An Experimental Rat Model for Studying Pulmonary Microcirculation by in Vivo Videomicroscopy

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Microvascular Research 62, 421–434 (2001)doi:10.1006/mvre.2001.2336, available online at http://www.idealibrary.com on

TECHNICAL REPORT

An Experimental Rat Model for Studying PulmonaryMicrocirculation by in Vivo Videomicroscopy

Paul Schneider, Thomas Foitzik, Sacha Kahrau,Andreas Podufal, and Heinz-J. BuhrDepartment of General, Vascular, and Thoracic Surgery, University Hospital Benjamin Franklin,Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany

Received August 22, 2000; published online August 21, 2001

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It is unclear what role pulmonary microcirculatory dis-orders play in the pathogenesis of adult respiratorydistress syndrome. The aim of this study was to estab-lish a rat model for the direct visualization of pulmo-nary microcirculation by in vivo fluorescence videomi-croscopy. The pulmonary terminal vascular bed wasvisualized and the microcirculatory parameters of leu-kocyte sticking, erythrocyte velocity, capillary perme-ability, and interalveolar septal diameter were quanti-fied. These parameters were examined simultaneously.The preparation was stable for 120 min. Under hyper-thermia, there was increased permeability with a rela-tive fluorescence of 0.39 6 0.19 compared to 0.16 6

0.13 in the control group, and interalveolar septal di-ameters were wider (30.7 6 2.9 mm) than in controlnimals (17.3 6 3 mm). Under hypothermia and hypo-

volemia, the erythrocyte velocity was lower (0.351 6

.063 and 0.378 6 0.044 mm/s) than in control groups0.527 6 0.07 mm/s). Under hypoventilation, we ob-erved a higher amount of leukocyte sticking (3.1 6

.1 vs 1.8 6 0.8 cells/alveolus) and increased perme-bility (relative fluorescence 1.03 6 0.37 vs 0.16 6

.13 in the control group). The model of rat lung ex-
osure for direct examination of microvascular struc-ures in living animals was valuable because it re-ained stable for 2 h under baseline conditions and
0026-2862/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

emonstrated distinct changes in microcirculatory pa-ameters following specific pathophysiologicalnterventions. © 2001 Academic Press

Key Words: microcirculation; videomicroscopy; lung; rat.

INTRODUCTION

Respiratory disorders such as adult respiratory dis-tress syndrome (ARDS) play a major role in severeseptic diseases. The individual pathogenetic factorscontributing to these pulmonary disorders are un-known, particularly on the microcirculatory level. In-creased leukocyte adherence to the endothelium ofalveolar capillaries has been proposed as an initialmechanism in the development of ARDS (24). Thismay lead to the disruption of the alveolar–capillarymembrane and subsequent capillary leakage with in-terstitial edema formation. Capillary blood flow isreduced, possibly due to an increased aggregation ofblood platelets.

Intravital microscopy has been used to further in-vestigate these pulmonary microcirculatory disorders,
which today are believed to be an important factor inthe pathogenesis of ARDS. Lien et al. showed neutro-phil sequestration in the alveolar capillaries of dogs
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(18). Groh et al. were the first to visualize hypoxicpulmonary vasoconstriction by intravital microscopyin a rabbit model (8). Kuebler et al. described thephysiological leukocyte kinetics in the arterioles, alve-olar capillaries, and venules of the lung (13). In 1997,both Kuebler et al. and Fingar et al. showed that ad-

esion molecules (selectin and ICAM-1) play an im-ortant role in leukocyte sequestration in the alveolarapillaries (7, 14). Fingar et al., who were the first to

introduce an intravital microscopic model for examin-ing permeability in the pulmonary microvasculatureof rats in 1994, used a pulmonary window chamber tovisualize the surface of the lung (6). Carter et al. werethe first to investigate subpleural arteriolar diametersin rats without implanting a window (4).

A rat model for visualizing pulmonary microcircu-lation would be the most useful because there arealready established disease models for this species likecolitis or pancreatitis in which ARDS develops as acomplication in the course of the systemic inflamma-tory response. In future studies, we intend to use thesemodels for studying the changes in pulmonary micro-circulation. Thus, the goals of this study were as fol-lows:

• Direct visualization and quantification of the pul-monary capillary bed enabling simultaneous measure-ment of various functional microcirculatory parame-ters by minithoracotomy for quick and nontraumaticlung exposure. To minimize the effects of surgery, wedid not resect the chest wall or implant a pulmonarywindow chamber.

• Quantitative assessment of leukocyte–endothelialinteraction, erythrocyte velocity in the alveolar capil-laries, and permeability of the alveolar–capillarymembrane as well as alveolar septal diameters andalveolar diameters.

• Verification of the stability of the preparation for120 min under physiological conditions (microcircula-tory parameters and macrohemodynamics).

• Since pulmonary microcirculation had not previ-ously been quantified using this technique, it wasnecessary to evaluate the method under well-definedand reproducible conditions of increased and im-

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paired organ blood flow and other conditions knownto change vascular resistance and endothelial func-tion. We thus assessed the changes in the above

Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

microcirculatory parameters induced by pathophysi-ological stimuli (hyper- and hypothermia, hypovole-mia, hypoxemia) (21).

MATERIALS AND METHODS

Preparation

Forty-nine male Sprague–Dawley rats (300–350 g)were housed individually in rooms maintained at21 6 1° with a 12-h dark cycle. The animals were feda standard rat chow and fasted overnight prior to theexperiment with free access to water. Care was pro-vided in accordance with the national guidelines forthe care and use of laboratory animals. The study wasapproved by the local ethics committee.

Surgical anesthesia was induced with vaporizedether followed by 20 mg/kg of intraperitoneal pento-barbital (Nembutal, Pharmazeutische Handelsgesell-schaft, Garbsen, Germany) and 40 mg/kg of intramus-cular ketamine (Ketanest, Parke Davis & Co., Berlin,Germany). The right internal jugular vein was cannu-lated for drugs and fluid infusion. Another catheter ofthe same type was placed in the left carotid artery forblood sampling and blood pressure measurements.

The animals were placed on a heating plate to main-tain body temperature at 37 6 1°. The animals weretracheotomized and the trachea was cannulated. Me-chanical ventilation with 35% O2–65% N2 using asmall-animal ventilator (KTR4, Hugo Sachs, March-Hugstetten, Germany) was installed. Oxygenationwas achieved by pressure-limited ventilation (70 cy-cles/min, 12.5 cm H2O). To prevent atelectasis, end-expiratory airway pressure was set at 3 cm H2O. Then,a left-sided thoracotomy was performed in the fifthintercostal space, and the left lung was exposed usinga thorax retractor (Aesculap, Tuttlingen, Germany). Asmall glass plate was directly placed on the lung sur-face, externally covered with commercially availablecontact gel (Sonogel, Bad Camberg, Germany) to op-tically couple the lung surface to the objective (Fig. 1).

Technical Report

To enable microscopic observation, the lung surfacehad to be kept immobile and expanded. Thus, theventilator was turned off and its valves were closed

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during the observation phase. Ventilation tubes con-ducted a continuous flow of gas (50% O2), maintainingan intratracheal pressure (continuous positive airwaypressure (CPAP)) of 12.5 cm H2O (Figs. 2A and 2B). In

FIG. 1. Exposure of the lung: thin glass plate coated with contact gelnserted between the lung surface and the objective of the microscope.

Technical Report

FIG. 2. CPAP system. A: Normal ventilation by a small-animal ventilator. B

this way, the lungs remained distended during theobservation period. Cardiac motion artifacts on thelung surface were eliminated by interposing a metalplate between the heart and the examined lung.

Quantitation of Microcirculatory Parametersby Intravital Microscopy

The animals were placed in right lateral decubitusposition under a fluorescence microscope (Leitz,Wetzlar, Germany). Epi-illumination was achievedwith a short xenon arc lamp (XBO 100W/2, Osram,Berlin, Germany) applying both heat-protecting andtwo excitation (530 to 560 nm, respectively, 450 to 490nm) filters. After immobilizing the lung with a CPAP

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: Continuous positive airway pressure during the observation period.


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of 12.5 cm H2O, 5 alveoli or 5 alveolar capillaries ineach of three different regions of the left lung wereexamined, corresponding to N 5 15 alveoli or capil-laries in each animal. The examined region was 400 3325 mm with 25 6 5 alveoli each.

Leukocytes were labeled in vivo by the iv adminis-ration of a bolus of 1 ml/kg 0.02% rhodamine 6GSigma Chemical, Deisenhofen, Germany) and illumi-ated by a 530- to 560-nm filter (1). Leukocyte stickingas recorded when a leukocyte remained motionless

n the alveolar wall for 30 s. Five alveoli in three fieldsesulted in 15 analyzed alveoli in each animal (N 55). Leukocyte sticking (Nr WBC) was defined as the

number of leukocytes per alveolus (leuko/alv).Erythrocytes were labeled in vitro with fluorescein iso-

thiocyanate (FITC) isomer I (No. F-7250, Sigma Chemi-cal, Deisenhofen, Germany) according to the methods ofButcher and Sarelius (2, 25). All animals received anintravenous injection of 0.5 ml/kg FITC-labeled erythro-cytes (hematocrit 50%) and were allowed to stabilize forat least 15 min. A 450- to 490-nm excitation filter wasused. To measure erythrocyte velocity, the passage of

FIG. 3. One-frame videomicrograph at 480-fold magnification illusthe cursor was fixed at the inner limit of the alveolus and then movdiameter of the alveolus. With regard to the interalveolar septum me

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FITC-labeled erythrocytes through the alveolar capillar-ies was observed. Capillary length and the time requiredfor passage were measured to calculate erythrocyte ve-


locity (VRBC) in millimeters per second. Five alveolarcapillaries in three fields were examined in each animal(N 5 15). The velocity of five erythrocytes was measuredin each capillary.

Thirty minutes after an injection of 0.3 ml 5% FITC-labeled dextran with a molecular weight of 70,000 Da(Sigma Chemical), the capillary permeability in thesubpleural capillary bed was determined by quantify-ing the increase in alveolar fluorescein intensity in thesame field (five fields per animal) over 30 min usingCAP-Image (12). This system calculates capillary per-meability from the changes in alveolar gray valuescaused by extravasation of FITC-labeled dextran. Per-meability (Perm CAP) was expressed as the relativefluorescence of the baseline gray value. Since contin-uous extravasation of FITC–dextran causes halation inthe microscopic picture, thus compromising furthermeasurements, capillary permeability was determinedonly once per animal at the end of the experiment.

Five alveolar septal diameters (DSEP) as an expres-sion of interstitial edema and five alveolar diameters(DALV) in three regions (N 5 15) were measured off-

morphometry following an injection of rhodamine. The position ofpendicular to the opposite inner limit. The length of this line is the

ent, the cursor is moved to the inner limit of the adjacent alveolus.

Technical Report

line with the image analysis system CAP-Image andgiven in micrometers. Figure 3 illustrates the mor-phometry of DSEP and DALV.

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Systemic Hemodynamics and LaboratoryParameters

Mean arterial pressure (MAP) was recorded continu-ously on an Electrodyne recorder (Becton–Dickinson,Sharon, MA) during intravital microscopy. Hematocritand arterial blood gases were measured before, during,and after the intravital microscopic recording. In partic-ular, blood gas samples were taken at the end of theCPAP period to demonstrate stability during the exam-ination. The total time needed for the exposure (15–20min), stabilization (15 min), and recording (45–55 min) ofthe circulatory beds was 75–90 min per animal.

Study Protocol

Part 1: Control Group

After preparation, animals (n 5 12) were allowed tostabilize for 15 min. Thereafter, systemic hemodynamicand laboratory parameters were assessed. Only datafrom animals with stable cardiorespiratory conditionswere included in the analysis of the microcirculatoryparameters. Exclusion criteria were an MAP of ,80 mmHg, a PaCO2 .50 mm Hg, a PaO2 ,80 mm Hg, or a pHof ,7.3 or .7.5 at any point during the experiment. Wehen determined leukocyte–endothelial interaction, sub-leural capillary erythrocyte velocity, alveolar–capillaryembrane permeability, and interalveolar septal diam-

ter. Measurements were taken during normal ventila-ion and at the end of the CPAP period.

Part 2: Stability of the Preparation over 2 h

To demonstrate the stability of the preparation,erythrocyte velocity in the subpleural capillaries andinteralveolar septal diameter was measured for 120min at 30-min intervals in three different regions of sixanimals. Simultaneously, macrocirculatory parame-ters and arterial blood gases were measured.

Part 3: Changes under Pathological Conditions

Technical Report

To investigate the microcirculatory parameters un-der pathological conditions (hyperthermia, hypother-mia, hypovolemia, hypoventilation), four groups of

six animals were examined. Microcirculatory mea-surements were taken immediately after attaining thedesired temperature, blood gases, or MAP. To verifystability during CPAP ventilation in this group, bloodgas samples were taken during normal pressure-lim-ited ventilation and after the 30-s CPAP ventilation.

Group A: Hyperthermia. Following surgical prep-aration, the body temperature of six animals was in-creased from 37–38° to 42.5–43.5° over 20–30 min byadjusting the surgical heating plate. The temperaturewas continuously monitored during the examinationwith a rectal thermometer and maintained at thislevel.

Group B: Hypothermia. Following surgical prepa-ration, six animals were cooled with coolpacks to arectal temperature of 35–35.5° from 37–38°. The tem-perature was continuously monitored during the ex-amination with a rectal thermometer and maintainedat this level.

Group C: Hypovolemia. Following surgical prepa-ration, the MAP of six animals was reduced to a levelof 50 6 5 mm Hg by withdrawing 6 ml of blood via therterial catheter. This level was maintained over 45in of observation either by withdrawing an addi-

ional 0.5 ml or by reperfusion.Group D: Hypoventilation. Following surgical

reparation, animals in this group were hypoventi-ated by reducing ventilatory pressure and CPAProm 12.5 to 8 cm H2O, which resulted in hypoxemia

(PaO2 of 60 6 10 mm Hg) and hypercapnia (PaCO2 of55 6 10 mm Hg).

Evaluation

The microscopic image was transferred to a monitorby video camera (CCD 4810, Cohu, San Diego, CA)and recorded on S-VHS videotape (Panasonic VCR,AG-7350 Japan) for subsequent blinded off-line anal-ysis by an independent observer using a computer-assisted videoframe analysis system (CAP-Image).This novel system for dynamic capillaroscopy allows

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the off-line analysis of a variety of microcirculatoryparameters, especially red blood cell velocity, perme-ability, and the determination of distances (12).


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Statistics

Data were presented as population mean and pop-ulation standard deviation. A P value of ,0.05 wasconsidered significant. The stability of parametersover time was assessed by repeated-measures analysisof variance (ANOVA). Data from groups exposed topathophysiological conditions were compared to con-trols by a Student t test. P values were corrected formultiple comparison according to Bonferroni, result-ing in a P , 0.0125 for each of the four comparisons.

RESULTS

General Observations

In the microscopic examination of the lung surface,the anatomic structures were identified as shown inFig. 4. The alveoli are displayed as dark round or oval

FIG. 4. Photomicrograph taken from the subpleural microvasculinjection of rhodamine. This example shows the alveoli (A), a venulecapillaries; the corner vessels (➜) are located at the intersection of

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structures. The interalveolar septa are seen as brightborders. As described by Gunderoth et al. (9) in alectron microscopy study, the interalveolar septa are


omposed of a meshwork of alveolar capillaries.arger vessels, so-called corner vessels, are found at

he intersections of the septa. The following fluores-ein leukocyte movements were observed: (1) Leuko-ytes traverse the capillaries at a steady velocity (con-inuous flow); (2) individual cells interrupt their

ovement for a short time and then resume it at theame speed; (3) leukocytes slow down without com-ng to a standstill (rolling leukocytes) and (4) remain

otionless for $30 s in the capillaries (sticking leuko-ytes). We examined the latter group to quantify leu-ocyte–endothelial interaction.The flow of FITC-labeled erythrocytes was homoge-

eous. Arterioles, alveolar capillaries, and postcapil-ary venules were identified based on flow directionnd velocity.

Part 1 (Control Group)

Mortality and Drop Outs

uring in vivo microscopy at 480-fold magnification following annd the interalveolar septa (3) composed of a meshwork of alveolarveolar septa.

Technical Report

One animal was excluded due to unstable cardiore-spiratory parameters; thus, 12 animals were evalu-ated. The systemic parameters during normal ventila-

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tion and at the end of the CPAP period aresummarized in Table 1. No difference was observedbetween the ventilation and CPAP period. We ob-served a capillary erythrocyte velocity (VRBC) of 0.5276 0.07 mm/s, a leukocyte–endothelial interaction(NrWBC) of 1.8 6 0.8 leuko/alv, a capillary permeability(PermCAP) of 0.16 6 0.13, an alveolar septal diameterDSEP) of 17.3 6 3 mm, and an alveolar diameter (DALV)

of 90.2 6 14.2 mm.

TABLE 1

Macrocirculatory Parameters of the Arterial Blood Gas Analysis inControl Groups (n 5 12)

Systemicparameters

Mean(during

ventilation) 6SD

Mean(duringCPAP) 6SD

AP (mm Hg) 114 16 110 18H 7.48 0.06 7.45 0.09aO2 (mm Hg) 162 42 153 29

PaCO2 (mm Hg) 30 4 30 9ase excess (mmol) 20.5 2.2 21.5 1.6ematocrit (%) 44 3 43 3

Note. The left side of the table shows the results when animalsere ventilated with 35% O2–65% N2 by pressure-limited ventila-

ion (70 cycles/min, 12.5 cm H2O, PEEP 3 cm H2O). Measurementson the right were taken at the end of the CPAP period (50% O2–50%N2, 12.5 cm H2O).

Technical Report

FIG. 5. Part 2 (stability): VRBC and DSEP during 120 min

Part 2 (Stability)

Mortality and Drop Outs

One of the eight animals died during the observa-tion period due to a pulmonary embolism, and an-other was excluded due to unstable cardiorespiratoryparameters; thus, six animals were evaluated. The sys-temic and microcirculatory parameters were recordedevery 15 min over 120 min. The standard deviation ofthe MAP was 15.8 mm Hg. The repeated-measuresANOVA was performed to determine whetherchanges took place over the course of the examination.A temporal effect could not be proven (P 5 0.72).Cardiorespiratory parameters did not change signifi-cantly during the observation period. Erythrocyte ve-locity and septal diameter during 120 min of observa-tion are presented in Fig. 5. Standard deviation was0.202 mm/s. The repeated-measures ANOVA wasperformed in order to determine whether changestook place over the course of the examination. A tem-poral effect could not be proven (P 5 0.29).

Erythrocyte velocity and septal diameter did notchange significantly during the 120-min observationunder stable cardiorespiratory conditions.

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of observation under physiological conditions.


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Part 3 (Changes under Pathological Conditions)

FIG. 6. Blood gas samples are taken during normal pressure-limventilation. A: PaO2; B: PaCO2.

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Blood gas samples under pathologic conditions areillustrated in Fig. 6. We found no difference in PaO2


and PaCO2 between normal pressure-limited ventila-

ntilation in Part 3. A control is done after the 30-s CPAP period

Technical Report

tion and CPAP period.All six animals survived under hyperthermia. No

significant changes in the cardiorespiratory parame-

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ters occurred compared to the control group (Figs. 6Aand 6B). There was a nonsignificant increase in VRBC,nd leukocyte–endothelial interaction remained un-

FIG. 7. Part 3: Changes of VRBC and NrWBC

Technical Report

FIG. 8. Part 3: Changes of PermCAP and DSEP under

changed (Fig. 7). PermCAP and DSEP increased signifi-cantly to 0.39 6 0.19 and 30.7 6 2.9 mm (Fig. 8). DALV

did not change (Fig. 9).

defined pathological conditions; *P , 0.05.

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defined pathological conditions, *P , 0.05.


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Under hypothermia, two of eight animals died ofcardiac arrest following bradycardia. We found aMAP of 105 6 8 mm Hg, a pH of 7.61 6 0.18, a basexcess of 23.5 6 2.2, and a PaO2 of 220 6 48 mm HgFig. 6A). The VRBC slowed significantly to 0.351 6

0.063 mm/s (Fig. 7). The leukocyte–endothelial inter-action and the Perm CAP did not change (Figs. 7 and 8).The alveolar septa thickened to 23.1 6 1.8 mm (Fig. 8).The DALV remained unchanged (Fig. 9).

Under hypovolemia, one of seven animals died ofardiac arrest following hypotension. We observed aystemic MAP of 46 6 9 mm Hg, a pH of 7.57 6 0.08,

a base excess of 21.5 6 4.0, and a PaO2 of 221 6 78m Hg. The VRBC slowed to 0.378 6 0.044 mm/s (Fig.

). No changes in leukocyte–endothelial interaction,ermCAP, DSEP, or DALV were observed (Figs 7, 8, and 9).One of seven animals died under hypoventilation.

We observed a MAP of 85 6 15 mm Hg, an acidosis ata pH of 7.25 6 0.07, a base excess of 23.0 6 4.1, a PaO2

of 62 6 13 mm Hg, and a PaCO2 of 55 6 4 mm Hg (Fig.). The VRBC decreased to 0.437 6 0.067 mm/s (Fig. 7);e observed an increase in leukocyte–endothelial in-

eraction to 3.17 6 1.1 leuko/alv as well as a signifi-

FIG. 9. Part 3: Changes of DALV unde

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ant increase in the PermCAP to 1.03 6 0.37 (Figs. 7 and). DSEP and DALV decreased to 13.6 6 1.6 and 63.0 6 8.4

mm (Figs. 8 and 9).


Capillary permeability correlated to the number ofsticking leukocytes (r 5 0.40, P 5 0.013) (Fig. 10).

DISCUSSION

The role of microcirculatory disturbances in respira-tory failure has not yet been clarified. This is partlydue to the limitations of the techniques and modelscurrently used for assessing pulmonary microcircula-tion. In vivo microscopy represents the best method forevaluating microcirculation, since it provides directcontinuous information about dynamic changes incapillary blood flow, permeability, and endothelial–leukocyte interaction. The lung IVM model in rabbitshas increased knowledge about pulmonary microcir-culation. The kinetics of leukocytes and erythrocyteswere measured under physiological and pathologicalconditions (13, 16). The role of adhesion molecules(selectin) in leukocyte sequestration in alveolar capil-laries was demonstrated by Kuebler et al. in 1997 (14).

We chose the rat because it is a robust laboratory

ed pathological conditions, *P , 0.05.

Technical Report

animal with numerous established disease models inwhich ARDS develops secondary in the course of thesystemic inflammatory response. For this reason and

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other practical considerations, many laboratories in-cluding our own utilize small rodent models for invivo studies of microcirculation (19).

Only a few intravital microscopic studies of the ratlung have been conducted. Fingar et al. were the firsto demonstrate interstitial edema in a rat model fol-owing the intravenous application of fatty acids (6).hanges in permeability and an increase in leukocyte–ndothelial interaction were observed in the lung afterhotodynamic therapy (27). Carter et al. observed aeduction in the diameter of subpleural arterioles afteropical application of endothelin and demonstrated aositive effect of PAF antagonists on pulmonary mac-omolecular leakage in cases of intestinal ischemia/eperfusion (4, 5).

Pulmonary windows were implanted by Kueblernd Kuhnle in rabbits and by Fingar in rats. Carter etl., on the other hand, decided not to implant a win-ow in his study of rats (4). We, too, were convinced

FIG. 10. Correlation between Pe

Technical Report

y the advantages of this model, namely, the fastxposure of the lung without time-consuming prepa-ation. The risk of trauma to the lung surface is thus

educed, preparation is considerably faster, and a neg-tive pressure suction system is not required. We onlysed a thin glass plate to cover the lung to create alane surface for intravital microscopy. Stability of thereparation was demonstrated in our second protocol.either erythrocyte velocity nor systemic hemody-amic parameters changed within 2 h after beginning

he preparation.To the best of our knowledge, the ventilation tech-

ique we used to observe the lung surface has noteen previously applied for intravital microscopy ofats. Continual gas flow at the preset level of 12.5 cm

2O excluded the periodic movements of inspiration/expiration during the observation. The lung surfaceremains immobile and, through the intratracheal pres-sure thus attained, oxygenation of the lung is possibleby diffusion (17). Repeated measurements of arterialblood gases during normal ventilation and at the endof the CPAP period demonstrated that neither PaO2

and NrWBC; r 5 0.40, P 5 0.013.

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dropped nor PaCO2 increased. Thus, we were able toprolong the examination of the subpleural capillarynetwork and observe the dynamics of erythrocytes


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and leukocytes for 30 s. Consequently, we definedleukocyte sticking for a period of 30 s. Other authorshave defined sticking leukocytes as cells remainingimmobile only for 5 s in a defined vascular segment(16).

The mean alveolar capillary velocity was 0.527 6.07 mm/s. Kuebler et al. measured a physiologicalevel of 0.470 6 0.049 mm/s in rabbits (13). This levelelates to the observation of the subpleural region of aulmonary segment under mechanical ventilation and

horacotomy. Using radioactively labeled micro-pheres, Kuhnle’s experiments in rabbits showed thatositive pressure ventilation and thoracotomy influ-nced blood flow in the lung but the pressure distri-ution in upper and lower pulmonary segments didot change (15). On the other hand, Short et al. dem-nstrated in rats that the recruitment of subpleuralapillaries accurately reflects that of centrally locatedapillaries (26). These observations suggest that sub-leural microcirculation is representative of bloodow in the entire lung and justify the use of in vivoicroscopy of subpleural capillaries.Under physiological conditions, we observed 1.8 6

0.8 immobile leukocytes per alveolar capillary for 30 s(leukocyte sticking). We thus confirmed the observa-tions of Minamiya et al. in rats (20). All of our mea-surements were obtained from the lung surface. How-ever, it has been demonstrated that the subpleuralobservations correspond to those taken from deeperlevels. Although the capillary density is higher in thecenter of the lung, electron microscopic examinationsof the rat lung showed that capillary diameters aresimilar in all regions of the lung (9). Capen et al.measured an identical capillary transit time in all seg-ments of the dog lung (3). Thus, it is reasonable tobelieve that the subpleural values are representative ofthe whole lung.

Intravital microscopy proved to be a valid quantita-tive examination method in cases of defined changesin physiological conditions. The level of immobile leu-kocytes in the interalveolar septum was elevated onlyunder hyperthermia. Erythrocyte velocity was signif-icantly increased under hyperthermia. On the otherhand, we observed a significant slowing of erythro-

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cytes in cases of hypothermia and hypovolemia. Theseobservations are in accordance with the findings ofKuhnle et al., who showed that mean erythrocyte ve-


locity correlates to cardiac output in a rabbit model(16). The slowing was not significant under hypox-emic conditions. The permeability for macromoleculesincreases under hyperthermia and hypoxemia. Perme-ability is an expression of damage to the alveolarcapillary membrane; that is, macromolecules such asFITC–dextran with a mean molecular weight of 70,000Da pass through the alveolar–capillary membraneand accumulate in the alveoli.

We found that capillary permeability correlated tothe number of sticking leukocytes. This is in accor-dance with findings of He et al., demonstrating in-creased microvessel permeability as early as 45 minfollowing leukocyte adhesion (11). Electron micros-copy studies by Montaner et al. suggest that the stick-ing leukocytes cause epithelial disruption, therebyleading to increased permeability and alveolar flood-ing 2 h after inducing low pressure pulmonary edema(22). However, increased permeability could be foundin the absence of leukocytes (10). Inflammatory medi-ators inducing a transient increase in endothelial cellcytoplasmatic Ca21 concentration may be an alterna-tive explanation for increased capillary permeabilitywithout pathological leukocyte endothelial interactionand microvascular disruption.

Septal diameter reflects the amount of interstitialfluid accumulation. An increase in permeability oc-curred very soon after induction of hyperthermia orhypoxemia. The alveolar diameter was the same in allexperiments using standard ventilation parameters. Itwas smaller only under hypoxemia and hypercapniadue to a reduction in airway pressure. We can use thisparameter as an indicator of alveolar ventilation.

ARDS is characterized by a disorder of the alveolar–capillary membrane, which separates the vascularspace from the air space. The mechanism of leukocyteaccumulation in the alveolar capillaries in interactionwith adhesion molecules has been well established.IVM allowed qualitative assessment of changes inpathophysiologically relevant microcirculatory pa-rameters associated with defined modulations of sys-temic parameters such as hypo- or hyperthermia, hy-povolemia, or hypoventilation. Our model showed the

Technical Report

cellular components of lung damage (leukocyte stick-ing) and their effects on capillary permeability. Weobserved such disorders immediately after inducing

1

1

1

1

1

1

1

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hypoxemia and hyperthermia. Erythrocyte velocityalso reacted promptly to these stimuli.

We can conclude from our experiments that pulmo-nary IVM in rats can be effectively performed withouta thoracic window chamber. The method enables si-multaneous quantitation of all important pulmonarysubpleural microcirculatory parameters, such as leu-kocyte sticking, erythrocyte velocity, permeability ofthe alveolar–capillary membrane, and septal diame-ter. This method can be applied to determine theinitial mechanisms of lung failure and facilitate thedevelopment of new therapeutic procedures aimed atpreventing ARDS.

ACKNOWLEDGMENT

The authors thank Prof. Helmut Habazettl, Institute of Physiol-ogy, Freie Universitat Berlin, for his valuable assistance and helpfuladvice with these studies.

REFERENCES

1. Baatz, H., Steinbauer, M., Harris, A. G., and Krombach, F.(1995). Kinetics of white blood cell staining by intravascularadministration of Rhodamine 6G. Int. J. Microcirc. 15, 85–91.

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An Experimental Rat Model for Studying Pulmonary Microcirculation by in Vivo Videomicroscopy