Techniques to Assess Cutaneous Microvascular Function in Vivo

Figure 2 Ions of soluble salt are introduced into the tissue by ion-tophoretic current. The amount of drug delivered to the skin is proportional to the magnitude and duration of the applied current. (see color insert)

Techniques to study cutaneous microvascular function will be discussed under three headings: techniques to introduce agents into the skin, techniques to assess blood flow and changes occurring therein, and techniques to recover biologically active chemicals from the dermis in order to investigate the biochemical and cellular mechanisms underlying changes in blood flow. Although many of the techniques originally developed in laboratory settings are either too invasive or technically unsuited for use in humans in vivo, over recent years a number of less invasive methods have been developed. Some of the most frequently used of these are the noninvasive imaging of blood flux and the delivery of exogenous vasoactive mediators to the skin and/or the recovery of endogenous mediators from the skin [2]. The remainder of this chapter will focus on these techniques and the type of information that has accrued using them.

Introduction of Agents into the Skin

Historically, pharmacologically active agents used to study vascular function have been introduced either into the systemic circulation, or locally via intradermal injection or topical application. Although both methods of local administration have their place, they both suffer from limitations. Intradermal injections have two potentially serious drawbacks. First, injection of even small volumes will cause major changes in local tissue pressure that may stimulate reflexes that confound or complicate the investigation under question. Second, agents diffuse a maximum of 2 to 3 mm from their site of injection and, hence, have no widespread actions. Topical application of agents to the skin also has limitations. The stratum corneum is permeable only to relatively small lipophilic molecules. Consequently, to get hydrophilic molecules into the skin requires the use of complex pharmaceutical preparations, or removal of the outer epidermis by tape stripping is necessary.

In recent years cutaneous iontophoresis has been developed as a method to deliver vasoactive agonists and antagonists to the skin, particularly in a clinical setting (Figure 2). The technique is noninvasive and avoids the confounding effects of local perturbation of the skin by intradermal injection or of systemic drug administration. Also, agents may be delivered to relatively large areas of the skin. Coupled with laser Doppler flowmetry, iontophoresis has been used to study the role of the endothelium in the modulation of blood flow in the skin. Examples of such studies are responses to acetylcholine (endothelium-dependent) and sodium nitro-prusside (endothelium-independent) in both healthy volunteers and in patients with a variety of disorders including diabetes by Dr. Shore's group, hypertension, systemic sclerosis, and Raynaud's phenomenon [3]. Furthermore, introduction of agents into the skin by iontophoresis has allowed studies of the physiology of dermal microvasculature in thermoregulation under heat stress and prolonged exercise [4], the response of the dermis to xenobiotics [5], and the modulation of neurogenic inflammation in the skin [6].

Imaging of Blood Flux

Intravital imaging using transmitted light is not generally possible in solid organs and accurate measurement of blood flow has proved somewhat problematic [7]. Some of the methods currently used include measurement of transcuta-neous oxygen, radionucleotide techniques, thermography, ultrasound, laser Doppler fluximetry, and infra red imaging and, most recently, the optical imaging technique of orthogonal polarization spectral analysis [8]. Of all the methods, those based on laser Doppler imaging, which use a measure of red blood cell movement to evaluate vascular flux, have proved the most suitable and best validated for use in a clinical setting.

High-resolution scanning laser Doppler imaging uses a low-power (~2mW), 633-nm red He-Ne laser to scan the skin in a raster pattern and build up a two-dimensional image of red blood cell flux up to a depth of approximately 0.6 to 1 mm. It is able to provide a real-time output of temporal and spatial changes in skin blood flux, particularly during dermal provocation. As the scanning head may be mounted 30 cm or more above the skin surface, it also allows space for manipulation within the scanned area. Thus, scanning laser Doppler imaging has been used by many groups to study the dermal microvasculature in a wide range of disease states including hypertension, diabetes, and peripheral vascular disease, in inflamed and damaged skin following burn injury, and in nonhealing wounds.

To illustrate the usefulness of scanning laser Doppler imaging in research, we will take the specific example of its application in investigating the mechanisms weal and flare responses to intradermal injections of histamine, bradykinin, and allergen. In these studies, changes in dermal blood flux were assessed by accumulating about 16,000 data points for analysis over a scanned area of skin of 5 cm x 5 cm using scanning laser Doppler imaging (SLDI, Moor LDI, Moor Instruments Ltd, Axminster, Devon, UK) (Figure 3). From these images, the areas of the weal and flare responses were calculated using the manufacturer's software. Our experience has shown that the changes in weal and flare areas may be measured to an accuracy of 0.05 cm2 and changes in perfusion to 5% [9].

Recovery of Vasoactive Mediators in the Skin

Until recently, the only way in which mechanistic studies could be performed in human skin in vivo was to use pharmacological agonists or antagonists to simulate or block, respectively, the response under investigation. However, such techniques yield only circumstantial evidence, which may not always be correct. What is needed is direct evidence, which may only obtained by sampling the interstitial space surrounding the microvasculature. A variety of techniques have been employed for this purpose, including needle or wick aspirate, tissue exudates such as blister fluid or wound fluid, and most recently microdialysis and ultrafiltration. Biological molecules recovered using these techniques range from metabolites such as glucose and lactate, vasoactive mediators such as histamine, nitric oxide, cyclic nucleotides, eicosanoids, and plasma proteins, and most recently cytokines, growth factors, and neuropeptides [10]. It is thus possible to identify and, if the characteristics of the recovery system are known and the assays sensitive enough, quantify changes in tissue levels of bioactive molecules.

Microdialysis, one of the most successful and widely used sampling techniques used in the skin, was originally developed to recover neurotransmitters from the brains of experimental animals. Subsequently, it has been modified to sample endogenous chemicals within the extravascular space in a number of organs in vivo and as a diagnostic tool to monitor tissue levels metabolites [11] (Figure 4). Micro-dialysis has the advantage over other dermal sampling techniques in that it causes minimal tissue trauma, is well tolerated, and can be used to follow the temporal variations in the generation and release of a substance at a discreet location within the tissue space. It has also been used to follow the pharmacokinetics of a wide range of xenobiotics within the dermis [12].

In an extension of its original application, the dialysis technique has also been used to investigate the balance and distribution of blood flow within tissues such as muscle and skin that are served by two different vascular beds (nutritive and nonnutritive). By measuring the extraction of highly diffusible markers from the dialysis perfusate, it is possible not only to determine changes in total blood flow but also to detect changes in the distribution of functional blood flow with a high level of sensitivity [13]. This opens the way to studies of the functional control of local tissue perfusion in humans in vivo under both physiological and pathophysio-logical conditions.

Figure 3 High-resolution scanning laser Doppler imaging uses a low-power (~2mW), 633-nm red He-Ne laser to scan the skin in a raster pattern and build up a two-dimensional image of red blood cell flux up to a depth of approximately 0.6 to 1 mm. (see color insert)

Figure 4 Principles of microdialysis. The recovery of a substance by microdialysis is governed by the same principles as that of exchange of small hydrophilic molecules across the microvascular wall, that is, passive diffusion down a concentration gradient as described by the Fick principle. It is influenced by a number of factors including the characteristics of the dialysis membrane and the recovered solute (its charge, size, solubility) and the concentration of the solute outside the membrane (which in turn may be influenced by tissue metabolism, hydration, and local blood flow). (see color insert)

Figure 4 Principles of microdialysis. The recovery of a substance by microdialysis is governed by the same principles as that of exchange of small hydrophilic molecules across the microvascular wall, that is, passive diffusion down a concentration gradient as described by the Fick principle. It is influenced by a number of factors including the characteristics of the dialysis membrane and the recovered solute (its charge, size, solubility) and the concentration of the solute outside the membrane (which in turn may be influenced by tissue metabolism, hydration, and local blood flow). (see color insert)

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