7 16.1 F 3.2
P < 0.001.
diameter of microvessels in which these measurements were made. Within roughly 20 min of PTX administration, intravascular RBC aggregation was clearly observed as clumps of RBCs flowing in the axial stream of the venules (Fig. 3). This was not quantified due to the limitations of our image analysis system. This aggregation of RBCs led to the margination of leukocytes towards the venular wall periphery resulting in an improved focus of these cells and enhanced image clarity. The effect of PTX administration was not found to be reversible during the imaging duration of approximately 30– 40 min after PTX infusion. We did not continue the experiments further and do not have data showing reversibility of the observed effect of PTX. Contrary to a smooth flow before PTX administration, leukocytes were now observed to stick in abundance to the postcapillary venules forming a pool of leukocytes along the venule margin. Leukocytes were not moving with a uniform velocity, rather they showed sticking and detaching phenomena. Leukocytes were observed to attach to the wall of the vessel, and then often dislodge from the attachment site with a jerk. Leukocytes were thus imparted with a step velocity after dislodgement into the sustained hemodynamic environment. Leukocytes that detached from the vessel wall due to cellular interaction in flow got a jerk of varying magnitude with an equal frequency of occurrence due to a transfer of momentum from flowing particles (leukocyte, RBC aggregates), thereby imparting a range of step velocities. These step velocities were calculated for leukocytes (n = 45) in 16 microvessels (two microvessels per rat) with approximately three leukocyte velocity determinations per microvessel. These results are shown in Table 2. The attachment of leukocytes to the endothelial lining of the vessel wall was observed to be stable Table 2 Leukocyte step velocities measured in postcapillary venules after 20 min of pentoxifylline bolus injection show equal number of occurrence
Fig. 3. Representative postcapillary venule images showing clumps of red cell aggregates in Panel B. Panel A images represent smooth blood flow before pentoxifylline administration and Panel B images are snaps from digital movie after pentoxifylline administration.
Ranges of leukocyte step velocities in postcapillary venules (Am/s)
Frequency of step velocities of leukocytes (n = 45)
<20 20 – 40 40 – 60
16 14 15
All the detaching leukocytes measured for their step velocities stuck to the venular wall only for fraction of a second (0.5 F 0.1 s).
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244 Table 3 Percentage reduction in the effective (flowing) diameter of microvessels after leukocyte adhesion and docking to microvascular wall No. of microvessels
Actual diameter (Am)
Reduced (effective) diameter (Am)
% Change
48 (six microvessels per rat)
20.1 F 3.2
12.8 F 2.0
36.2 F 3.2
P < 0.001.
if it remained attached for a duration approximating 1 s, thereby forming a pool of leukocytes. However, most leukocytes were seen to detach as a result of collision with other blood cells within a duration of half a second (0.5 F 0.1 s as calculated for n = 45 leukocytes). Due to the marginating pool of leukocytes, the effective diameter of the vessel was observed to decrease by 32 – 44% as presented in Table 3. The reduction in the effective diameter of microvessel is sketched from a representative image (Fig. 4). We also measured a population of microvasucular diameters before and after PTX administration to determine if PTX imparted any major change in the actual diameter of microvessels. The histograms showing the distribution of microvascular diameters before and after PTX infusion are very similar as presented in Fig. 5. This shows that vessel diameters did not change with PTX administration but the effective flowing diameter was reduced by the marginating pool of leukocytes.
Discussion Video stored in digital form has several advantages over its analog counterpart. These include: (a) digital recordings are almost immune to signal problems, and deliver the highest picture quality; (b) digital video has approximately twice the horizontal resolution of a standard VHS videocassette recorder. The resolution of a digital video standard image is approximately 25% better than that of an S-VHS or Hi-8 camcorder or VCR; (c) color resolution (or rendition) can be a problem for analog video, producing color blur and
241
color noise. Digital video does not exhibit this problem, delivering a more life-like video image on the screen, with sharper subject edges and clearer color reproduction; (d) video quality of analog video is reduced each time a tape is played, whereas digital video does not degrade with time; (e) digital copies of digital videos are indistinguishable from the original, which makes editing and image manipulation easier and with higher quality than that delivered by analog video technology; (f) digital video can be subjected to sophisticated digital image processing techniques for extracting information. We have carried out extensive measurements on digital video, which would not have been possible with analog video; (g) digital video is nonlinear, meaning that any scene or video clip can be accessed randomly without regard for its location in the footage. Analog video is linear, meaning that to locate a scene at the end of the tape, the user must bypass all preceding footage; (h) the digital video process is nondestructive. Experimentation on it can be carried out in an unlimited manner without risking the loss or destruction of the original footage; (i) digitally encoded video can be stored on DVD or other formats that have extended archival lives. This type of media is also more compact, and more durable than videotape, and finally, (j) video capture in digital format can be controlled more easily and efficiently by using the record or capture function in the digital video editing software. Certainly, these great benefits of digital video imaging warrants its application in variety of biological research. Biologists traditionally have been utilizing the video imaging for their research especially in hemodynamics of microcirculation (Kamm, 2002). In the present work, we have been able to extract valuable information from the digitized video images demonstrating the effect of PTX on microvasucular flow parameters at cellular level. In arterioles, where shear stresses are generally higher than those in venules, RBCs are kept dynamically disaggregated in the flow. Consequently, leukocytes are the largest moving particles and travel near the vessel axis. On the other hand, in the venules, shear stresses are much lower. Therefore, RBCs can rapidly form large aggregates and displace leukocytes away from the preferred axial layers
Fig. 4. Representative image showing peripheral marginating leukocyte pool, causing reduction of effective postcapillary venular diameter. (A) Actual image; (B) tracing of image A showing marginating leukocyte pools.
242
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
towards the wall as these RBC aggregates are larger than individual leukocytes. Under pathological conditions (i.e., when the tendency of RBCs to aggregate is increased), the aggregates persist even in the presence of very high shearing forces. Further, a high plasma concentration of aggregating colloids can enhance this effect (e.g., fibrinogen can enhance leukocyte margination by exaggerating and accelerating RBC rouleaux formation). A hemoconcentration experiment that is conducted by stepwise increase in hematocrit has simulated this margination phenomenon of leukocytes (Abbitt and Nash, 2003). In this work, the RBC aggregate forming tendency of PTX (Singh and Kumaravel, 1996) is used to simulate a pathological condition of RBC aggregability, thus facilitating the margination of leukocytes from axial blood flow. This study further demonstrates the associated reduction in effective vessel diameter due to leukocyte pool formation along the vessel wall. The mechanisms of this rheological margination have been studied in detail (Abbitt and Nash, 2003; Goldsmith and Spain, 1984; Nobis et al., 1985; Schmid-Scho¨nbein et al., 1980). Before PTX infusion, however, we observed leukocytes to appear occasionally
Fig. 5. Histograms showing distribution of microvascular diameter measurements (A) before and (B) after PTX administration. A total of (A) 72 and (B) 74 microvessels were analyzed.
near the microvessel wall and flow smoothly. As we were just interested in single cell velocity measurements, we did not feel it necessary to tag leukocytes with fluorescent dyes and hence the relative change in leukocyte distribution from axial to marginal flow was not quantified in this study. It is clear from the present study that a qualitative increase in RBC aggregation after PTX bolus infusion caused a displacement of leukocytes toward the vessel wall and thus favored margination and adhesion. The movements of leukocytes in terminal vasculature are passively coordinated by the interactions with moving RBCs. A dynamic RBC aggregation – disaggregation would determine which type of blood cell (individual leukocyte or aggregated RBC) would comprise the largest particle in flow. Leukocytes tend to get displaced away from the axial stream to the vessel wall apparently because of formed RBC aggregates in the flow. A variable number of leukocytes that come to an effective standstill and are classified as sticking, forming a pool in this study, may be attributed to several factors such as leukocyte and endothelial ‘‘stickiness’’ (adhesive energies), expression of adhesion molecules on endothelial cells, cytokine production by leukocytes, maintenance of endothelial cell continuity, local hemodynamic forces, shear stresses, and transmural pressure in addition to the red cell aggregation and/or red cell rouleaux formation. PTX increases the deformability of RBCs (Dawson et al., 2002; Seiffge and Kiesewetter, 1981) and also induces RBC aggregation (Singh and Kumaravel, 1996). In this work, PTX facilitated the formation of large clumps of RBCs that pushed leukocyte into the marginal layers close to microvessel wall. Although the extent of this intravascular RBC aggregation as observed in this study was not quantified, it may be possible to do so in vitro using indirect methods, for example, photometric or ultrasound aggregometers (Schmid Scho¨nbein, 1996) or a low shear viscometer (Hussain and Puniyani, 1999; Hussain et al., 1994, 1995). The present use of PTX made it easy to watch marginating leukocytes in postcapillary venules and to calculate leukocyte velocities. To initiate an active contact with endothelial cells, it appears that the attachment duration must be close to 1 s. Most often, leukocytes that were observed to detach had half a second (0.5 F 0.1 s) attachment duration. However, the leukocytes that succeeded in remaining attached for a longer duration (usually at least close to a second) were seen to continue sticking in place contributing to the leukocyte pool, possibly due to firm binding with adhesion molecules expressed on endothelial cell surface (Ley et al., 1989; Warnock et al., 1998) after which these may show the rolling phenomena. Interestingly, our observation of leukocyte arrest on the endothelial wall with a duration of close to 1 s provides direct evidence of effective bond formation between receptor and ligands that has been reported to occur on a time scale of less than 1 s (Hughes and Pfaff, 1998; Shimaoka et al., 2002). All detaching leukocytes investigated for step velocities in this work stayed attached for nearly half a second before they were detached by collision with
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
other flowing cells, indicating poor adhesion of leukocytes during this time span. However, beyond this duration, all leukocytes remained docked along the microvessel wall indicating firm adhesion. Therefore, we postulate that for a leukocyte to become firmly attached to endothelium or to establish an active contact between ligand and receptor, more than half a second is required. The mechanism that is required within this time frame for a firm receptor ligand bond formation is the activation of intracellular signaling pathways that impinge on integrin cytoplasmic domains, and make the extracellular domain competent for ligand binding (Hughes and Pfaff, 1998; Shimaoka et al., 2002). This is a very potent mechanism demonstrating the close interrelation between molecular events of receptor-ligand binding and micro-hemorheology. Detachment after the attachment duration was quantified in terms of step velocities of individual leukocytes. The equal occurrence of varying magnitudes of leukocyte step velocities could readily be attributed to transfer of momentum from other flowing leukocytes or RBC aggregates in the microhemodynamic environment of microvascular blood flow. However, there could be other possible reasons for these differences in step velocities that were not explored in the present study. For instance, leukocyte type may determine these differences, as PTX-mediated reactive oxygen species (ROS) production in leukocytes varies with leukocyte subsets (Elbim et al., 1998). Specific labeling of leukocyte subset may answer this. Similarly, a well-characterized inhibitory effect of PTX on leukocytes and endothelial cells could be another reason, in terms of cell adhesion molecule expression and activation, by blocking the synthesis of several cytokines, including tumor necrosis factor (TNF) (Bahra et al., 2001; Dominguez-Jimenez, 2002). The net result of different cytokine actions and adhesion molecule expressions in the event of inflammation appears in terms of receptor-ligand binding strength. However, PTX interferes with such bond formation, thereby affecting the total binding strength. In the complex in vivo setup, there exists a complex interplay of such biochemical responses with hemodynamic factors that may result in variable binding strength between leukocytes and endothelial cells at different time points. Therefore, it is conceivable that such biochemical responses together with biophysical factors determine leukocyte step velocities. In conclusion, understanding the causes of leukocyte migration has great potential in promoting new methods for regulating immune responses. Leukocyte margination from the axial stream followed by attachment to the endothelial wall is a prerequisite of the final migration step and therefore, is an important field of investigation. However, to achieve this goal, one must proceed with a strategy that combines the capability of maximum possible optical resolution of the biofluid dynamics with cell-physiological investigations in the artifact-free setting of in vivo rheoscope. Efforts to utilize progress in the understanding of microcirculatory cell biology have been stalled by the fact
243
that intravital microscopic data on the microcirculatory domain does not have the required resolution. High-resolution imaging of RBC aggregation and subsequent leukocyte displacements in the hemodynamic environment deserves special emphasis since the mere transition from normal to aggregated RBCs achieved by PTX bolus injection plays such a pivotal role in this process, displacing leukocytes in a purely passive manner to the wall. PTX induces intravascular RBC aggregation and sedimentation (Singh and Kumaravel, 1996), which has been visually verified in this work. We are currently involved in the development of an algorithm to quantitatively assess the extent of intravascular RBC aggregation using image-processing techniques.
Acknowledgments The authors gratefully acknowledge the financial support for this work from the DST project under Young Scientist Scheme New Delhi (HR/OY/E-05/97). Partial support comes from Council of Scientific and Industrial Research (CSIR) New Delhi, Scientist Pool Scheme (No. 7233A). The valuable support extended by Dr. C.S. Harendranath of RSIC, IIT Mumbai, to use the Soft Imaging System is highly appreciated and acknowledged. We are also thankful to Dr. K.R. Milner (Pennsylvania, USA) for going through the manuscript and providing valuable suggestions.
References Abbitt, K.B., Nash, G.B., 2003. Rheological properties of the blood influencing selectin-mediated adhesion of flowing leukocytes. Am. J. Physiol.: Heart Circ. Physiol. 285, H229 – H240. Bagge, U., Blixt, A., Braide, M., 1986. Macromodel experiments on the effect of wall-adhering white cells on flow resistance. Clin. Hemorheol. 6, 365 – 372. Bahra, P.S., Rainger, G.E., Wautier, J.L., Nash, G.B., 2001. Effects of pentoxifylline on the different steps during adhesion and transendothelial migration of flowing neutrophils. Cell Biochem. Funct. 19, 249 – 257. Dawson, D.L., Zheng, Q., Worthy, S.A., Charles, B., Bradley Jr., D.V., 2002. Failure of pentoxifylline or cilostazol to improve blood and plasma viscosity, fibrinogen, and erythrocyte deformability in claudication. Angiology 53, 509 – 520. Dominguez-Jimenez, C., Sancho, D., Nieto, M., Montoya, M.C., Barreiro, O., Sanchez-Madrid, F., Gonzalez-Amaro, R., 2002. Effect of pentoxifylline on polarization and migration of human leukocytes. J. Leukocyte Biol. 71, 588 – 596. Elbim, C., Hakim, J., Gougerot-Pocidalo, M.A., 1998. Heterogeneity in Lewis-X and sialyl-Lewis-X antigen expression on monocytes in whole blood: relation to stimulus-induced oxidative burst. Am. J. Pathol. 152, 1081 – 1090. Gerszten, R.E., Lim, Y.C., Ding, H.T., Snapp, K., Kansas, G., Dichek, D.A., Cabanas, C., Sanchez-Madrid, F., Gimbrone Jr., M.A., Rosenzweig, A., Luscinskas, F.W., 1998. Adhesion of monocytes to vascular cell adhesion molecule-1-transduced human endothelial cells: implications for atherogenesis. Circ. Res. 82, 871 – 878. Goldsmith, H.L., Spain, S., 1984. Margination of leukocytes in blood flow through small tubes. Microvasc. Res. 27, 204 – 222.
244
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
Hughes, P.E., Pfaff, M., 1998. Integrin affinity modulation. Trends Cell Biol. 8, 359 – 364. Hussain, M.A., Puniyani, R.R., 1999. Relationship between power law coefficients and major blood constituents affecting the whole blood viscosity. J. Biosci. 24, 329 – 337. Hussain, M.A., Puniyani, R.R., Kar, S., 1994. Quantification of blood viscosity using power law model in cerebrovascular accidents and high-risk controls. Clin. Hemorheol. 14, 685 – 696. Hussain, M.A., Puniyani, R.R., Kar, S., 1995. Blood viscosity autoregulation factor in cerebrovascular accidents and high risk cases—A new approach. Clin. Hemorheol. 15, 61 – 71. Kamm, R.D., 2002. Cellular fluid mechanics. Annu. Rev. Fluid Mech. 34, 211 – 232. Krakauer, T., 2000. Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells. Immunopharmacology 46, 253 – 261. Krakauer, T., Stiles, B.G., 1999. Pentoxifylline inhibits superantigen-induced toxic shock and cytokine release. Clin. Diagn. Lab. Immunol. 6, 594 – 598. Ley, K., Meyer, J.U., Intaglietta, M., ArFors, K.E., 1989. Shunting of leukocytes in rabbit tenuissimus muscle. Am. J. Physiol. 256, H85 – H93. Lipowski, H.H., Usami, S., Chien, S., 1980. In vivo measurement of ‘‘apparent viscosity’’ and microvessel hematocrit in the mesentery of the cat. Microvasc. Res. 19, 297 – 319. Mohan, S., Mohan, N., Valente, A.J., Sprague, E.A., 1999. Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. Am. J. Physiol. 276 (5 Pt 1), C1100 – C1107. Nobis, U., Pries, A.R., Cokelet, G.R., Gaehtgens, P., 1985. Radial distribution of white cells during blood flow in small tubes. Microvasc. Res. 29, 295 – 304. Ogawa, T., Yorioka, N., Ito, T., Ogata, S., Kumagai, J., Kawanishi, H., Yamakido, M., 1997. Precise ultrastructural localization of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in patients with IgA nephropathy. Nephron 75, 54 – 64. Pearson, M.J., Lipowsky, H.H., 2000. Influence of erythrocyte aggregation on leukocyte margination in postcapillary venules of rat mesentery. Am. J. Physiol.: Heart Circ. Physiol. 279, H1460 – H1471. Quezado, Z.M.N., Hoffman, W.D., Banks, S.M., Danner, R.L., Eichacker, P.Q., Susla, G.M., Natanson, C., 1999. Increasing doses of pentoxifylline as a continuous infusion in canine septic shock. J. Pharmacol. Exp. Ther. 288, 107 – 113.
Schmid Scho¨nbein, H., 1996. Heart and circulation: hemorheology. In: Greger, R., Windhorst, U. (Eds.), Comprehensive Human Physiology, vol. 2. Springer-Verlag, Berlin, pp. 1747 – 1792. Schmid-Scho¨nbein, G.W., Usami, S., Skalak, R., Chien, S., 1980. The interaction of leukocytes and erythrocytes in capillary and postcapillary vessels. Microvasc. Res. 19, 45 – 70. Seiffge, D., Kiesewetter, H., 1981. Filtrability investigations with red blood cell (RBC) suspensions: effects of different blood components and pentoxifylline on RBC flow rate. Ric. Clin. Lab. 11, 117 – 123. Shimaoka, M., Takagi, J., Springer, T.A., 2002. Conformational regulation of integrin structure and function. Annu. Rev. Biophys. Biomol. Struct. 31, 485 – 516. Singh, M., Kumaravel, M., 1996. Influence of pentoxifylline and dispirin on aggregation and deformability of erythrocytes under in vitro conditions. Indian J. Biochem. Biophys. 33, 199 – 205. Sliwa, K., Woodiwiss, A., Candy, G., Badenhorst, D., Libhaber, C., Norton, G., Skudicky, D., Sareli, P., 2002. Effects of pentoxifylline on cytokine profiles and left ventricular performance in patients with decompensated congestive heart failure secondary to idiopathic dilated cardiomyopathy. Am. J. Cardiol. 90, 1118 – 1122. Springer, T.A., 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57, 827 – 872. Stein, J.V., Cheng, G., Stockton, B.M., Fors, B.P., Butcher, E.C., von Andrian, U.H., 1999. L-Selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity. J. Exp. Med. 189, 37 – 50. Warnock, R.A., Askari, S., Butcher, E.C., von Andrian, U.H., 1998. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187, 205 – 216. Weitz, J.I., Byrne, J., Clagett, G.P., Farkouh, M.E., Porter, J.M., Sackett, D.L., Strandness Jr., D.E., Taylor, L.M., 1996. Diagnosis and treatment of chronic arterial insufficiency of the lower extremities: a critical review. Circulation 94, 3026 – 3049. William, E.S., 1995. Drugs used in treatment of asthma. In: Hardman, J.G., Limbird, L.E. (Eds.), Goodman and Gilman’s the Pharmacological Basis of Therapeutics, seventh ed. McGraw-Hill Intl., New York, pp. 659 – 682. Wojciak-Stothard, B., Williams, L., Ridley, A.J., 1999. Monocyte adhesion and spreading on human endothelial cells is dependent on rho-regulated receptor clustering. J. Cell Biol. 145 (6), 1293 – 3107.
A decrease in effective diameter of rat mesenteric venules due to leukocyte margination after a bolus injection of pentoxifylline—digital image $ analysis of an intravital microscopic observation M.A. Hussain, a,* S.N. Merchant, b L.S. Mombasawala, c and R.R. Puniyani a a School of Biomedical Engineering, IIT Bombay, Powai, Mumbai 400-076, India Advanced Centre for Research in Electronics, IIT Bombay, Powai, Mumbai 400-076, India c Regional Sophisticated Instrumentation Centre, IIT Bombay, Powai, Mumbai 400-076, India b
Received 29 July 2003 Available online 11 March 2004
Abstract The ability of leukocytes to adhere to endothelial cells (EC) and then to migrate out of the blood stream into tissues enable them to perform their surveillance functions. Adhesion of leukocytes to EC is, however, only possible if the cells have marginated as a result of rheological interaction with other blood cells in flow. Using Pentoxifylline (PTX), a rheologically active drug, to manipulate this interaction, we have imaged and quantified this margination phenomenon in vivo. A system has been developing to perform this imaging via an intravital microscope connected to an image processing system. Albino rats were anesthetized and cannulated for intravenous bolus injection (0.5 ml) of PTX (1.25 mg/ml) through the femoral vein. A longitudinal incision exposed the mesentery, part of which was observed under microscope to visualize microcirculation. The image of interest was then stored on computer hard drive. Individual leukocyte velocities were determined before and after PTX infusion. The leukocytes, marginating and sticking after PTX infusion either remained attached, constituting the peripheral marginating leukocyte pool in the postcapillary venules, or detached with different step velocities. The reduction in effective venular diameters as a result of leukocyte margination was estimated to be 32 – 44%. These results demonstrate the biological importance of hemodynamic displacement leading to docking, adhesion, rolling and migration processes of leukocytes in blood. D 2004 Elsevier Inc. All rights reserved. Keywords: Intravital microscopy; Digital image processing; Microcirculation; Leukocyte; Pentoxifylline
Introduction Leukocytes are seen to adhere to the vascular endothelium of postcapillary venules under a variety of conditions. The molecular mechanisms involved in leukocyte adherence to and subsequent rolling on the endothelial cell (EC) lining of microvessel walls have been extensively studied (Bahra et al., 2001; Dominguez-Jimenez et al., 2002; Gerszten et al., 1998; Mohan et al., 1999; Ogawa et al., 1997; Pearson and Lipowsky, 2000; Stein et al., 1999; Warnock et al., 1998; Wojciak-Stothard et al., 1999). Biophysical aspects of $
Leukocyte margination event observed in rat venular microcirculation. * Corresponding author. Department of Surgery, Biomedical Engineering Institute, H 151, Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, PA 17033. Fax: +1-717-531-4464. E-mail address: [email protected] (M.A. Hussain). 0026-2862/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2004.01.005
the EC – leukocyte interaction leading to hemorheological manifestation in vivo are no less important, and although in vitro investigations exist, there is little in vivo data. Model studies have shown (Bagge et al., 1986) that adhering cells may cause a significant increase in flow resistance by reducing the effective diameter of the vessel, and this is supported by direct measurements in the cat microvessels (Lipowski et al., 1980). This aspect of leukocyte adhesion biology, although of great significance, is only observed after margination from the axial stream primarily because of rheological interaction with other blood cells in flow. An interesting study on this influence of blood rheological properties on the adhesion of leukocytes in vitro has recently been published (Abbitt and Nash, 2003). From a hemodynamic perspective, a leukocyte tends to remain in axial flow in the blood vessel, as it has greater mass than a red blood cell (RBC). However, when RBC
238
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
aggregates are formed and exceed the mass of a leukocyte, the latter becomes marginated. In essence, when as few as five RBCs unite, their mass is much higher than that of an individual leukocyte. Hence, any physical or chemical factor leading to RBC aggregate formation in vivo would automatically tend to displace leukocytes away from the axial stream (Schmid Scho¨nbein, 1996). One such chemical factor widely used clinically is 1-[5-oxohexyl]-3,7-dimethylxanthine, a derivative of theobromine and commercially known as Pentoxifylline (PTX), shown in vitro to induce RBC aggregation (Singh and Kumaravel, 1996). Clinical responses to long-term oral administration of PTX are thought to result primarily from improved erythrocyte flexibility and reduced blood viscosity (Weitz et al., 1996). Newer uses of PTX at the molecular level have been investigated, demonstrating that PTX has inhibitory effects ex vivo on various inflammatory mechanisms, including the complement cascade, neutrophil adherence and cytokine production (Krakauer, 2000; Krakauer and Stiles, 1999; Sliwa et al., 2002; William, 1995). Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF) promote adhesion and migration of leukocytes on endothelial cells (Springer, 1995). As a response to inflammation, the cytokines cause expression of E- and P-selectin, which support adhesion and rolling of leukocytes. Cytokines also upregulate endothelial intercellular adhesion molecule-1 (ICAM-1), which binds integrins on leukocytes and facilitates migration (Springer, 1995). Studies have shown that PTX has a definitive effect on the interaction of different types of leukocytes and endothelial cells at various stages of adhesion and migration (Bahra, 2001; Dominguez-Jimenez, 2002). At the cellular level, however, the effects of PTX on in vivo microhemodynamic behavior of leukocytes and RBCs remain obscure. In this study, we have used PTX to manipulate and an intravital microscopy to visualize leukocyte adhesion events in the microvascular blood flow in vivo. Intravital microscopy provides unique opportunities for visualizing cellular events in microvascular flow at the micrometer scale. Intravital cellular microscopy when combined with digital image processing allows easy handling of the resulting image data. Digital image processing is a rapidly evolving field with growing applications in science and engineering, and generally refers to the processing of a two-dimensional picture by computer. In a broader context, it implies digital processing of any two-dimensional data. An image is first digitized and stored as a matrix of binary digits in computer memory. The digitized image can then be processed and/or displayed on a high-resolution color monitor. Personal Computers (PCs) can be used to communicate and control the digitization, storage, processing, and display operations via a computer network. Digital image processing can be classified broadly into four areas: image enhancement, restoration, coding and understanding. In this work, tools from the first three areas have been extensively utilized. We specifically describe the quantitative measurement of a decrease in effective (flowing) diameter of the microvessel
due to leukocyte margination and subsequent docking along the venular wall. Our results show attaching and detaching events of leukocytes to the venular walls and quantify the step velocities of dislodged cells after detachment. In our analysis and discussion, we interpret the attachment duration of leukocytes to the endothelial cell lining of vascular wall in terms of ligand-receptor activation. Individual leukocyte velocity before and after PTX administration in the microvessel is reported using a semiautomatic method with the help of the digital image processing system.
Methods Animal preparation Albino rats (n = 8) were anesthetized by intraperitoneal injection of Pentothal Sodium (0.1 mg/kg body weight). Femoral veins were cannulated for a 0.5-ml intravenous bolus injection of 1.25 mg/ml PTX (Hoechst), this dose being selected from a published report (Quezado et al., 1999) and also from unstable effects on animals used in screening experiments, performed with higher dose to establish experimental protocol. A longitudinal incision was made to open the belly and a part of mesentery was placed under the microscope (Meiji, Japan) to visualize microcirculation via 40 objective. The mesentery was continuously flushed with normal saline to keep it wet. All experiments were performed in accordance with institutional guidelines. Intravital microscopic setup A CCD camera (Pulnix, USA) was attached to the microscope (Meiji, Japan) and connected with the power supply and image grabber card (Dazzle, Multimedia). The image grabber card, installed in a Personal Computer, was also connected to a VCR to record the experiment on videocassette (Fig. 1). Data acquisition Using the software supplied with the image grabber card, the image data of interest were digitized and stored on the computer hard disk while conducting experiments. A simultaneous recording was also made on videocassette through out the experimental duration to allow offline replay and further digitization of any data that might have been omitted during online digitization. Image analysis Image analysis was carried out using Soft Imaging System—analySIS software. The leukocyte velocities and microvessel diameters were calculated as described below.
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
239
their flow was no longer smooth. Leukocytes were now observed to flow in steps, such that an individual leukocyte appeared to form a contact at one location on the venular endothelial wall, detached to step up and attached again to another location in the same microvessel under focus. Such individual leukocytes were tracked by replaying digital video frame by frame and observed to stick to the venular wall before detachment with a jerk. The velocity of each leukocyte that detached after sticking was measured using the same semiautomatic method as described above and was termed the step velocity of an individual leukocyte. Duration of attachment of leukocyte to the microvessel wall
Fig. 1. Block diagram showing the scheme of image grabbing, editing and analysis systems.
The duration of attachment for a given leukocyte to the venular wall was determined by replaying the digital movie frame by frame. The ratio of the number of frames elapsed before an adhering leukocyte detached from the venular wall with a jerk, to the frame rate of the image grabbing system, determined the attachment duration of the adhering leukocyte.
Velocity determination of leukocytes Vessel diameter measurement The velocities of flowing leukocytes were determined before and after PTX administration using a semiautomatic method with the help of Soft Imaging System, analySIS. Two frames, namely, frame-1 and frame-n, were snapped by replaying the digital movie of the mesenteric microcirculation stored on the hard disk frame by frame in such a way that the movement of the leukocyte in question was visually tracked in each frame. The location of the leukocyte in the first frame (i.e., frame-1) and nth frame (frame-n) was determined by assigning a common reference point in both frames. This reference point was chosen to be present in both frames and was a static structure in the matrix of interstitial spaces in the microvascular bed. The distance blood cell moved was calculated by drawing a line between the cell and reference point in fame-1. The length of this line and the angle between this line and a vertical line were measured. This length and angle relative to the reference were then used to relocate the initial blood cell position in frame-n. The distance between the relocated postion-1 and the position-2 of the moving blood cell in the frame-n may then be determined. Representative images depicting this method for a leukocyte velocity calculation are shown in Fig. 2. The time was calculated by the number of frames elapsed (i.e., n frames) divided by the frame rate at which the image was grabbed (25 frames/s). Hence, with the distance traveled and time elapsed, leukocyte velocities were determined. The experiments were focused at measuring individual leukocyte velocities near the microvessel wall and not the flux of leukocytes flowing through the microvessel. Before PTX administration, leukocytes were occasionally found flowing smoothly along the microvessel wall and their velocities were determined as above. After PTX infusion, more leukocytes started appearing on the venular wall but
Two diameters were calculated: one, the ‘actual’ diameters of the microvessel including the marginating pool of leukocytes, and, two, the ‘reduced’ diameter of the flowing red cell column excluding the marginating pool of leukocytes. They were calculated by taking the average of five diameters measured at points within a length equal to 2.5 times the microvessel radius. The percentage of change in diameter was calculated using the following equation. Percentage diameter change ¼ ðda dr Þ=da 100% where da is actual diameter measured; dr is the reduced diameter.
Fig. 2. Representative image depicting the method for leukocyte velocity calculation. From a common reference points in two given images, a vertical line is drawn. In the given frame-1, another line is drawn from the reference point connecting it to the leukocyte and the angle that this line makes with vertical line is noted. In the frame-n now at this measured angle, a line of the same length is drawn to relocate the leukocyte position in this frame corresponding to the frame-1. A broken line with two-sided arrow shown in frame-n gives the distance traveled by the leukocyte during the time lapse from frame-1 to frame-n. The velocity was calculated from this time and distance data.
240
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
Statistical analysis Means and standard deviations are reported. The level of significance was set to P < 0.001 at 95% of confidence.
Table 1 Leukocyte velocity measured in postcapillary venules and capillaries before infusing pentoxifylline. Sixty leukocytes were analyzed from digitized movie of 16 venules and capillaries, roughly seven velocity measurements per two microvessels per rat Diameter ranges
Results Before administration of PTX, leukocytes were often observed passing through capillaries but occasionally appeared to crawl along venular walls. The flow of leukocytes observed in venules was smooth with a uniform velocity, although this was not always the case in capillaries. Rather, leukocytes would often obstruct the flow and even plug the capillaries momentarily. We measured the velocities of 60 smoothly flowing leukocytes from digitized movies of 16 venules and capillaries, with roughly seven velocity measurements per two microvessels per rat. The results of leukocyte velocity measurements in venules and capillaries are shown in Table 1. We did not find any significant correlation between leukocyte velocities and
Venules (n = 30) 15 – 25 Am Capillaries (n = 30) 3 – 8 Am
Leukocyte velocity (Am/s) 50.3 F 3.7 16.1 F 3.2
P < 0.001.
diameter of microvessels in which these measurements were made. Within roughly 20 min of PTX administration, intravascular RBC aggregation was clearly observed as clumps of RBCs flowing in the axial stream of the venules (Fig. 3). This was not quantified due to the limitations of our image analysis system. This aggregation of RBCs led to the margination of leukocytes towards the venular wall periphery resulting in an improved focus of these cells and enhanced image clarity. The effect of PTX administration was not found to be reversible during the imaging duration of approximately 30– 40 min after PTX infusion. We did not continue the experiments further and do not have data showing reversibility of the observed effect of PTX. Contrary to a smooth flow before PTX administration, leukocytes were now observed to stick in abundance to the postcapillary venules forming a pool of leukocytes along the venule margin. Leukocytes were not moving with a uniform velocity, rather they showed sticking and detaching phenomena. Leukocytes were observed to attach to the wall of the vessel, and then often dislodge from the attachment site with a jerk. Leukocytes were thus imparted with a step velocity after dislodgement into the sustained hemodynamic environment. Leukocytes that detached from the vessel wall due to cellular interaction in flow got a jerk of varying magnitude with an equal frequency of occurrence due to a transfer of momentum from flowing particles (leukocyte, RBC aggregates), thereby imparting a range of step velocities. These step velocities were calculated for leukocytes (n = 45) in 16 microvessels (two microvessels per rat) with approximately three leukocyte velocity determinations per microvessel. These results are shown in Table 2. The attachment of leukocytes to the endothelial lining of the vessel wall was observed to be stable Table 2 Leukocyte step velocities measured in postcapillary venules after 20 min of pentoxifylline bolus injection show equal number of occurrence
Fig. 3. Representative postcapillary venule images showing clumps of red cell aggregates in Panel B. Panel A images represent smooth blood flow before pentoxifylline administration and Panel B images are snaps from digital movie after pentoxifylline administration.
Ranges of leukocyte step velocities in postcapillary venules (Am/s)
Frequency of step velocities of leukocytes (n = 45)
<20 20 – 40 40 – 60
16 14 15
All the detaching leukocytes measured for their step velocities stuck to the venular wall only for fraction of a second (0.5 F 0.1 s).
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244 Table 3 Percentage reduction in the effective (flowing) diameter of microvessels after leukocyte adhesion and docking to microvascular wall No. of microvessels
Actual diameter (Am)
Reduced (effective) diameter (Am)
% Change
48 (six microvessels per rat)
20.1 F 3.2
12.8 F 2.0
36.2 F 3.2
P < 0.001.
if it remained attached for a duration approximating 1 s, thereby forming a pool of leukocytes. However, most leukocytes were seen to detach as a result of collision with other blood cells within a duration of half a second (0.5 F 0.1 s as calculated for n = 45 leukocytes). Due to the marginating pool of leukocytes, the effective diameter of the vessel was observed to decrease by 32 – 44% as presented in Table 3. The reduction in the effective diameter of microvessel is sketched from a representative image (Fig. 4). We also measured a population of microvasucular diameters before and after PTX administration to determine if PTX imparted any major change in the actual diameter of microvessels. The histograms showing the distribution of microvascular diameters before and after PTX infusion are very similar as presented in Fig. 5. This shows that vessel diameters did not change with PTX administration but the effective flowing diameter was reduced by the marginating pool of leukocytes.
Discussion Video stored in digital form has several advantages over its analog counterpart. These include: (a) digital recordings are almost immune to signal problems, and deliver the highest picture quality; (b) digital video has approximately twice the horizontal resolution of a standard VHS videocassette recorder. The resolution of a digital video standard image is approximately 25% better than that of an S-VHS or Hi-8 camcorder or VCR; (c) color resolution (or rendition) can be a problem for analog video, producing color blur and
241
color noise. Digital video does not exhibit this problem, delivering a more life-like video image on the screen, with sharper subject edges and clearer color reproduction; (d) video quality of analog video is reduced each time a tape is played, whereas digital video does not degrade with time; (e) digital copies of digital videos are indistinguishable from the original, which makes editing and image manipulation easier and with higher quality than that delivered by analog video technology; (f) digital video can be subjected to sophisticated digital image processing techniques for extracting information. We have carried out extensive measurements on digital video, which would not have been possible with analog video; (g) digital video is nonlinear, meaning that any scene or video clip can be accessed randomly without regard for its location in the footage. Analog video is linear, meaning that to locate a scene at the end of the tape, the user must bypass all preceding footage; (h) the digital video process is nondestructive. Experimentation on it can be carried out in an unlimited manner without risking the loss or destruction of the original footage; (i) digitally encoded video can be stored on DVD or other formats that have extended archival lives. This type of media is also more compact, and more durable than videotape, and finally, (j) video capture in digital format can be controlled more easily and efficiently by using the record or capture function in the digital video editing software. Certainly, these great benefits of digital video imaging warrants its application in variety of biological research. Biologists traditionally have been utilizing the video imaging for their research especially in hemodynamics of microcirculation (Kamm, 2002). In the present work, we have been able to extract valuable information from the digitized video images demonstrating the effect of PTX on microvasucular flow parameters at cellular level. In arterioles, where shear stresses are generally higher than those in venules, RBCs are kept dynamically disaggregated in the flow. Consequently, leukocytes are the largest moving particles and travel near the vessel axis. On the other hand, in the venules, shear stresses are much lower. Therefore, RBCs can rapidly form large aggregates and displace leukocytes away from the preferred axial layers
Fig. 4. Representative image showing peripheral marginating leukocyte pool, causing reduction of effective postcapillary venular diameter. (A) Actual image; (B) tracing of image A showing marginating leukocyte pools.
242
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
towards the wall as these RBC aggregates are larger than individual leukocytes. Under pathological conditions (i.e., when the tendency of RBCs to aggregate is increased), the aggregates persist even in the presence of very high shearing forces. Further, a high plasma concentration of aggregating colloids can enhance this effect (e.g., fibrinogen can enhance leukocyte margination by exaggerating and accelerating RBC rouleaux formation). A hemoconcentration experiment that is conducted by stepwise increase in hematocrit has simulated this margination phenomenon of leukocytes (Abbitt and Nash, 2003). In this work, the RBC aggregate forming tendency of PTX (Singh and Kumaravel, 1996) is used to simulate a pathological condition of RBC aggregability, thus facilitating the margination of leukocytes from axial blood flow. This study further demonstrates the associated reduction in effective vessel diameter due to leukocyte pool formation along the vessel wall. The mechanisms of this rheological margination have been studied in detail (Abbitt and Nash, 2003; Goldsmith and Spain, 1984; Nobis et al., 1985; Schmid-Scho¨nbein et al., 1980). Before PTX infusion, however, we observed leukocytes to appear occasionally
Fig. 5. Histograms showing distribution of microvascular diameter measurements (A) before and (B) after PTX administration. A total of (A) 72 and (B) 74 microvessels were analyzed.
near the microvessel wall and flow smoothly. As we were just interested in single cell velocity measurements, we did not feel it necessary to tag leukocytes with fluorescent dyes and hence the relative change in leukocyte distribution from axial to marginal flow was not quantified in this study. It is clear from the present study that a qualitative increase in RBC aggregation after PTX bolus infusion caused a displacement of leukocytes toward the vessel wall and thus favored margination and adhesion. The movements of leukocytes in terminal vasculature are passively coordinated by the interactions with moving RBCs. A dynamic RBC aggregation – disaggregation would determine which type of blood cell (individual leukocyte or aggregated RBC) would comprise the largest particle in flow. Leukocytes tend to get displaced away from the axial stream to the vessel wall apparently because of formed RBC aggregates in the flow. A variable number of leukocytes that come to an effective standstill and are classified as sticking, forming a pool in this study, may be attributed to several factors such as leukocyte and endothelial ‘‘stickiness’’ (adhesive energies), expression of adhesion molecules on endothelial cells, cytokine production by leukocytes, maintenance of endothelial cell continuity, local hemodynamic forces, shear stresses, and transmural pressure in addition to the red cell aggregation and/or red cell rouleaux formation. PTX increases the deformability of RBCs (Dawson et al., 2002; Seiffge and Kiesewetter, 1981) and also induces RBC aggregation (Singh and Kumaravel, 1996). In this work, PTX facilitated the formation of large clumps of RBCs that pushed leukocyte into the marginal layers close to microvessel wall. Although the extent of this intravascular RBC aggregation as observed in this study was not quantified, it may be possible to do so in vitro using indirect methods, for example, photometric or ultrasound aggregometers (Schmid Scho¨nbein, 1996) or a low shear viscometer (Hussain and Puniyani, 1999; Hussain et al., 1994, 1995). The present use of PTX made it easy to watch marginating leukocytes in postcapillary venules and to calculate leukocyte velocities. To initiate an active contact with endothelial cells, it appears that the attachment duration must be close to 1 s. Most often, leukocytes that were observed to detach had half a second (0.5 F 0.1 s) attachment duration. However, the leukocytes that succeeded in remaining attached for a longer duration (usually at least close to a second) were seen to continue sticking in place contributing to the leukocyte pool, possibly due to firm binding with adhesion molecules expressed on endothelial cell surface (Ley et al., 1989; Warnock et al., 1998) after which these may show the rolling phenomena. Interestingly, our observation of leukocyte arrest on the endothelial wall with a duration of close to 1 s provides direct evidence of effective bond formation between receptor and ligands that has been reported to occur on a time scale of less than 1 s (Hughes and Pfaff, 1998; Shimaoka et al., 2002). All detaching leukocytes investigated for step velocities in this work stayed attached for nearly half a second before they were detached by collision with
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
other flowing cells, indicating poor adhesion of leukocytes during this time span. However, beyond this duration, all leukocytes remained docked along the microvessel wall indicating firm adhesion. Therefore, we postulate that for a leukocyte to become firmly attached to endothelium or to establish an active contact between ligand and receptor, more than half a second is required. The mechanism that is required within this time frame for a firm receptor ligand bond formation is the activation of intracellular signaling pathways that impinge on integrin cytoplasmic domains, and make the extracellular domain competent for ligand binding (Hughes and Pfaff, 1998; Shimaoka et al., 2002). This is a very potent mechanism demonstrating the close interrelation between molecular events of receptor-ligand binding and micro-hemorheology. Detachment after the attachment duration was quantified in terms of step velocities of individual leukocytes. The equal occurrence of varying magnitudes of leukocyte step velocities could readily be attributed to transfer of momentum from other flowing leukocytes or RBC aggregates in the microhemodynamic environment of microvascular blood flow. However, there could be other possible reasons for these differences in step velocities that were not explored in the present study. For instance, leukocyte type may determine these differences, as PTX-mediated reactive oxygen species (ROS) production in leukocytes varies with leukocyte subsets (Elbim et al., 1998). Specific labeling of leukocyte subset may answer this. Similarly, a well-characterized inhibitory effect of PTX on leukocytes and endothelial cells could be another reason, in terms of cell adhesion molecule expression and activation, by blocking the synthesis of several cytokines, including tumor necrosis factor (TNF) (Bahra et al., 2001; Dominguez-Jimenez, 2002). The net result of different cytokine actions and adhesion molecule expressions in the event of inflammation appears in terms of receptor-ligand binding strength. However, PTX interferes with such bond formation, thereby affecting the total binding strength. In the complex in vivo setup, there exists a complex interplay of such biochemical responses with hemodynamic factors that may result in variable binding strength between leukocytes and endothelial cells at different time points. Therefore, it is conceivable that such biochemical responses together with biophysical factors determine leukocyte step velocities. In conclusion, understanding the causes of leukocyte migration has great potential in promoting new methods for regulating immune responses. Leukocyte margination from the axial stream followed by attachment to the endothelial wall is a prerequisite of the final migration step and therefore, is an important field of investigation. However, to achieve this goal, one must proceed with a strategy that combines the capability of maximum possible optical resolution of the biofluid dynamics with cell-physiological investigations in the artifact-free setting of in vivo rheoscope. Efforts to utilize progress in the understanding of microcirculatory cell biology have been stalled by the fact
243
that intravital microscopic data on the microcirculatory domain does not have the required resolution. High-resolution imaging of RBC aggregation and subsequent leukocyte displacements in the hemodynamic environment deserves special emphasis since the mere transition from normal to aggregated RBCs achieved by PTX bolus injection plays such a pivotal role in this process, displacing leukocytes in a purely passive manner to the wall. PTX induces intravascular RBC aggregation and sedimentation (Singh and Kumaravel, 1996), which has been visually verified in this work. We are currently involved in the development of an algorithm to quantitatively assess the extent of intravascular RBC aggregation using image-processing techniques.
Acknowledgments The authors gratefully acknowledge the financial support for this work from the DST project under Young Scientist Scheme New Delhi (HR/OY/E-05/97). Partial support comes from Council of Scientific and Industrial Research (CSIR) New Delhi, Scientist Pool Scheme (No. 7233A). The valuable support extended by Dr. C.S. Harendranath of RSIC, IIT Mumbai, to use the Soft Imaging System is highly appreciated and acknowledged. We are also thankful to Dr. K.R. Milner (Pennsylvania, USA) for going through the manuscript and providing valuable suggestions.
References Abbitt, K.B., Nash, G.B., 2003. Rheological properties of the blood influencing selectin-mediated adhesion of flowing leukocytes. Am. J. Physiol.: Heart Circ. Physiol. 285, H229 – H240. Bagge, U., Blixt, A., Braide, M., 1986. Macromodel experiments on the effect of wall-adhering white cells on flow resistance. Clin. Hemorheol. 6, 365 – 372. Bahra, P.S., Rainger, G.E., Wautier, J.L., Nash, G.B., 2001. Effects of pentoxifylline on the different steps during adhesion and transendothelial migration of flowing neutrophils. Cell Biochem. Funct. 19, 249 – 257. Dawson, D.L., Zheng, Q., Worthy, S.A., Charles, B., Bradley Jr., D.V., 2002. Failure of pentoxifylline or cilostazol to improve blood and plasma viscosity, fibrinogen, and erythrocyte deformability in claudication. Angiology 53, 509 – 520. Dominguez-Jimenez, C., Sancho, D., Nieto, M., Montoya, M.C., Barreiro, O., Sanchez-Madrid, F., Gonzalez-Amaro, R., 2002. Effect of pentoxifylline on polarization and migration of human leukocytes. J. Leukocyte Biol. 71, 588 – 596. Elbim, C., Hakim, J., Gougerot-Pocidalo, M.A., 1998. Heterogeneity in Lewis-X and sialyl-Lewis-X antigen expression on monocytes in whole blood: relation to stimulus-induced oxidative burst. Am. J. Pathol. 152, 1081 – 1090. Gerszten, R.E., Lim, Y.C., Ding, H.T., Snapp, K., Kansas, G., Dichek, D.A., Cabanas, C., Sanchez-Madrid, F., Gimbrone Jr., M.A., Rosenzweig, A., Luscinskas, F.W., 1998. Adhesion of monocytes to vascular cell adhesion molecule-1-transduced human endothelial cells: implications for atherogenesis. Circ. Res. 82, 871 – 878. Goldsmith, H.L., Spain, S., 1984. Margination of leukocytes in blood flow through small tubes. Microvasc. Res. 27, 204 – 222.
244
M.A. Hussain et al. / Microvascular Research 67 (2004) 237–244
Hughes, P.E., Pfaff, M., 1998. Integrin affinity modulation. Trends Cell Biol. 8, 359 – 364. Hussain, M.A., Puniyani, R.R., 1999. Relationship between power law coefficients and major blood constituents affecting the whole blood viscosity. J. Biosci. 24, 329 – 337. Hussain, M.A., Puniyani, R.R., Kar, S., 1994. Quantification of blood viscosity using power law model in cerebrovascular accidents and high-risk controls. Clin. Hemorheol. 14, 685 – 696. Hussain, M.A., Puniyani, R.R., Kar, S., 1995. Blood viscosity autoregulation factor in cerebrovascular accidents and high risk cases—A new approach. Clin. Hemorheol. 15, 61 – 71. Kamm, R.D., 2002. Cellular fluid mechanics. Annu. Rev. Fluid Mech. 34, 211 – 232. Krakauer, T., 2000. Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells. Immunopharmacology 46, 253 – 261. Krakauer, T., Stiles, B.G., 1999. Pentoxifylline inhibits superantigen-induced toxic shock and cytokine release. Clin. Diagn. Lab. Immunol. 6, 594 – 598. Ley, K., Meyer, J.U., Intaglietta, M., ArFors, K.E., 1989. Shunting of leukocytes in rabbit tenuissimus muscle. Am. J. Physiol. 256, H85 – H93. Lipowski, H.H., Usami, S., Chien, S., 1980. In vivo measurement of ‘‘apparent viscosity’’ and microvessel hematocrit in the mesentery of the cat. Microvasc. Res. 19, 297 – 319. Mohan, S., Mohan, N., Valente, A.J., Sprague, E.A., 1999. Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. Am. J. Physiol. 276 (5 Pt 1), C1100 – C1107. Nobis, U., Pries, A.R., Cokelet, G.R., Gaehtgens, P., 1985. Radial distribution of white cells during blood flow in small tubes. Microvasc. Res. 29, 295 – 304. Ogawa, T., Yorioka, N., Ito, T., Ogata, S., Kumagai, J., Kawanishi, H., Yamakido, M., 1997. Precise ultrastructural localization of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in patients with IgA nephropathy. Nephron 75, 54 – 64. Pearson, M.J., Lipowsky, H.H., 2000. Influence of erythrocyte aggregation on leukocyte margination in postcapillary venules of rat mesentery. Am. J. Physiol.: Heart Circ. Physiol. 279, H1460 – H1471. Quezado, Z.M.N., Hoffman, W.D., Banks, S.M., Danner, R.L., Eichacker, P.Q., Susla, G.M., Natanson, C., 1999. Increasing doses of pentoxifylline as a continuous infusion in canine septic shock. J. Pharmacol. Exp. Ther. 288, 107 – 113.
Schmid Scho¨nbein, H., 1996. Heart and circulation: hemorheology. In: Greger, R., Windhorst, U. (Eds.), Comprehensive Human Physiology, vol. 2. Springer-Verlag, Berlin, pp. 1747 – 1792. Schmid-Scho¨nbein, G.W., Usami, S., Skalak, R., Chien, S., 1980. The interaction of leukocytes and erythrocytes in capillary and postcapillary vessels. Microvasc. Res. 19, 45 – 70. Seiffge, D., Kiesewetter, H., 1981. Filtrability investigations with red blood cell (RBC) suspensions: effects of different blood components and pentoxifylline on RBC flow rate. Ric. Clin. Lab. 11, 117 – 123. Shimaoka, M., Takagi, J., Springer, T.A., 2002. Conformational regulation of integrin structure and function. Annu. Rev. Biophys. Biomol. Struct. 31, 485 – 516. Singh, M., Kumaravel, M., 1996. Influence of pentoxifylline and dispirin on aggregation and deformability of erythrocytes under in vitro conditions. Indian J. Biochem. Biophys. 33, 199 – 205. Sliwa, K., Woodiwiss, A., Candy, G., Badenhorst, D., Libhaber, C., Norton, G., Skudicky, D., Sareli, P., 2002. Effects of pentoxifylline on cytokine profiles and left ventricular performance in patients with decompensated congestive heart failure secondary to idiopathic dilated cardiomyopathy. Am. J. Cardiol. 90, 1118 – 1122. Springer, T.A., 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57, 827 – 872. Stein, J.V., Cheng, G., Stockton, B.M., Fors, B.P., Butcher, E.C., von Andrian, U.H., 1999. L-Selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity. J. Exp. Med. 189, 37 – 50. Warnock, R.A., Askari, S., Butcher, E.C., von Andrian, U.H., 1998. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187, 205 – 216. Weitz, J.I., Byrne, J., Clagett, G.P., Farkouh, M.E., Porter, J.M., Sackett, D.L., Strandness Jr., D.E., Taylor, L.M., 1996. Diagnosis and treatment of chronic arterial insufficiency of the lower extremities: a critical review. Circulation 94, 3026 – 3049. William, E.S., 1995. Drugs used in treatment of asthma. In: Hardman, J.G., Limbird, L.E. (Eds.), Goodman and Gilman’s the Pharmacological Basis of Therapeutics, seventh ed. McGraw-Hill Intl., New York, pp. 659 – 682. Wojciak-Stothard, B., Williams, L., Ridley, A.J., 1999. Monocyte adhesion and spreading on human endothelial cells is dependent on rho-regulated receptor clustering. J. Cell Biol. 145 (6), 1293 – 3107.