Evaluation method for vascular sympathetic nerve injury caused by neuronal intracellular inclusion body disease

Abstract

The invention discloses an evaluation method for vascular sympathetic nerve injury caused by a neuronal intracellular inclusion body disease. The method belongs to the technical field of biological science and comprises the following operation steps of skin biopsy, immunofluorescence staining, slide scanning and calculation of the density of the blood vessel adrenal gland energetic small fibers. Tissue is obtained through skin biopsy, shooting is carried out after immunofluorescence staining, and the density of the sympathetic adrenergic minor nerve marked by the TH antibody in the dermal superficial blood vessel is calculated by using Image J software, so that whether a neuron intracellular inclusion body disease receptor has sympathetic nerve injury or not and the severity of the sympathetic nerve injury are accurately evaluated.

Description

Technical Field

[0001] The present invention belongs to the field of biological science and technology, and relates to a method for evaluating vascular sympathetic nerve injury in neuronal intranuclear inclusion disease (a method for evaluating vascular sympathetic nerve injury in neuronal intranuclear inclusion disease). Background Art

[0002] Neuronal intranuclear inclusion disease is a rare disease, and its pathological feature is the presence of eosinophilic inclusions in the cell nuclei of the central and peripheral nervous systems as well as visceral organs. The clinical manifestations of this disease are complex and diverse. Cognitive impairment, movement disorders, autonomic nervous system dysfunction, etc. are all common symptoms. Among them, vasomotor dysfunction has a serious impact on the quality of life and survival time of patients. Vasomotor function is mainly regulated by the sympathetic and parasympathetic nerves, and the sympathetic nerve has a wider range of effects on blood vessels. However, there are still some deficiencies in the current examination methods for measuring vascular sympathetic nerve function. The supine-to-standing blood pressure test is difficult to accurately judge vascular autonomic nerve function due to large individual differences and many interfering factors; quantitative sensory testing has problems such as strong subjectivity and being easily interfered by environmental factors, thus affecting the reliability of test results; the stability and specificity of skin sympathetic nerve responses are poor, and various diseases or pathological states may affect its waveform, latency, and amplitude, and there are large variations within and between individuals. None of these three methods can accurately evaluate the vascular sympathetic nerve function of the body. Summary of the Invention

[0003] In view of the above problems, the object of the present invention is to propose a method for evaluating vascular sympathetic nerve injury in neuronal intranuclear inclusion disease, aiming to quantitatively evaluate the number of vascular sympathetic small fibers in neuronal intranuclear inclusion disease.

[0004] The technical solution of the present invention is as follows: A method for evaluating vascular sympathetic nerve injury in neuronal intranuclear inclusion disease according to the present invention conducts skin biopsies on neuronal intranuclear inclusion disease and control recipients without nervous system diseases. After sectioning, immunofluorescence staining is performed using TH antibody, and then Image J software is used to quantitatively calculate the density of sympathetic adrenergic small fibers labeled by TH antibody in the superficial dermis blood vessels, and analyze whether there are differences in this density between the two groups, so as to evaluate the sympathetic nerve injury condition and its severity;

[0005] The operation steps are as follows:

[0006] 1. Skin biopsy:

[0007] a. Select a suitable biopsy site on the outer side of the recipient's forearm for disinfection. After local anesthesia with 2% lidocaine, use a skin biopsy needle to take a 3-mm diameter skin sample;

[0008] b. The skin samples were immersed in 4% paraformaldehyde overnight, washed with 0.01% PBS, then immersed in 25% sucrose solution overnight, and finally embedded in OCT compound.

[0009] 2. Immunofluorescence staining:

[0010] a. Discontinuous sections with a thickness of 30 μm were prepared using a cryostat.

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

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

[0013] d. The cell nuclei were labeled with DAPI (Abcam Cat#ab104139, RRID:no).

[0014] 3. Slide scanning:

[0015] The sections were photographed using a confocal laser scanning microscope. The scanning parameters were selected based on the best signal - to - noise ratio. The slice interval was set to 2 μm, and the magnification was ×200 to acquire three - dimensional digital images.

[0016] 4. Calculate the density of vascular adrenergic small fibers:

[0017] a. According to the TH (red) and DAPI (blue) stained images (a), the regions of interest (ROIs) of blood vessels were manually circled using Image J software.

[0018] b. The original fluorescence image stained with TH antibody was converted to a grayscale image.

[0019] c. The background was removed from the grayscale image to obtain a basic image. The basic image was blurred by Gaussian blur to obtain a defocused image, and the composite image was obtained by subtracting the defocused image from the basic image.

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

[0021] f. The areas of adrenergic nerve fibers and the areas of blood vessel ROIs were calculated respectively, and the density of adrenergic small fibers labeled with TH antibody in blood vessels was obtained by dividing the former by the latter.

[0022] g. Calculate the average density of adrenergic small fibers in 3 blood vessels for each receptor.

[0023] The beneficial effects of the present invention are as follows: The present invention has developed a simple and feasible method, which is specifically applied to the evaluation of vascular sympathetic nerve injury in neuronal intranuclear inclusion disease; skin biopsies are performed on receptors with neuronal intranuclear inclusion disease and control receptors without neurological diseases. After obtaining samples by biopsy and making them into sections, immunofluorescence staining is carried out. Subsequently, the density of adrenergic small fibers in the superficial dermis vessels labeled with TH antibody is quantitatively calculated using Image J software. By analyzing whether there are differences in this index between these two groups of receptors, the presence and severity of adrenergic nerve injury are evaluated. This invention has opened up a new way for accurately evaluating the condition and severity of sympathetic nerve injury in neuronal intranuclear inclusion disease. Brief Description of the Drawings

[0024] Figure 1 It is a schematic diagram of the quantitative calculation method for the innervation of adrenergic small fibers in the vessels of the control receptor in the embodiment of the present invention; for the merged image (a) stained with DAPI and TH antibody, the region of interest of the vessel is manually selected; the original TH-stained image is converted into a grayscale image (b); the background of the grayscale image (b) is removed to obtain the basic image (c); the defocused image (d) is obtained through Gaussian blur; the basic image (c) minus the defocused image (d) to obtain the composite image (e); the light threshold is adjusted to highlight the nerve fibers labeled with TH antibody (f); in the figure (f), the density of TH adrenergic small nerve fibers in the vessel is 3.036%, scale bar = 100μm, magnification: ×200;

[0025] Figure 2 It is a statistical chart of the innervation of adrenergic small fibers labeled with TH antibody in the vessels of the receptor with neuronal intranuclear inclusion disease and the control receptor without neurological diseases in the embodiment of the present invention; through quantitative analysis of the density of TH adrenergic small fibers, it is found that the NIID receptor is significantly lower than the control receptor; **: p < 0.01. Detailed Embodiments

[0026] The following further elaborates on the specific technical solutions of the present invention in combination with specific examples.

[0027] As shown in the figure, the present invention selects 20 cases of neuronal intranuclear inclusion disease and 21 control receptors without neurological diseases that are matched in terms of gender and age. A suitable biopsy site is determined and disinfected at the distal part of the upper limb of the receptor (10 cm above the wrist on the lateral side of the forearm). After local anesthesia with 2% lidocaine, a 3-mm diameter skin sample is taken with a skin biopsy needle;

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

[0029] Discontinuous sections with a thickness of 30 μm were made using a cryostat. The sections were incubated with the corresponding primary antibodies overnight. The primary antibodies included rabbit tyrosine hydroxylase (TH) (Novus Biologicals Cat#NB300 - 109, RRID:AB_10077691) that labels adrenergic small fibers;

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

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

[0032] Three - dimensional digital images were acquired using a confocal laser scanning microscope (Nikon A1R HD25, Tokyo, Japan). The layer spacing was set at 2 μm and the magnification was ×200. The three - dimensional digital images were acquired; the scanning parameters were selected based on the best ratio of the staining signal intensity to the noise,

[0033] Quantitative analysis was performed using Image J software. Blood vessels were identified based on DAPI and TH staining;

[0034] The density of adrenergic small fibers labeled with TH antibody in blood vessels was quantitatively calculated using Image J software, which is the ratio of the area of small nerve fibers labeled with TH antibody to the area of blood vessels;

[0035] The out - of - focus image of the blood vessel was subtracted from its base image to obtain a composite image to highlight nerve fibers;

[0036] The density of adrenergic small fibers labeled with TH antibody in blood vessels was quantitatively calculated, which is the ratio of the area of small nerve fibers labeled with TH antibody to the area of blood vessels;

[0037] Generally, the average density of adrenergic small nerve fibers in 3 blood vessels per subject was calculated.

[0038] The experimental procedures are as follows:

[0039] In this invention, 20 subjects with neuronal intranuclear inclusion disease receptors and 21 control subjects without neurological diseases, matched in terms of gender and age, were selected for skin biopsies.

[0040] (1): In this invention, 20 subjects with neuronal intranuclear inclusion disease and 21 control subjects without neurological diseases, matched in terms of gender and age, were included for skin biopsies;

[0041] (2): Acquisition of specimens:

[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 tissue fixative, marker pen, tape;

[0043] (b): First, disinfect the selected biopsy site of the recipient with iodophor cotton balls;

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

[0045] (d): Press and rotate a sterile, 3 mm diameter skin biopsy needle to take a sample, with a depth of approximately 3 mm;

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

[0047] (3): Fixation of specimens:

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

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

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

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

[0052] (4): Preparation of sections:

[0053] (a): Use a cryostat (-20°C) to section, with a section thickness of 30 μm. When sectioning, the epidermis layer faces the blade. Cut discontinuous sections and wash with 0.01% PBS three times, 5 minutes each time;

[0054] (5): Immunofluorescence staining:

[0055] (a): Immerse in 0.3% PBST for 15 minutes;

[0056] (b): Block with 100 μl 5% BSA at room temperature for 1 hour;

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

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

[0059] (e): Dilute the secondary fluorescent antibody anti-rabbit Alexa Fluor 594 with 0.1% BSA (Abcam Cat# ab150080, RRID: ab_2650602) at a concentration of 1:500, and incubate in the dark at room temperature for 2 hours;

[0060] (f): Wash with 0.3% PBST three times, 5 minutes each time;

[0061] (g): After mounting the sections, add 1 - 2 drops of DAPI (Abcam Cat# ab104139, RRID: no), cover the slides, and store at 4°C;

[0062] (6): Slide scanning:

[0063] (a): Use a confocal microscope to acquire three-dimensional digital images, and identify blood vessels based on the DAPI and TH antibody staining images;

[0064] (b): Set the scanning parameters according to the best ratio of staining signal intensity to noise, set the layer spacing to 2 μm, and the magnification to ×200;

[0065] (7): Calculate the density of adrenergic small fibers:

[0066] (a): Use Image J software to manually circle the regions of interest of blood vessels based on the merged images stained with DAPI (blue) and TH (red) antibodies;

[0067] (b): Convert the original fluorescence image of adrenergic small fibers labeled with TH antibody into a grayscale image;

[0068] (c): First, remove the background from the grayscale image to obtain a base image, perform Gaussian blur on the base image to obtain a defocused image, and then subtract the defocused image from the base image to obtain a composite image;

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

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

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

Claims

1. A method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease, characterized in that: Skin biopsies were performed on subjects with neuronal nuclear inclusion disease and controls without neurological diseases. After sectioning, immunofluorescence staining was performed using TH antibodies. Image J software was then used to quantitatively calculate the density of sympathetic adrenergic fibers labeled with TH antibodies in superficial dermal vessels. The density was analyzed to determine whether there was a difference between the two groups, in order to assess the severity of sympathetic nerve damage.

2. The method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 1, characterized in that: The operation steps are as follows: (1): Skin biopsy; (2): Immunofluorescence staining; (3): Slide scanning; (4): Calculate the vascular adrenergic fiber density.

3. A method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 2, characterized in that: In step (1), the steps of skin biopsy are as follows: a. Select the biopsy site on the outside of the recipient's forearm and disinfect it. After local anesthesia with 2% lidocaine, use a skin biopsy needle to obtain skin. b. The skin was soaked in 4% paraformaldehyde overnight, washed with 0.01% PBS, soaked in 25% sucrose solution overnight, and then embedded in OCT glue.

4. A method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 3, characterized in that: In step a, the diameter of the skin sampled is 3 mm.

5. The method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step (2), the immunohistochemical staining procedure is as follows: a. Use a freezing microtome to perform discontinuous sectioning; b. Incubate sections overnight with rabbit tyrosine hydroxylase, which labels adrenergic fibrils, as the primary antibody; c. Use anti-rabbit Alexa Fluor 594 antibody as secondary antibody and incubate the sections at room temperature; d. DAPI was used to label cell nuclei.

6. A method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 5, characterized in that: In step a, the thickness of the discontinuous sections is 30 μm.

7. A method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 5, characterized in that: In step c, the room temperature is 20-25°C, and the slice incubation time is 2 hours.

8. The method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step (3), the slide scanning specifically includes: photographing the slices with the aid of a confocal laser scanning microscope, and selecting scanning parameters based on an optimal ratio of signal intensity to noise.

9. A method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 8, characterized in that: The inter-layer spacing was set to 2 μm, the magnification was ×200, and three-dimensional digital images were collected.

10. The method for evaluating vascular sympathetic nerve damage in neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step (4), the operation steps for calculating the vascular adrenergic fiber density are as follows: a. Based on TH and DAPI staining images, the vascular region of interest was manually circled using Image J software; b. Convert the original fluorescence image stained with TH antibody into a grayscale image; c. removing the background from the grayscale image to obtain a base image, performing Gaussian blurring on the base image to obtain a defocused image, and subtracting the defocused image from the base image to obtain a composite image; d. Adjust the light threshold to highlight the small adrenergic nerve fibers labeled by TH antibody; e. Calculate the adrenergic nerve fiber area and the vascular region of interest area respectively, and divide the two to obtain the density of adrenergic small fibers labeled by TH antibody in the blood vessels; f. Calculate the average adrenergic fiber density of the three vessels for each receptor.

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