Morphological changes in blood vessels produced by hyperosmotic agents and measured by optical coherence tomography[para]

ABSTRACT

Optical tissue clearing by hyperosmotic chemical agents significantly increases light depth penetration in skin and may improve light-based therapeutics such as laser treatment of cutaneous vascular lesions. A feasibility study was conducted to evaluate the potential role of optical clearing by glycerol in laser treatment of cutaneous vessels. Optical imaging was performed to investigate the morphological effects of glycerol on blood vessels of skin. Blood vessels were imaged using Doppler optical coherence tomography in in vivo hamster skin treated with glycerol. Images were obtained from the subdermal side to assess morphological changes in the blood vessels caused by glycerol and from the epidermal side to assess enhanced Doppler imaging of blood vessels. Application of glycerol to the subdermis resulted in venule stasis and for prolonged treatment times, arteriole stasis. In cases where flow remained in arterioles, an improved Doppler signal was detected from blood vessels when imaging transepidermally compared with the native condition. Intensity images indicated changes in blood optical properties and improved contrast of skin cross sections after glycerol application. The observed optical and morphological effects were reversed upon hydration of the skin with phosphate-buffered saline. The combination of increased depth of light penetration and the temporary slowing or cessation of flow in blood vessels could mean improved laser treatment of vessels.

Abbreviations: DOCT, Doppler optical coherence tomography; OCT, optical coherence tomography.

INTRODUCTION

Hyperosmolic solutions of glycerol and glucose, among others, substantially increase the penetration depth of light in skin (1-6). The increase in light penetration depth is accomplished by reducing light scattering in the tissue (4,6). A potential benefit of the optical clearing technique is the improvement of laser therapeutic techniques that rely on sufficient light penetration to a target embedded in tissue. Combining optical clearing with laser radiation could reduce the laser fluences required for a therapeutic effect. A specific application that may benefit from the optical clearing technique is blood vessel photocoagulation. Significantly lower laser irradiances (watts per square centimeter) may be required to irreversibly damage targeted blood vessels in skin treated with a hyperosmotic clearing agent. Also, because the method of optical clearing enables aiming or focusing laser light more directly onto a given blood vessel, damage to collateral tissue may be minimized.

Successful blood vessel coagulation using the optical clearing technique will be influenced not only by the decrease in light scattering in overlying tissue but also by changes in blood vessel morphology and functionality such as changes in diameter and flow velocity. In this study, Doppler optical coherence tomography (DOCT) is used to identify changes in cutaneous vessel diameter and flow velocity after application of glycerol to the surrounding skin. The likelihood of improved vessel coagulation after tissue optical clearing is discussed using the results of this study.

DOCT provides noninvasive imaging of tissue in depth and allows the identification of blood vessels by detecting frequency-shifted light backscattered from tissue constituents (7,8). DOCT uses phase information not used in intensity-based optical coherence tomography (OCT), in which only coherently backscattered light is used to form a spatially resolved image of a sample. Flow velocity of moving objects is computed from the Doppler shift ([Delta][function of]^sub D^) of the modulation frequency. The velocity of a moving object is given as

where [lambda]^sub 0^ is the illumination source center frequency, n is the refractive index and [theta] is the angle between the sample probe and the flow direction. DOCT has been used in the past to image blood vessels of the skin for evaluating changes in flow and size after application of laser radiation (9-12).

MATERIALS AND METHODS

Animal model: Hamster dorsal skin flap window preparation. Syrian Golden hamsters, approximately 90 g in weight, were anesthetized with a 4:3 mixture of ketamine (100 mg/mL):xylazine (20 mg/mL), and given 0.1 mL/100 g body weight. The dorsal area of each hamster was shaved and epilated to remove hair roots. The dorsal skin flap window preparation (13,14) was implanted using the following procedure. The skin flap was pulled away from the body at the dorsal midline and sutured to a vertical C-Clamp. A circle 1 cm in diameter was removed from a single layer of skin, exposing the opposing layer. This allowed direct observation of blood vessels of the subdermal skin. Native blood vessels ranged in diameter from 100 to 350 [mu]m. Arterioles and venules imaged in this study were present in the subdermal fat and connective tissue layers beneath the dermis. The hamster dorsal skin flap window preparation allowed the simultaneous exposure of the epidermal surface of the skin as well as the subdermal surface thus imaging could be performed from both sides.