CC BY
3DOI 10.24412/CL-37135-2023-1-43-46
PILOT STUDY OF A LOW-INVASIVE EX VIVO METHOD OF OPTICAL CLEARING SKIN USING NEEDLE-FREE INJECTION OF OPTICAL CLEARING AGENTS
YURIY SURKOV12, ISABELLA SEREBRYAKOVA1,2, ELINA GENINA1'2 AND V.V. TUCHIN1,
2, 3
1Saratov National Research State University, N.G. Chernyshevsky, Russia 2National Research Tomsk State University, Russia 3 Institute of Precision Mechanics and Control Problems of the Russian Academy of Sciences, Russia
e-mail: [email protected]
ABSTRACT
Optical imaging techniques have some advantages, such as non-invasiveness, high resolution, relative simplicity and minimization of side effects, which are not achievable by classical medical imaging, such as computed tomography, magnetic resonance imaging and ultrasound. [1, 2]
It is important to note that optical imaging techniques, such as confocal microscopy, optical coherence tomography (OCT), photoacoustic and multiphoton microscopy [1, 2], and phototherapy techniques, such as laser, photodynamic and photothermal therapy, are playing an increasingly important role in modern medical care and research practice [3,4]. However, due to the low transparency of biological tissues, which arises due to the heterogeneity of the refractive indices of the structural components of the tissue and intercellular fluid, as well as due to the absorption of probing radiation by chromophores that are part of biological tissues [1, 5]. As a consequence, optical imaging and phototherapy are applied only to the superficial tissues of the body.
Increasing the intensity of incident radiation can increase the penetration depth of the probing light beam, but this approach will inevitably cause additional stress or damage to surrounding tissues [4]. Optical clearing (OC) of biological tissues is a method of increasing the transparency of biological tissues, designed to increase the efficiency and quality of imaging and the effective depth of phototherapeutic methods [6].
The classical approach to OC uses the application of optical clearing agents (OCAs) to the target area of biological tissue. Alcohols, sugars, organic acids, and others are usually used as OCAs. In case of OC of the skin, the barrier and heterogeneous characteristics of the skin layers prevent the penetration of OC, which leads to low effectiveness of OC and/or requires prolonged application/soaking. [6] Several approaches have been proposed to overcome the barrier properties of the skin, such as mechanical and laser perforations, subcutaneous injection of OCA, sonophoresis, microdermabrasion, and the use of microneedle patches [6, 7]. In this work, we propose a method that allows for rapid and efficient delivery of OCA into the dermal layers using a needle-free injection.
A needle-free injector allows the target substance to be injected under high pressure into the skin. The jet pierces the upper layers of the skin and penetrates the biological tissue through a small hole. In addition, with this method of administration, OCA is distributed throughout the volume of the dermis, in contrast to traditional injection, in which OCA forms a drop under the skin, and more time is required for its uniform distribution [8].
Aqueous 40% solutions of Omnipaque-300 (iohexol 300 mg/ml), polyethylene glycol (PEG-300), polypropylene glycol (PPG), 100% Omnipaque-300 and 100% PEG-300 were used as OCA. The injection volume for each OCA was 50 pl. The object of study was 5 areas of skin on the right and left sides of the back, 1.5 cm away from the spine, on the carcass of a male outbred laboratory rat, obtained from the vivarium of the Center for Collective Use of the State Medical University named after V.I. Razumovsky. On the studied areas of the skin, hair was removed using depilatory cream.
To monitor the skin condition before and immediately after needle-free injection of OCA, dermatoscopy with 10x magnification and high-frequency ultrasound (ultrasound) using the DUB SkinScanner device (tpm taberna pro medicum GmbH, Germany) with two probes operating at central frequencies were used 33 and 75 MHz with a scanning depth of 3.2 mm and axial resolution of 48 and 21 pm, respectively. To record changes in the optical characteristics of the skin, a GAN930V2-BU spectral optical coherence tomograph (Thorlabs, USA), with an axial resolution of 5.34, was used.
Artifact-free regions of interest (ROIs) were selected to analyze B-scan OCT images before and after needle-free injection. The width of such ROIs was from 150 to 650 ^m, the number of ROIs for each OCA was at least 5. The B-scan within the ROI was averaged into one A-scan. Because the skin has an uneven surface, we used a program written in Matlab for horizontal alignment before averaging the B-scan (Figure 1).
To restore the attenuation coefficient of the OCT signal, the algorithm proposed in [9] was used. This method is based on a single scattering model and two assumptions: i. almost all radiation is attenuated within the depth range of the OCT scan; ii. backscattered light, which is recorded by the OCT system, constitutes a fixed fraction of the attenuated probing radiation. These assumptions allow us to estimate the attenuation coefficients for each pixel in the data set. Multiple scattered light is not taken into account. According to this approach, the attenuation coefficient - ^0CT, is determined by the equation:
1 ( KO \ MocrCO-^lo^l+^rTôjj.«!)
where A is the pixel size, I(i) is the signal intensity of the i-th pixel, N is the number of pixels in the axial direction.
Figure 1: Schematic representation of obtaining an averaged A-scan within the region of interest, highlighted with a red
rectangle
Equation (1) was used to calculate the attenuation coefficient of each pixel in the depth profiles for each B-scan. For each ROI selected for B-scan analysis, |iOCT(z) profiles were constructed with a step of 50 ^m.
Figure 2 shows dermoscopy images before and after needle-free OCA injection. It can be seen that the OCA injection site becomes whiter, which is associated with an increase in scattering of the tissue area. However, after injection of 100% Omnipaque-300, a decrease in scattering can be observed, which makes the underlying layers of biological tissue visible even to the naked eye - muscle tissue. Figure 2 shows typical images obtained using high-frequency ultrasound before and after OCA injection. It is easy to notice a change in the geometry of biological tissue and the echogenicity of the ultrasound signal after injection of OCA. The volume of tissue that has changed as a result of injection has a fuzzy boundary, the echo signal of which is indistinguishable from the signal of the intact area, which makes it difficult to measure the size of the volume of biological tissue exposed to the action of OCA. The average width and depth of the volume of tissue affected by the change can be estimated at approximately 5.9 and 1.2 mm, respectively. Figure 2 3 column shows typical OCT images before and after OCA injection. You can notice a change in the geometry of the skin after injection and relatively small traces of violation of the integrity of the epidermis.
PEG-300 (100%)
Figure 2: Representative images of dermoscopy, ultrasound, and OCT before and after free-needle OCA injection
In Figure 3 you can see the location of the breakdown of biological tissue by the OCA jet; the width and depth were approximately 163 and 748 ^m, respectively.
Figure 3: Images of the site ofpuncture of rat skin during needle-free injection of 40% PPG solution, (A) macrophoto, (B)
OCT scan
Figure 4 shows a typical macro image, B-scan and reconstructed OCT signal attenuation coefficient image of the skin of a laboratory rat before and after OCA injection. In the attenuation coefficient image, contrast structures that are not visible on the original B-scan can be easily seen. In addition, the attenuation coefficient of the intact dermis has a fairly uniform depth profile; inhomogeneities ^OCT can arise for several reasons: i. violation of the key assumption of the model [9]. It is easy to notice that a needle-free injection of 50 ^l of a 40% aqueous solution of PEG-300 causes a significant increase in the attenuation coefficient of the OCT signal compared to the intact area. Injection of undiluted PEG-300 causes a local increase in ^OCT, but less compared to 40% PEG-300. Injection of undiluted Omnipaque leads to a decrease in the attenuation coefficient and intensity of the OCT signal compared to intact skin, however, small areas of increase in ^OCT can be seen.
Intact PEG-300 (40%)
PEG-300 (100%) Omnipaque (100%)
Figure 4: Images of (A) macrophoto with the plane and direction of OCT scanning marked with a red arrow, (B) B-scan of OCT, (C) calculated attenuation coefficient of the OCT signal, skin of a laboratory rat before and after injection of OCA
Figure 5 shows the dependences of the OCT signal depth and |xOCT, calculated from several ROIs for each OCA and intact skin. It can be noted that for intact skin, in Figure 5 it is designated as "Control", and for undiluted Omnipaque-300 the dependence of the attenuation coefficient has a more horizontal form, while for the remaining OCA (xOCT tends to increase to a depth of ~ 400 - 500 ^m, which can be explained by a larger proportion of multiply scattered photons in the case of injection of the OCA used, with the exception of 100% Omnipaque. The decrease in OCT signal amplitude and ^OCT upon injection of 100% Omnipaque compared to the intact area at a depth of up to ~ 350 ^m demonstrates the potential of this method of delivering OCA into the skin for effective and rapid OC.
o o
0 200 400 600 800 1000 1200 1400 1600 Depth, microns
intact
0mnipaque-300 (40%) PEG-300 (40%) PPG (40%)
0mnipaque-300 (100%) PEG-300 (100%
0 200 400 600 800 1000 1200 1400 1600
Depth, microns
(A) (B)
Figure 5 OCT signal (A) profiles and OCT signal attenuation coefficient (B) from depth before (Intact) and after the introduction of
various OCAs into the rat casing
150-
100-
50-
0-
A new approach to the rapid delivery of OCA into the casing using the function of fast and effective optical clearing of biological tissues is presented. This method is based on needle-free injection of OCA into the casing. The size of the area changed immediately after injection is approximately 5.9 mm in the lateral direction and 1.2 mm in the direction from the surface of the skin. During a dermoscopic examination, we noticed that a small part of the OCA is removed from the injection area through a breakdown in the skin, resulting from rupture of the upper layers of tissue under the influence of the injector jet. The maximum breakdown size that we observed in the experiments was estimated using OCT, the force width and depth being 163 and 748 ^m, respectively. The effect of instantaneous OC upon injection of undiluted Omnipaque-300 has been shown.
The reported study was funded by the grant of RFBR (#20-52-56005) and the grant under the Degree of the Government of the Russian Federation No. 220 of 09 April 2010 (Agreement No.075-15-2021-615 of 04 June 2021).
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