Changes in Vascular Permeability

In a classical electron microscopy study by Majno and Palade (1961), it was shown that postcapillary venules of 20 to 30 mm diameter leaked carbon particles through gaps formed between endothelial cells as a result of histamine stimulation and endothelial cell contraction. Such gaps have been confirmed to exist in the HCP after bradykinin stimulation. Studies combining intravital microscopy with subsequent electron microscopy of the same cheek pouch showed that interendothelial gaps had formed at sites where extravasation of FITC-dextran already had been observed in vivo, as in Figure 1B. The advantage of FITC-dextran as a marker is that it can first be observed in living tissue and then can be traced as dark precipitates after the fixation of the tissue, which demonstrates the pathway through interendothelial gaps taken by macromolecules in inflammation. The gaps are formed as a result of mediator-induced (e.g., bradykinin) contraction of endothelial cells in the postcapillary venules. Supportive evidence for endothelial cell contraction as a mechanism for gap formation can be found in the pharmacological studies of smooth muscle relaxant drugs that maintain the intracellular level of cAMP (adenyl cyclase activators, ß2-adrenoreceptor stimulants) or block its break

Figure 2 Schematic illustration of the experimental setup for the hamster cheek pouch model. The bicarbonate buffer superfusing the cheek pouch is heated and equilibrated with 5 percent CO2 and 95 percent N2. A peristaltic pump keeps buffer flow to microscope stage and fluorimeter constant.

down (phosphodiesterase inhibitors), which counteracts the mediator-induced contraction, the subsequent gap formation, and thus, the macromolecular leakage (Table II).

There has been some controversy in the past on the nature of endothelial gaps, intracellular or intercellular, but the controversy was resolved in a study by McDonald et al (1999) on the formation of interendothelial gaps and their function as a route for plasma leakage in inflamed rat trachea. Using five different methods to examine normal and inflamed tissue samples, the researchers concluded that most of the openings in leaky venules were intercellular gaps, not transcellular holes, and that the formation and closure of gaps are likely to be energy dependent, while the process of plasma leakage is not, provided there is adequate driving force for extravasation. The cellular mechanisms of gap opening and closure still remain to be elucidated.

The mean diameter of postcapillary venules in the HCP most prone to leak on mediator stimulation varies between 8 and 15 mm, and the interendothelial gaps formed after stimulation with bradykinin vary between 0.08 and 1.4 mm in width.

For studies of macromolecular leakage at postcapillary venules, it is sufficient to use a magnification of 40x to 50x and for measurements of arteriolar diameter and for studies of leukocyte rolling and adhesion a water-immersible lens is recommended with a magnification of 200x to 400x. Illumination with a 50- to 100-W mercury lamp and filtering of the light with proper excitation and emission filters clearly shows the intravascular FITC-dextran and its extravasation after stimulation (see Figure 1). Epi-illumination gives better contrast between fluorescent and nonfluorescent tissue. Inflammatory mediators and drugs that may affect macro-molecular leakage can be added to the superfusion buffer before it flows over the cheek pouch. The microvascular permeability increase or macromolecular leakage following the addition of bradykinin to the superfusion buffer is immediate; can be seen as early as at 30 seconds after its application; and reaches a maximum between 2 and 5 minutes after its application. The macromolecular leakage is measured by counting the number of leaking postcapillary venules (leaks) per cm2 with 2-minute intervals until the maximal value is reached. The maximal number of leaks per cm2 correlates with the fluorescent light intensity as measured with a photomultiplier on top of the microscope and also with the amount of FITC-dextran eliminated by the superfusing buffer and measured by in fluorimetry. The HCP is cleared

Table II Inhibitors of Mediator-Induced Macromolecular Leakage in the HCP.

Inhibitor

Concentration, M or dose

Mediator or procedure

ß2-adrenoceptor agonist (terbutaline, salbutamol)

10-6M

Histamine, bradykinin, LTB4, adenosine, phorbol ester, oxidant injury, I/R

Phophodiesterase inhibitors (Rolipram, milrinone)

10-5M

Bradykinin

Theophylline (adenosin receptor agonist)

10-5M

Histamine

Glucocorticoids (budesonide, methylprednisolone, dexamethasone)

10-7M

Histamine, bradykinin, LTB4, LTC4, PAF, immune aggregates, oxidant injury, I/R, phorbol ester, endotoxin

Calcium antagonist (verapamil)

10-5M

Histamine, bradykinin

Bradykinin-2-receptor antagonists (HOE 140, NPC 17647)

10-5M

Bradykinin

Triglycylvasopressin, 1-desamino-8D-argininvasopressin

10-8M

Histamine, bradykinin

H1-receptor antagonist (mepyramine, dimethpyrindene)

2 ■ 10-6M

Histamine

5HT2-receptor antagonist (ketanserin)

5 ■ 10-7M

Serotonin, histamine

Staurosporin (protein kinase C inhibitor)

10-9M

Phorbol ester (PDBu)

NOS-inhibitors (L-NA, L-NAME, L-NMMA)

10-5M

Histamine, bradykinin, I/R, PAF, endotoxin

Prostacyclin, iloprost

10-10M

I/R

CuZn-SOD, EC-SOD (superoxid dismutases)

25 mg/kg, i.v.

I/R, oxidant injury

Tocopherol

10-5M 1 mg/kg/day

I/R, oxidant injury (TBOOH)

Ascorbic acid

10-5M

I/R, oxidant injury (TBOOH)

Flavonoids:hydroxylrutoside, diosmin, ruscus extract (Cyclo 3 Fort)

5-80 mg/kg/day

I/R, oxidant injury (TBOOH)

Glibenclamide, gliclazide (sulfonylureas, diabetes drugs)

10-8M

I/R

WEB 2170 (PAF-antagonist)

10-5M

PAF, oxLDL

Ropivacain (local anestetic)

10-5M

LTB4

Melatonin

I/R

Ketoprofen (COX-inhibitor)

> 10-5M

IL-1 ß, bradykinin

Nedocromil

Histamine, LTB4

Lipoxin

LTB4

Dextran sulfate

1, 75 mg/kg

LTB4, polylysine

from the extravasated FITC-dextran at 30 minutes after a permeability increase and appears as before the increase (see Figure 1C). A linear dose-response relationship has been shown to exist between the number of leaks and the logarithmic dose of bradykinin, histamine, and several leukotrienes and also between the maximal number of leaks and the amount of FITC-dextran eliminated by the superfusion buffer over the course of 30 minutes.

Another characteristic of the response to certain inflammatory mediators such as bradykinin, histamine, and leukotriene B4 is that on repeated stimulation with the same submaximal dose at 30-minute intervals, there is apparently no downregulation of receptor function and thus only slight reduction in the macromolecular leakage response over a time period when measured as number of leaks. Therefore, the HCP can be used with the first application of the mediator as a control followed by as many as seven subsequent applications together with different inhibitors. Certain mediators and procedures can make the HCP resistant to a secondary stimulation of the same kind. Thus a secondary application of ischemia/reperfusion (I/R), platelet activating factor (PAF), or parasites to the cheek pouch will result in a response which is 30% or less of the first. There is no complete understanding of the mechanisms behind this state of preconditioning to secondary stimulation, but a detailed study on the subject has been published by Korthuis et al. (2003).

Altogether, these data suggest that the HCP can be very useful in physiological and pharmacological studies on the regulation (inhibition or potentiation) of macromolecular leakage via interendothelial gaps induced by a mediators acting on the endothelial cells in postcapillary venules.

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