NEFROLOGÍA. Vol. XXIII. Suplemento 3. 2003 III. FISIOPATOLOGÍA DE LA RESPUESTA PERITONEAL A LA DIÁLISIS Intravital videomicroscopy in peritoneal dialysis research N. Lameire, A. De Vriese and S. Mortier Renal Division. University Hospital. Ghent. Belgium. INTRODUCTION Long-term exposure to peritoneal dialysis (PD) solutions may exert deleterious effects on the peritoneal membrane, ultimately leading to ultrafiltration failure, inadequate dialysis or increased susceptibility to infection 11. A number of experimental techniques are available for the study of the effects of dialysate exposure, usually focusing on a particular aspect of peritoneal structure or function. The present paper describes an integrated evaluation of the functional and structural characteristics of the peritoneal membrane using an intravital microscopy technique, with off-line analysis by a multifunctional computer-assisted image analysis system. The intravital microscopy set-up and the potential applications of the technique for the study of PD solutions are discussed. Some of the results that have been obtained with this technique are described. INTRAVITAL MICROSCOPY This technique has previously been described in detail 2, 5. Briefly, female Wistar rats are anaesthetized with subcutaneous thiobutabarbital 100 mg/kg (Inactin, RBI, Natick, MA, USA). After intubation, a jugular vein is cannulated for continuous infusion of isotonic saline and a carotid artery is cannulated for continuous monitoring of mean arterial blood pressure. A small midline abdominal incision is made with an electrocauter and a short segment of the small bowel Correspondence: Norbert Lameire Renal Division University Hospital 185, De Pintelaan 9000 Ghent, Belgium E-mail: norbert.lameire@rug.ac.be is exteriorized, carefully avoiding stretching. The visceral peritoneum is spread over a plexiglas plate and superfused continuously with an isotonic, isocolloidal solution (Haemaccel, Hoechst Marion Roussel, Marburg/Lahn, Germany) maintained at 37° C. The preparation is allowed to stabilize for 30 min after completion of surgery. Observations are made with an Axiotech Vario 100 HD microscope (Zeiss, Jena, Germany) using water immersion objectives (Achroplan 10x, 40x, 63x). The microscopical stage is driven by a stepping motor control MCL-2 (Lang, Huttenberg, Germany), operated by a joystick or a software program (Wincommander, MarzhauserWetzlar, Wetzlar, Germany) via an RS-232 interface. The tissue is transilluminated via a fibreoptic using a light source (KL 1500, Schott, Wiesbaden, Germany) equipped with a 150 W halogen lamp. Epifluorescence is performed with a mercury lamp HBO 50 W and a fluorescein isothiocyanate (FITC) filter set. The resulting image is displayed on a television monitor by a TK-1281 camera or a high-speed video camera (Kodak Motioncorder Analyser, Eastman Kodak Company, San Diego, CA, USA) and recorded by a videorecorder (S-VHS Panasonic AG-7355, Matsushita, Japan) for off-line analysis. All automatic gain controls are switched off during the experiments. The video images are digitized with an IP8/AT Matrox image processing board and analysed with a multifunctional image analysis software program (Cap-Image, Ingenieurburo Zeintl, Heidelberg, Germany). APPLICATIONS Blood flow rate Blood flow rate is generally studied in arterioles or postcapillary venules. Red blood cell velocity is measured with the line-shift-diagram method 7. In ar- 28 INTRAVITAL VIDEOMICROSCOPY IN PERITONEAL DIALYSIS RESEARCH terioles, red blood cell velocity exceeds 2 mm/s, which is the upper limit of velocity that can be analysed off-line using standard video framing rates. Therefore, recordings are made with a high-speed video camera at a rate of up to 600 images per second, extending the measurement range to 40 mm/s 5. Luminal diameter is measured with the perpendicular line method 7. Blood flow rate (BFR) is calculated from the equation: BFR = VRBC x irD2/4 where VRBC = red blood cell velocity and D = luminal diameter. A possible application of these measurements is the study of local haemodynamic changes induced by acute exposure to fresh dialysate and the possible interactions of the different dialysate components (pH, buffer, glucose degradation products) in provoking these effects. Permeability to macromolecules Microvascular permeability is studied in a venule, using an FITC-labelled macromolecule. The molecule- generally FITC-albumin-is administered as an intravenous bolus and epifluorescence recordings are made every 10 min for 120 min. On the digitized image of the venule, two intraluminal areas and two contiguous areas of perivenular interstitium are defined. The average grey scale value, ranging from 0 for black to 255 for white, is calculated for each area. Escape of the fluorescent molecule from the circulation will cause a decrease of the average grey scale value within the venule (Gv) and an increase of the average grey scale value in the perivenular interstitium (Gi). Macromolecular leakage is defined as the change of the Gv:Gi ratio over time. Development of peritoneal hyperpermeability is a common problem in PD patients and is associated with an adverse prognosis 12. Intravital microscopy permits the study of peritoneal permeability in direct relation to other microvascular parameters, including microvascular proliferation and leukocyte behaviour (see below). A better understanding of the aetiology of peritoneal hyperpermeability may enable the design of novel approaches to prevent or reverse its development. Leukocyte endothelial interactions The recruitment of a circulating leukocyte into a tissue follows a multi-step process, directed by adhesive interactions. Selections and their carbohy- drate-containing ligands mediate the initial and transient contact between the leukocyte and the vascular endothelium, the socalled rolling. This is followed by firm adherence and transendothelial migration, mediated by interaction of integrins with immunoglobulin-like molecules. Intravital microscopy permits the direct visualization of these leukocyte-endothelial interactions. Leukocyte rolling and adhesion are usually studied in the same venule selected for the study of macromolecular permeability. Rolling leukocytes are defined as those that move at a velocity lower than that of the red blood cells and are in contact with the endothelial surface. The flux of rolling leukocytes is determined by counting the number of rollers crossing an imaginary line perpendicular to the axis of the venule, per minute. The flux of rolling leukocytes may be corrected by blood flow rate to avoid confounding by concomitant changes in blood flow. The number of leukocytes adhering to the venular endothelial lining and not moving during a 30 s period is counted and expressed as the number per 100-lm length of venule. It is well known that PD solutions profoundly affect different aspects of leukocyte behaviour 6. Intravital microscopy studies, allowing direct visualization of leukocyte kinetics in response to dialysate exposure, may be ancillary to a better understanding of the effects of PD solutions on in vivo leukocyte recruitment in different pathophysiological conditions. Microvascular density The microvascular density of the visceral peritoneum increases progressively towards the distal ileum. Hence, a standardized selection of measurement areas is of great importance. The objective is positioned at random within several pre-defined segments of the visceral peritoneum of the distal ileum. With the aid of the Wincommander software, the microscopical stage is driven through a meander consisting of five steps of 1 mm in the x-direction and five steps of 1 mm in the y-direction. The microscopic image is recorded at each of the 30 positions. The vessel length per area for each microscopic image is calculated, with the use of the Capimage software. The angiogenetic potential of PD solutions has been demonstrated by a recent study 1 and its role in peritoneal ultrafiltration failure has been discussed by us before 3. Therefore, direct measurement of microvascular density by intravital microscopy is a valuable tool in the evaluation of the long-term effects of PD solutions. 29 N. LAMEIRE y cols. The dynamic nature of microvascular networks can be studied by only including perfused capillaries in the analysis. The capability of PD solutions to recruit capillary reserves and thus additional transport surface may be a determinant of the efficiency of solute clearance. The direct measurement of peritoneal capillary recruitment by intravital microscopy may thus contribute to the understanding of this phenomenon and allow a more in-depth study of the physiology of peritoneal transport. Lymph vessels In the visceral peritoneum, lymph vessels are generally found in the immediate vicinity of the fat axes. Lymphatic pumping occurs at an irregular rate and is guided by an internal pacemaker. Lymph vessel diameter and constriction frequency, and their responsiveness to local application of pharmacological agents can be measured. Excessive lymphatic absorption may contribute to loss of ultrafiltration and solute clearance in PD patients 8, 9. A potential application of intravital microscopy may be the study of lymph vessel kinetics during inflammatory conditions or during dialysate exposure. RESULTS OF STUDIES PERFORMED WITH INTRAVITAL VIDEOMICROSCOPY OF THE PERITONEAL CIRCULATION Long-term exposure to the high glucose concentrations in conventional peritoneal dialysate has been implicated in the pathogenesis of peritoneal hyperpermeability and neoangiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that potently stimulates microvascular permeability and proliferation. High glucose exposure upregulates VEGF expression in various cell types and tissues. This study investigated whether VEGF plays a pathogenetic role in hyperglycemia-induced microvascular dysfunction in the peritoneal membrane 4. The peritoneal microcirculation of streptozotocininduced diabetic rats and age-matched controls was studied in vivo with a combination of functional and morphologic techniques. The diabetic microcirculation was characterized by an elevated transport of small solutes, indicating the presence of an increased effective vascular surface area. The leakage of FITC-albumin was more rapid in diabetic vessels, suggesting hyperpermeability for macromolecules. Structurally, an increased vascular density with focal 30 areas of irregular capillary budding was found in the diabetic peritoneum. The hyperglycemia-induced structural and functional microvascular alterations were prevented by long-term treatment with neutralizing anti-VEGF monoclonal antibodies, whereas treatment with isotype-matched control antibodies had no effect. VEGF blockade did not influence microvascular density or macromolecular leakage in control rats, demonstrating specificity for the hyperglycemia-induced alterations. The present results thus support an causative link among high glucose exposure, upregulation of VEGF, and peritoneal microvascular dysfunction. The hemodynamic effects of conventional and new PD solutions were then explored 10. These results showed that a conventional, acidic pH, lactate-buffered 4.25% glucose PDF induced maximal vasodilation of mesenteric arteries, resulting in a doubling of the arteriolar flow and a 20% increase of the perfused capillary length per area. The hemodynamic effects of conventional PDF were similar after pH-adjustment with NaOH, indicating that acidity per se is not essential for the changes. Superfusion by a pH-neutral, lactate-buffered PDF with low GDP content caused only a transient arterial vasodilation despite continuous exposure, with a commensurate effect on arteriolar flow and capillary recruitment. Application of a pH-neutral, bicarbonate-buffered PDF with low GDP content did not affect the hemodynamic parameters. Resterilization of the bicarbonate solution increased GDP lev-els and completely restored the vasodilatory capacity. The corresponding 1.5% glucose PDF induced similar but less pronounced changes. Conventional PDF have important vasoactive effects on the peritoneal circulation, mainly because of the presence of GDP and transiently because of high lactate concentrations. Capillary recruitment may increase effective peritoneal vascular surface area. In addition, chronic vasodila-tion may induce structural adaptations in the blood vessel wall, contributing to vascular sclerosis. PDF with reduced GDP content induce no major hemodynamic effects and may thus have the potential to better preserve peritoneal vascular integrity. A third series of experiments involved the investigation of several peritoneal dialysis fluids on the leukocyte response to two toxins derived from Gramnegative and Gram positive microbes. Both LPS and a stapylococcal --derived toxin induced an increase in leukocyte rolling, endothelial adherence and vascular transmigration when added to a physiologic buffer solution, EBSS. In contrast the classical PD fluids strongly suppressed these leukocyte reactions. INTRAVITAL VIDEOMICROSCOPY IN PERITONEAL DIALYSIS RESEARCH A GDP-free bicarbonate containing PD fluid showed a much less suppressive effect. In order to delineate which components in the classical PD fluids were responsible for the suppression, artifical fluids containing either D-Mannitol, or lactate were tested and it could be concluded that both the hyperosmolaity and the lactate buffer were mostly responsible for the suppression of the normal behaviour of the leukocytes in the peritoneal capillaries when challenged with microbial-derived toxins. (Mortier et al, unpublished results.) CONCLUSION Intravital microscopy coupled to computer-assisted image analysis, as described in the present communication, is a versatile technique that allows the integrated assessment of peritoneal structure and function under different pathophysiological conditions. The technique permits in vivo and direct visualization of peritoneal arterioles, capillaries, venules and lymphatic vessels. Functional parameters such as blood flow rate, vessel diameter, permeability to macromolecules, leukocyte-endothelial interactions, capillary recruitment and lymph vessel kinetics can be studied and quantified. In addition, the architecture and density of the microvascular network can be evaluated. The strength of the technique is the potential to evaluate different parameters concomitantly in the same experimental animal, allowing an integration of the experimental findings. The method is easy to implement and uses commercially available hardware and software. There is compelling evidence that the actions of PD solutions on peritoneal function and structure are complex and by no means limited to direct cytotoxic effects. 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