A method for assessing vascular sympathetic nerve injury in a neuronal intranuclear inclusion disease

Abstract

The application discloses a kind of for neuron intranuclear inclusion disease vascular sympathetic nerve injury evaluation method;Belong to the field of biological science and technology, its operating steps are as follows: skin biopsy, immunofluorescence staining, slide scanning, calculate vascular adrenergic small fiber density.The application obtains tissue by skin biopsy, is photographed after being given immunofluorescence staining, and the density of the sympathetic adrenergic small nerve of the superficial layer of dermis blood vessel marked by TH antibody is calculated using Image J software, to accurately assess whether the receptor of neuron intranuclear inclusion disease exists sympathetic nerve injury and its severity.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological science and technology, and relates to a method for evaluating vascular sympathetic nerve injury of neuron intranuclear inclusion disease (a method for evaluating vascular sympathetic nerve injury of neuron intranuclear inclusion disease). BACKGROUND

[0002] Neuron intranuclear inclusion disease is a rare disease, and its pathological feature is that there are acidophilic inclusions in the nuclei of cells in the central and peripheral nervous systems and internal organs. The disease has complex and diverse clinical manifestations, and cognitive impairment, movement disorder and autonomic nervous function disorder are common symptoms, among which vascular tone dysfunction has a serious impact on the quality of life and survival time of patients. Vascular tone is mainly regulated by sympathetic nerves and parasympathetic nerves, and the range of action of sympathetic nerves on blood vessels is wide. However, the current examination methods for determining vascular sympathetic nerve function still have some deficiencies. The supine and standing blood pressure detection is difficult to accurately determine the vascular autonomic nerve function due to individual differences, many interference factors and the like; the quantitative sensory test has strong subjectivity and is easily disturbed by environmental factors, thereby affecting the reliability of the test results; and the stability and specificity of the skin sympathetic nerve response are poor, and various diseases or pathological states can affect the waveform, latency and amplitude thereof, and there is great variation within and between individuals. The three methods cannot accurately evaluate the vascular sympathetic nerve function of the body. SUMMARY

[0003] In view of the above problems, the application aims to provide a method for evaluating vascular sympathetic nerve injury of neuron intranuclear inclusion disease, which is used to quantitatively evaluate the number of vascular sympathetic small fibers of neuron intranuclear inclusion disease.

[0004] The technical scheme of the application is as follows: the method for evaluating vascular sympathetic nerve injury of neuron intranuclear inclusion disease according to the application is used to carry out skin biopsy on a control subject with no nervous system disease and a subject with neuron intranuclear inclusion disease, perform immunofluorescence staining on the section using a TH antibody, quantitatively calculate the density of TH antibody-labeled sympathetic adrenergic small fibers in the superficial layer of the dermis by using Image J software, and analyze whether there is a difference in the density between the two groups, so as to evaluate the sympathetic nerve injury and its severity.

[0005] The operation steps are as follows:

[0006] 1. Skin biopsy:

[0007] a. Disinfect the appropriate biopsy site on the lateral forearm of the subject, and after local anesthesia with 2% lidocaine, use a skin biopsy needle to take a 3mm-diameter skin;

[0008] b. The skin was soaked in 4% paraformaldehyde overnight, washed with 0.01% PBS, and then soaked in 25% sucrose solution overnight. The skin was embedded with OCT glue;

[0009] 2. Immunofluorescence staining:

[0010] a. Discontinuous sections with a thickness of 30 pm were made using a freezing microtome;

[0011] b. The sections were incubated overnight with rabbit tyrosine hydroxylase (TH) (Novus Biologicals Cat# NB300-109, RRID: AB_10077691) labeled adrenergic small fibers as a primary antibody;

[0012] c. The sections were incubated for 2 hours at room temperature with an anti-rabbit Alexa Fluor 594 antibody (Abeam Cat# ab150080, RRID: ab_2650602) as a secondary antibody;

[0013] d. DAPI (Abeam Cat# ab104139, RRID: no) was used to label the cell nucleus;

[0014] 3. Slide scanning:

[0015] The sections were photographed by a confocal laser scanning microscope. The scanning parameters were selected according to the best ratio of signal intensity to noise. The interlayer distance was set to 2 pm, the magnification was x200, and three-dimensional digital images were collected;

[0016] 4. Calculation of vascular adrenergic small fiber density:

[0017] a. According to the TH (red) and DAPI (blue) staining images (a), the region of interest of the blood vessels was manually circled by Image J software;

[0018] b. The original fluorescence image stained by TH antibody was converted into a gray-scale image;

[0019] c. The background was removed from the gray-scale image to obtain a basic image. The basic image was blurred by Gaussian blur to obtain an out-of-focus image. The basic image minus the out-of-focus image obtained a composite image;

[0020] d. The light threshold was adjusted to highlight the adrenergic nerve fiber small fibers labeled by TH antibody;

[0021] f. The area of the adrenergic nerve fiber and the area of the region of interest of the blood vessels were calculated respectively. The vascular TH antibody labeled adrenergic small fiber density was obtained by dividing the two;

[0022] g. The average value of the adrenergic small fiber density of 3 blood vessels of each subject was calculated.

[0023] The beneficial effects of the present application are: the present application develops a simple and easy method, which is specially applied to the evaluation of sympathetic nerve injury of neuronal intranuclear inclusion disease; the skin biopsy is performed on the neuronal intranuclear inclusion disease receptor and the control receptor without nervous system disease, after the biopsy sample is obtained and the section is prepared, immunofluorescence staining is performed. Then, the Image J software is used to quantitatively calculate the density of TH antibody labeled dermal superficial layer blood vessel adrenergic small fibers. By analyzing whether there is a difference between the two groups of receptors in this indicator, whether the adrenergic nerve injury exists and its severity are evaluated. The present application opens up a new way for accurately evaluating the sympathetic nerve injury of neuronal intranuclear inclusion disease and its severity. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 1 is a schematic diagram of the quantitative calculation method of the control receptor blood vessel adrenergic small fiber innervation in the embodiment of the present application; the merged image dyed by DAPI and TH antibody (a) is manually selected as the blood vessel region of interest; the TH dyeing original image is converted into a gray scale image (b); the background of the gray scale image (b) is removed to obtain a base image (c); the defocus image (d) is obtained by Gaussian blur; the base image (c) is subtracted from the defocus image (d) to obtain a composite image (e); the light threshold value is adjusted to highlight the TH antibody labeled nerve fibers (f); the TH adrenergic small nerve fiber density of the blood vessel in (f) is 3.036%, the scale bar = 100 μm, and the magnification is × 200;

[0025] Figure 2 Figure 2 is a statistical chart of the blood vessel TH antibody labeled adrenergic small fiber innervation of the neuronal intranuclear inclusion disease receptor and the control receptor without nervous system disease in the embodiment of the present application; quantitative analysis of the TH adrenergic small fiber density shows that the NIID receptor is significantly lower than the control receptor; **: p < 0.01. DETAILED DESCRIPTION

[0026] The specific technical solutions of the present application will be further described in detail below in combination with specific examples.

[0027] As shown in the figure, 20 cases of neuronal intranuclear inclusion disease and 21 cases of control receptors without nervous system disease are selected according to gender and age matching, the appropriate biopsy site is determined on the distal upper limb (10 cm above the wrist on the lateral forearm) of the receptor and is sterilized, after local anesthesia with 2% lidocaine, a 3mm diameter skin is taken with a skin biopsy needle;

[0028] The taken skin is soaked in 4% paraformaldehyde general tissue fixing solution overnight, washed with 0.01% PBS for 3 times, 5 minutes each time, soaked in 25% sucrose solution overnight, and then embedded with OCT glue;

[0029] Discontinuous sections with a thickness of 30 pm were made using a cryostat microtome, and the sections were incubated with the corresponding primary antibody overnight, the primary antibody including rabbit tyrosine hydroxylase (TH) (Novus Biologicals Cat# NB300-109, RRID: AB_10077691) labeled adrenergic small fibers;

[0030] The sections were incubated with the secondary antibody for 2 hours at room temperature, the secondary antibody including an anti-rabbit Alexa Fluor 594 antibody (Abeam Cat# ab150080, RRID: ab_2650602);

[0031] DAPI (Abeam Cat# ab104139, RRID: no) was added to the sections to label the cell nuclei;

[0032] Three-dimensional digital images were collected using a confocal laser scanning microscope (Nikon A1R HD25, Tokyo, Japan), with an interlayer distance of 2 pm and a magnification of x200; the scanning parameters were selected according to the best ratio of staining signal intensity to noise,

[0033] Quantitative analysis was performed using Image J software, and blood vessels were identified according to DAPI and TH staining;

[0034] The density of adrenergic small nerve fibers labeled by TH antibody was quantitatively calculated using Image J software, i.e., the ratio of the area of TH antibody-labeled small nerve fibers to the area of blood vessels;

[0035] The out-of-focus images of the blood vessels were subtracted from the base images to obtain composite images to highlight the nerve fibers;

[0036] The density of adrenergic small nerve fibers labeled by TH antibody was quantitatively calculated, i.e., the ratio of the area of TH antibody-labeled small nerve fibers to the area of blood vessels;

[0037] The average value of the density of adrenergic small nerve fibers of 3 blood vessels per subject was usually calculated.

[0038] The experimental operation was as follows:

[0039] The present application selected 20 cases of neuronal intranuclear inclusion disease and 21 cases of control subjects without nervous system diseases matched in gender and age for skin biopsy.

[0040] (1): The present application includes 20 cases of neuronal intranuclear inclusion disease and 21 cases of control subjects without nervous system diseases matched in gender and age for skin biopsy;

[0041] (2): Specimen acquisition:

[0042] (a): Preparation of materials: disposable surgical dressing kit, sterile gloves, 2 ml syringe, 3 mm skin biopsy needle, EP tube, 2% lidocaine, epinephrine hydrochloride injection, 4% paraformaldehyde general-purpose tissue fixative, marker pen, adhesive tape;

[0043] (b): First, use iodophor cotton ball to disinfect the selected recipient biopsy site;

[0044] (c): Wear sterile gloves and use 2% lidocaine with a small amount of epinephrine for subcutaneous local anesthesia;

[0045] (d): Press and rotate the sterile, 3 mm diameter skin biopsy needle to sample, about 3 mm deep;

[0046] (e): Press and bandage the biopsy site;

[0047] (3) Fixation of the specimen:

[0048] (a): Place the skin specimen in 4% paraformaldehyde general-purpose tissue fixative and store at 4°C overnight;

[0049] (b): Wash with 0.01% PBS for 3 times, 5 minutes each time;

[0050] (c): Dehydrate in 25% sucrose solution (25g sucrose + 100ml 0.01% PBS) at 4°C overnight;

[0051] (d): Embed the tissue with OCT glue, with the skin epidermis perpendicular to the mold base, and mark the epidermis position;

[0052] (4) Preparation of sections:

[0053] (a): Use a freezing microtome (-20°C) to cut sections, with a thickness of 30μm, cut the epidermis layer towards the blade, and use 0.01% PBS to wash for 3 times, 5 minutes each time;

[0054] (5) Immunofluorescence staining:

[0055] (a): Soak with 0.3% PBST for 15 minutes;

[0056] (b): Use 100ul 5% BSA to block at room temperature for 1 hour;

[0057] (c): Dilute rabbit TH (Novus Biologicals Cat# NB300-109, RRID: AB_10077691) antibody with 0.1% BSA, concentration 1:200, 4°C overnight;

[0058] (d): Wash with 0.3% PBST for 3 times, 5 minutes each time;

[0059] (e): 0.1% BSA diluted fluorescent secondary antibody anti-rabbit Alexa Fluor 594 (Abeam Cat# ab150080, RRID: ab_2650602) at 1:500, incubated at room temperature for 2 hours in the dark;

[0060] (f): 0.3% PBST wash for 3 times, 5 minutes each time;

[0061] (g): 1-2 drops of DAPI (Abeam Cat# ab104139, RRID: no) were added after the patch, and the slide was sealed and stored at 4°C;

[0062] (6): Slide scanning:

[0063] (a): Three-dimensional digital images were collected using a confocal microscope, and blood vessels were identified according to DAPI and TH antibody staining images;

[0064] (b): The scanning parameters were set according to the best ratio of staining signal intensity to noise, the interlayer distance was set to 2 pm, and the magnification was set to x200;

[0065] (7): Calculation of adrenergic small fiber density:

[0066] (a): The blood vessel region of interest was manually circled using Image J software according to the merged image of DAPI (blue) and TH (red) antibody staining;

[0067] (b): The original fluorescence image of TH antibody labeled adrenergic small fibers was converted to a gray scale image;

[0068] (c): First, the gray scale image was removed from the background to obtain a basic image, and the basic image was processed by Gaussian blur to obtain an out-of-focus image, and then the composite image was obtained by subtracting the out-of-focus image from the basic image;

[0069] (d): Adjust the light threshold to highlight the nerve fibers, and obtain the TH antibody labeled adrenergic small fibers;

[0070] (e): Calculate the area of TH antibody labeled adrenergic small fibers divided by the area of the blood vessel region of interest, which is the TH antibody labeled adrenergic small fiber density;

[0071] (d): Calculate the average value of the TH antibody labeled adrenergic small fiber density of 3 blood vessels of each subject, which is the dermal superficial layer sympathetic adrenergic small fiber density of each receptor.

Claims

1. A method for assessing vascular sympathetic nerve injury in neuronal intranuclear inclusion body disease for non-disease diagnostic purposes, characterized in that; Skin biopsies were performed on recipients with neuronal intranuclear inclusion body disease and control groups without neurological disease. After sectioning, immunofluorescence staining with TH antibody was performed. ImageJ software was then used to quantitatively calculate the density of TH antibody-labeled sympathetic adrenergic small fibers in the superficial dermal vessels, and the difference in density between the two groups was analyzed to assess the extent and severity of sympathetic nerve damage. The procedure is as follows: (1): Skin biopsy; the steps are as follows: a. After disinfecting the selected biopsy site on the outer side of the recipient's forearm and administering local anesthesia with 2% lidocaine, skin is taken using a skin biopsy needle; In step a, the diameter of the skin sample is 3mm; b. The skin samples were soaked in 4% paraformaldehyde overnight, washed with 0.01% PBS, soaked in 25% sucrose solution overnight, and then embedded in OCT gel. (2): Immunofluorescence staining; the procedure is as follows: a. Perform discontinuous slicing using a cryostat; b. Incubate sections overnight with rabbit tyrosine hydroxylase labeled with adrenergic filaments as primary antibody; c. Use anti-rabbit Alexa Fluor 594 antibody as a secondary antibody and incubate the sections at room temperature; d. Label cell nuclei using DAPI; In step a, the thickness of the discontinuous slice is 30 μm; In step c, the room temperature is 20-25℃, and the incubation time for the slices is 2 hours. (3): Slide scanning; specifically: using a confocal laser scanning microscope to photograph the slide, and selecting scanning parameters based on the optimal ratio of signal intensity to noise; Set the interlayer spacing to 2 μm and the magnification to × 200 to acquire three-dimensional digital images; (4): Calculate the density of vascular adrenergic small fibers; the operation steps are as follows: a. Based on the TH and DAPI staining images, manually delineate the regions of interest for blood vessels using ImageJ software; b. Convert the original fluorescent image stained with TH antibody to a grayscale image; c. Remove the background from the grayscale image to obtain the base image, apply Gaussian blur to the base image to obtain the defocused image, and subtract the defocused image from the base image to obtain the composite image; d. Adjust the light threshold to highlight the small fibers of adrenergic nerve fibers labeled with TH antibody; e. Calculate the area of ​​adrenergic nerve fibers and the area of ​​the region of interest in blood vessels, and divide the two to obtain the density of adrenergic small fibers labeled with TH antibody in blood vessels; f. Calculate the average density of adrenergic small fibers in three vessels for each receptor.

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