A skin pain sensation quantification detection system based on optical coherence tomography imaging and application thereof

Abstract

The application discloses a skin pain sensation quantitative detection system based on optical coherence tomography imaging and application thereof. The application adopts optical coherence tomography imaging technology to perform in-vivo noninvasive imaging on the skin, can dynamically and quantitatively detect the epidermis thickness, and completes histological verification in combination with hematoxylin-eosin staining, and it is proved that the technology can objectively reflect the skin pain sensation related state through the skin thickness change. Through in-vivo detection verification of various pain sensation modeling mice such as ultraviolet irradiation damage, TRPV1 receptor activation / inhibition intervention and formalin treatment, the technology can accurately and quantitatively detect the epidermis thickness, and judge the skin structure change and damage degree related to the pain sensation. The application can efficiently and accurately detect the epidermis thickness, real-time evaluate the skin damage process and severity, has the advantages of noninvasiveness, real-time, simple operation and the like, and has a wide application prospect in clinical evaluation and scientific research monitoring.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a skin pain quantification detection system based on optical coherence tomography imaging and its application. Background Technology

[0002] Optical coherence tomography (OCT) is a high-resolution, non-invasive optical imaging technique. Its core principle is to use low-coherence near-infrared light to perform lateral scanning of biological tissues or scattering media, and then obtain micrometer-level depth-resolution tomographic images through coherent demodulation. It is currently widely used in ophthalmology, dermatology, neurovascular imaging, and other fields.

[0003] As the body's largest defense organ, the skin is richly supplied with sensory receptors and nerve endings, making it susceptible to damage from dermatitis, sunburn, burns, or chemical irritants, leading to varying degrees of pain. Current clinical and research settings suffer from significant shortcomings in pain assessment: clinical practice primarily relies on subjective rating scales such as the Numerical Rating Scale (NRS) and the Visual Analogue Scale (VAS), which are highly influenced by emotions, cultural background, and individual threshold differences, exhibiting strong subjectivity, lacking objectivity, and heavily dependent on patient cooperation. In animal experiments, behavioral tests such as the Von-Frey mechanical pain threshold test have poor universality, neuronal electrophysiology / calcium imaging techniques are complex and often invasive, and while machine learning-based behavioral recognition methods have emerged in recent years, they can only indirectly reflect pain states and cannot reveal microscopic pathological changes in tissues.

[0004] Therefore, there is an urgent need to develop a non-invasive, universally applicable, easy-to-operate, and real-time dynamic objective pain assessment technique to overcome the shortcomings of existing methods, such as high subjectivity, dependence on cooperation, poor universality, or complex operation, and to provide more reliable technical support for clinical pain assessment and research on animal pain models. Summary of the Invention

[0005] This invention utilizes optical coherence tomography (OCT) technology to accurately and non-invasively image the skin in vivo and quantify the thickness between different skin layers. Based on this, it was further discovered that the epidermis significantly thins when the skin is stimulated and experiences pain. This finding reveals a new application prospect for OCT technology: by accurately quantifying epidermal thickness through real-time skin imaging, it can determine the presence and degree of pain. It has broad application prospects in clinical and research settings.

[0006] This invention proposes a skin pain quantification detection system based on optical coherence tomography (OCT) imaging, comprising an OCT imaging module and an image processing and analysis module; the OCT imaging module is used to acquire OCT images of the sample to be tested; the image processing and analysis module is used to extract features and analyze parameters from the tomographic images and to determine the presence or absence of pain and to quantify and grade the degree of pain.

[0007] Furthermore, the sample to be tested includes either human skin or skin from a model animal.

[0008] Furthermore, the optical coherence tomography imaging module includes a light source unit, an optical fiber interference path, an optical scanning unit, and a spectral detection unit.

[0009] Furthermore, the scanning wavelength of the imaging module is 800-900nm.

[0010] Furthermore, the imaging module has a lateral resolution of 8-10 μm and a longitudinal resolution of 1.5-2 μm.

[0011] Furthermore, the image processing and analysis module measures the image thickness of the sample to be 90-120 mm.

[0012] Furthermore, the system detection includes the following steps:

[0013] S1: The optical coherence tomography (OCT) imaging module is used to perform tomographic scanning on the skin area to be tested, and to obtain two-dimensional / three-dimensional tomographic images of the skin. S2: Based on two-dimensional / three-dimensional tomographic images, identify the skin surface and epidermal-dermal boundary, calculate the vertical distance from the stratum corneum surface to the basal layer, and obtain quantitative data on epidermal thickness; S3: Compare and analyze the thickness of the normal epidermis with that of the area to be tested. Based on the differences in epidermal thickness, determine the presence or absence of pain and quantify and grade the degree of pain.

[0014] This invention provides an application of the aforementioned skin pain quantification detection system in the determination and quantitative assessment of skin pain intensity.

[0015] Furthermore, the pain includes skin damage pain caused by ultraviolet radiation. In some embodiments, optical coherence tomography can detect a significant thinning of the skin after ultraviolet radiation.

[0016] The present invention also provides an application of the aforementioned skin pain quantification detection system in screening or preparing anti-pain drugs.

[0017] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The present invention is highly objective and eliminates subjective errors: it does not rely on the patient's subjective description or the experimenter's subjective judgment. It uses the epidermal thickness quantified by OCT as an objective indicator, avoids the evaluation errors caused by individual pain threshold differences, insufficient patient cooperation, and experimenter operation deviations, and achieves objective determination and quantification of pain.

[0018] (2) The present invention is non-invasive and real-time, and easy to operate: it adopts OCT imaging technology, which does not require invasive sampling or complicated operation, and can perform real-time tomographic imaging of the skin and quickly complete the integral calculation of epidermal thickness, which meets the convenience needs of clinical and scientific research scenarios.

[0019] (3) The present invention has a wide range of applications and strong universality: it not only solves the problem of pain assessment for patients who cannot express themselves in clinical practice, but also adapts to pain monitoring of skin model mice in scientific research, without limiting the modeling site and avoiding the limitations of traditional methods. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the optical coherence tomography imaging device and imaging method of the present invention.

[0022] Figure 2 The diagram shows the chemical structures of capsaicin and GSK205 added in this invention; (A) is capsaicin, and (B) is GSK205.

[0023] Figure 3 The results of measuring the thickness of the epidermal layer of the skin using optical coherence tomography (OCT) technology in Example 1 of this invention are as follows: (A) Quantitative measurement of epidermal thickness using OCT imaging; (B) Quantitative measurement of the epidermal layer after sampling and HE staining of the OCT imaging area and imaging with an optical microscope; (C) Image of the skin structure after HE staining, with the yellow line indicating the epidermal thickness. The data are presented as mean ± SEM, with p < 0.05 indicating significant difference, and statistical analysis was performed using a standard t-test.

[0024] Figure 4This is the measurement result of the epidermal layer thickness of skin treated with capsaicin using optical coherence tomography (OCT) in Example 2 of the present invention. (A) OCT was used to image and quantify the epidermal layer thickness of the left foot (with the drug solvent applied) and the right foot (with capsaicin applied). (B) The pain threshold was measured using a Von-Frey device. (C) HE staining was used to quantify the epidermal layer thickness; the yellow line indicates the quantification of thickness. Data are presented as mean ± SEM, with p < 0.05 considered statistically significant, and a standard t-test was used for statistical analysis.

[0025] Figure 5 The results of measuring the thickness of the skin epidermis after injection of formalin or CFA in Example 3 of the present invention are as follows: (A) Quantitative measurement of epidermal thickness by optical coherence tomography at different times after CFA injection; (B) Quantitative measurement of epidermal thickness by imaging at different times after formalin injection.

[0026] Figure 6 The results of measuring the thickness of the skin epidermis after ultraviolet light irradiation using optical coherence tomography (OCT) in Example 4 of this invention are as follows: (A) Quantitative epidermal thickness measured by OCT imaging before irradiation and at 24h and 28h after irradiation; (B) Measurement of mechanical pain threshold 24h after irradiation. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0028] Transient receptor potential vanillic acid subtype 1 (TRPV1) is a key cation channel mediating thermal pain, inflammatory pain, and chemical irritant pain, while transient receptor potential vanillic acid subtype 4 (TRPV4) is mainly involved in the signal regulation of edema pain and neuropathic pain. Both can be activated under external damage conditions such as prolonged ultraviolet radiation, high temperature burns, and stimulation by harmful chemicals.

[0029] By specifically activating TRPV1 with chemical reagents, or by establishing an inflammation model through local injection of complete Freund's adjuvant or formalin into the skin, mice can be induced to exhibit distinct mechanodystrophic and thermohyperalgesic phenotypes, which are highly consistent with the pain behavior characteristics induced by spontaneous skin inflammation in mice. Excessive ultraviolet radiation exposure can directly activate TRPV4 in skin tissue, thereby inducing pain responses, hyperalgesia, and sunburn pathological changes, ultimately leading to a state of skin hyperalgesia. This phenotype is similar to the pain behavior exhibited by mice under normal physiological conditions when subjected to harmful mechanical stimuli.

[0030] After pain modeling, mice exhibit typical biological characteristics, displaying significant nociceptive pain behaviors such as rapid paw lifting, paw swinging, and persistent licking of the modeled skin area. Simultaneously, the expression levels of inflammatory factors in the modeled skin area are upregulated, local redness and swelling worsen, and temperature perception and pain sensitivity are further enhanced. At the same time, transient receptor potential (TRP) family receptors in keratinocytes are activated, promoting extracellular calcium ion influx and initiating downstream pain and inflammation-related signaling pathway cascades.

[0031] This invention employs optical coherence tomography (OCT) to perform tomographic imaging of skin tissue. Hematoxylin-eosin staining is used to pathologically quantify the thickness of different layers of mouse skin, thereby verifying the accuracy of OCT in detecting epidermal thickness. Simultaneously, the Von-Frey behavioral test is used to assess pain levels in mice with various induced pain models, including those induced by ultraviolet radiation-induced skin damage, chemically activated TRPV1 receptors simulating inflammatory pain, and those induced by subcutaneous injection of formalin or complete Freund's adjuvant. Through the mutual corroboration of these various experimental methods, it is confirmed that OCT can objectively detect and determine the occurrence and state of pain by quantitatively detecting changes in skin tissue thickness.

[0032] Detection methods Optical tomography (CT) to test skin thickness: Figure 1This is a schematic diagram of the optical coherence tomography (OCT) equipment and imaging. The imaging method is as follows: Mice were anesthetized by intraperitoneal injection of 120 μL / 10 g of 0.25% tribromoethanol. The mice were placed on the stage and their paws were fixed. Frequency domain OCT (SD-OCT) was used to image them. A small amount of refractive index-matched gel (n≈1.38-1.42) was dropped between the window and the skin to reduce skin surface reflection and microslippage. The field of view was set to 6 mm × 6 mm, A-scan rate of 100 kHz, volume scan of 512 × 512 A-lines; axial sampling interval Δzair≈1 μm (in air); epidermal refractive index ne=1.40; scanning wavelength of 800-900 nm, lateral resolution of 8-10 μm, and longitudinal resolution of 1.5-2 μm were set. The thickness of the sample image within the 90-120 μm range of the mice was measured. The epidermal thickness was calculated by tracing the skin surface curve S by hand (along the upper edge of the strongest surface reflection band, with a maximum curvature limit set). Draw the epidermal bottom boundary curve D (along the reflection / contrast transition zone at the epidermal-dermal junction). Read the corresponding axial sampling indices zS and zD of the two surfaces, calculate the depth difference Δzair = (zD - zS) × Δzair_step, and apply refractive index correction: t(x,y) = Δzair / ne, where ne is the refractive index of the epidermal group. The average epidermal thickness is the arithmetic mean of all effective t(x,y).

[0033] Von-Frey behavioral test: Mice are placed on a wire mesh platform and separated by small chambers. Pressure is applied to the soles of the mice's feet with Vonfrey filaments of different specifications. The specifications of the Vonfrey filaments corresponding to the withdrawal of the mouse's paw are recorded to calculate the mechanical pain threshold.

[0034] Hematoxylin-eosin staining method: Skin tissue samples from the mouse paw model site were fixed overnight in 4% paraformaldehyde, then dehydrated in 30% sucrose solution until the tissue settled. After embedding with OCT, the tissue was frozen and sectioned to a thickness of 20 μm. Sections were stained with hematoxylin and eosin (HE) (Beyotime, C0105S) according to the manufacturer's instructions. After staining, the sections were photographed and observed using an inverted optical microscope, and the average thickness of the epidermal layer of each section was measured.

[0035] Unless otherwise specified, the raw materials involved in the following specific embodiments of the present invention are all conventional materials in the art, and can be purchased from commercially available products. Some raw material information is as follows: Capsaicin: a TRPV1 receptor agonist, CAS number 404-86-4, its chemical structural formula is as follows Figure 2 As shown in A, DMSO was used to prepare a solution of 1 mg / mL for application to mice, and it was prepared and used immediately. GSK205: A TRPV1 receptor inhibitor, CAS number 1263068-83-2, its chemical structure is as follows Figure 2 As shown in B, DMSO was used to prepare 5mM for application to mice, and it was prepared and used immediately.

[0036] Example 1: Determination of the thickness of the epidermal layer of skin using optical coherence tomography (OCT) imaging technology Mice were anesthetized by intraperitoneal injection of 300 μL of 0.25% tribromoethanol. After complete anesthesia, the mice's feet were fixed to the stage, and the skin of the feet was imaged. After imaging, the mice were euthanized by cervical insemination, and the skin of the imaged area on the feet was fixed overnight in 4% paraformaldehyde. The tissue was dehydrated with 30% sucrose aqueous solution until the tissue block settled, then embedded in OCT for cryosectioning, and cut into 20 μm thick sections. After section preparation, staining was performed using the Beyotime hematoxylin and eosin staining kit (Beyotime, C0105S), following the instructions of the HE staining kit. Final imaging was performed using an inverted optical microscope.

[0037] Figure 3 The results from AC showed that the accuracy of epidermal thickness quantification after in vivo skin imaging using optical coherence tomography (OCT) technology can reach about 80% of that of HE staining, proving that this technology can quantify skin epidermal thickness without damage.

[0038] Example 2: Capsaicin-induced pain response and detection of skin epidermal thickness using optical coherence tomography (OCT) imaging. TRPV1 knockout mice and wild-type mice were used as control groups. DMSO was applied to the left paw of both groups of mice, and 5 μL (5 μg) of capsaicin was injected into the right paw. One hour after application, pain behavior was tested using a Von-Frey device. After the behavioral experiment, optical coherence tomography (OCT) imaging was performed on the modeling site. The mice were then euthanized by cervical indwelling, and skin from the paws was collected for HE staining.

[0039] like Figure 4 As shown in A, capsaicin significantly reduced the epidermal thickness of wild-type mice (p<0.001, extremely significant), but this effect disappeared in TRPV1 knockout mice. Figure 4 The results of measuring the pain threshold using the Von-Frey device in B further verified that capsaicin can induce significant mechanical hyperalgesia in mice, which is consistent with the results of optical coherence tomography. Figure 4 The histological HE staining results of C in the study confirmed that OCT imaging could detect that capsaicin treatment significantly reduced the thickness of the mouse epidermis, which highly correlated with the occurrence of pain response.

[0040] The above experimental results demonstrate that capsaicin can activate TRPV1 receptors, induce mechanical hyperalgesia in mice, and lead to a significant reduction in epidermal thickness; optical coherence tomography (OCT) imaging technology can accurately measure changes in epidermal thickness.

[0041] Example 3: Injection of CFA or formalin to induce pain response, followed by detection of skin epidermal thickness using optical coherence tomography (OCT). Optical coherence tomography (OCT) imaging was performed on the skin of the mouse paws. After imaging, mice were generally anesthetized with isoflurane, and 10 μL of complete Freund's adjuvant (CFA) or formalin was injected into the paw. Time-gradient imaging was performed after injection. OCT imaging of the injection site was performed at 48 hours, 96 hours, and 7 days after CFA injection; for formalin injection, OCT imaging was performed at 24 hours after injection.

[0042] In the early stages of CFA injection, skin redness and swelling, and epidermal thickening may occur; as the inflammation progresses, the epidermis undergoes structural remodeling and a decrease in thickness.

[0043] Formalin treatment of ex vivo tissues causes the skin tissue to shrink as a whole and collagen fibers to become denser and aggregated, resulting in a decrease in the measured thickness of both the epidermis and dermis.

[0044] like Figure 5 As shown in AB, optical coherence tomography (OCT) was used to detect a decrease in skin thickness in mice treated with CFA at 96 h and 7 days. The thickness decreased after 24 h of formalin treatment, indicating that the mouse epidermis was thinner, which was consistent with the results of the treatment and verified the accuracy of OCT in detecting epidermal thickness.

[0045] Example 4: Ultraviolet radiation induces pain response, and skin epidermal thickness is detected using optical coherence tomography (OCT). Wild-type mice were used. The drug solvent was applied to the left paw, and the TRPV4 receptor inhibitor GSK205 (5 mM, 10 μL) was applied to the right paw. One hour after application, the mice were irradiated with UVB at an energy of 1200 mJ / cm². 2 Optical coherence tomography (OCT) images of the skin on the left and right feet were performed before irradiation, 24 hours after irradiation, and 48 hours after irradiation. The Von-Frey mechanical pain threshold was measured at 24 hours after irradiation.

[0046] In a mouse model of UV-induced injury, we also observed significant sensitization to mechanical stimuli after UV irradiation, with pronounced pain responses. Figure 6 Optical coherence tomography (OCT) imaging of A in the mouse showed significant thinning of the epidermis. In contrast, mice treated with the TRPV4 inhibitor GSK205 exhibited significantly lower pain sensitization after irradiation compared to the control paws, and no significant thinning of the epidermis was observed. Figure 6As shown in B in the diagram.

[0047] Based on the results of the above embodiments, it was found that optical coherence tomography (OCT) imaging technology can accurately quantify the thickness of the skin epidermis. The accuracy and reliability of this technology were verified by HE staining and pain behavior testing experiments using a Von-Frey device.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A skin pain sensation quantification detection system based on optical coherence tomography imaging, characterized in that, It includes an optical coherence tomography (OCT) imaging module and an image processing and analysis module; the OCT imaging module is used to acquire OCT images of the sample to be tested; the image processing and analysis module is used to extract features and analyze parameters from the tomographic images and to determine the presence or absence of pain and to quantify and grade the degree of pain.

2. The skin pain quantification detection system of claim 1, wherein, The sample to be tested includes either human skin or skin from a model animal.

3. The skin pain quantification detection system of claim 1, wherein, The optical coherence tomography imaging system includes a light source unit, a fiber optic interferometer, an optical scanning unit, and a spectral detection unit.

4. The skin pain quantification detection system of claim 1, wherein, The scanning wavelength of the imaging module is 800-900nm.

5. The skin pain quantification detection system of claim 1, wherein, The imaging module has a horizontal resolution of 8-10 μm and a vertical resolution of 1.5-2 μm.

6. The skin pain quantification detection system of claim 1, wherein, The image processing and analysis module measures the sample imaging thickness to be 90-120 μm.

7. The skin pain quantification detection system of claim 1, wherein, The system detection includes the following steps: S1: The optical coherence tomography (OCT) imaging module is used to perform tomographic scanning on the skin area to be tested, and to obtain two-dimensional / three-dimensional tomographic images of the skin. S2: Based on two-dimensional / three-dimensional tomographic images, identify the skin surface and epidermal-dermal boundary, calculate the vertical distance from the stratum corneum surface to the basal layer, and obtain quantitative data on epidermal thickness; S3: Compare and analyze the thickness of the normal epidermis with that of the area to be tested. Based on the differences in epidermal thickness, determine the presence or absence of pain and quantify and grade the degree of pain.

8. The application of the skin pain quantification detection system according to any one of claims 1-7 in the determination and quantitative assessment of skin pain.

9. Use according to claim 8, characterized in that, The pain includes skin damage pain caused by ultraviolet radiation.

10. The use of the skin pain quantification detection system according to any one of claims 1-7 in screening or preparing anti-pain drugs.

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