Quantitative detection method for sweat gland cholinergic nerves of neuron intranuclear inclusion body disease

Abstract

The invention discloses a quantitative detection method for sweat gland cholinergic nerves of a neuron intracellular inclusion body disease. Belongs to the technical field of biological science. Performing immunohistochemical staining; scanning a slide; and calculating the density of the cholinergic nerve fiber. The invention relates to a simple and feasible method for quantitatively evaluating cholinergic nerve fibers of a neuron intracellular inclusion body disease. The method comprises the following steps: carrying out skin biopsy on control receptors of a neuron intranuclear inclusion body disease and a nervous system-free disease, slicing, carrying out immunofluorescent staining by using a VIP antibody, quantitatively calculating the density of cholinergic nerve fibers labeled by the sweat gland VIP antibody in dermis by using Image J software, analyzing whether the cholinergic nerve fibers have differences in the two groups of receptors, and if so, determining whether the cholinergic nerve fibers have differences in the two groups of receptors. Evaluating the nervous injury and severity of the cholinergic nerve; the invention provides a new way for accurately evaluating the autonomic nerve injury and severity of the neuronal intracellular inclusion body disease.

Description

Technical Field

[0001] The present invention belongs to the field of biological science technology, and relates to a method for quantitatively detecting cholinergic nerves in sweat glands with neuronal intranuclear inclusion disease (a method for quantitatively detecting cholinergic nerve damage in sweat glands with neuronal intranuclear inclusion disease); specifically, it relates to a technology for quantitatively and accurately detecting cholinergic nerve damage in patients with neuronal intranuclear inclusion disease. Background Art

[0002] Neuronal intranuclear inclusion disease is a rare, slowly progressive neurodegenerative disease with highly heterogeneous clinical manifestations. Sudoresis can be the initial symptom or occur at different stages of the disease course, seriously reducing the patient's quality of life. Sympathetic nerves that control sweat gland secretion include cholinergic nerves and adrenergic nerves. There are few related detection technologies available. Skin sympathetic nerve response is mainly used to detect sympathetic postganglionic C-type nerve fibers, but it is affected by skin temperature, stimulation intensity and patient adaptability, and the positioning significance is unclear, and it is impossible to distinguish between cholinergic nerves and adrenergic nerves. Sudoresis nerve function testing is easy to operate and inexpensive, but it is unclear whether it directly or simultaneously stimulates sudomotor nerve fibers or sweat glands. Thermoregulation sweat test is used to evaluate overall sweat function, but this technology has not yet been widely used in China. Therefore, in order to detect sweat gland nerve damage early and intervene as soon as possible, it is of great significance to find a simple and inexpensive method to quantitatively evaluate the status of sweat gland nerves. Summary of the invention

[0003] In view of the above problems, the purpose of the present invention is to propose a quantitative detection method for cholinergic nerves in sweat glands of neuronal intranuclear inclusion disease, aiming to develop a simple and easy method for quantitatively evaluating cholinergic nerve fibers in neuronal intranuclear inclusion disease; skin biopsy is performed on neuronal intranuclear inclusion disease and control receptors without neurological diseases, and VIP antibody is used for immunofluorescence staining after sectioning, and Image J software is used to quantitatively calculate the density of cholinergic nerve fibers labeled with VIP antibody in the sweat glands in the dermis, analyze whether there is a difference between the two groups of receptors, and evaluate the damage and severity of cholinergic nerves.

[0004] The technical solution of the present invention is: a quantitative detection method for sweat gland cholinergic nerves of neuronal intranuclear inclusion disease described in the present invention, the operation steps of which are as follows:

[0005] 1. Skin biopsy:

[0006] a. Inclusion of matched control recipients with neuronal intranuclear inclusion disease and without neurological disease;

[0007] b. After applying 2% lidocaine for local anesthesia, use a skin biopsy needle to obtain a sample 10 cm above the wrist on the outer side of the forearm, with a skin diameter of 3 mm;

[0008] c. The specimens were immediately immersed in 4% paraformaldehyde overnight, dehydrated with 25% sucrose solution, and then embedded in OCT embedding medium;

[0009] 2. Immunohistochemical staining:

[0010] a. Use a freezing microtome to discontinuously slice the skin sample with a thickness of 30 μm;

[0011] b. The sections were incubated overnight with the corresponding primary antibodies; the primary antibodies included mouse PGP9.5 antibody (Abcam Cat#ab72911, RRID: ab_1269733) for marking myelinated and unmyelinated nerve fibers and rabbit vasoactive intestinal peptide antibody (VIP) (ImmunoStar Cat#20077, RRID:AB_572270) for marking sweat gland cholinergic fibers;

[0012] c. Sections were incubated with secondary antibodies, including anti-rabbit Alexa Fluor 594 antibody (Abcam Cat#ab150080, RRID:ab_2650602) and anti-mouse Alexa Fluor 488 antibody (Abcam Cat#ab150105, RRID:ab_2732856);

[0013] d. Add DAPI (Abcam Cat#ab104139, RRID: no) to label cell nuclei;

[0014] 3. Slide scanning:

[0015] a. Use confocal microscopy to collect three-dimensional digital images, identify sweat glands based on DAPI and PGP 9.5 staining and circle them as regions of interest;

[0016] b. Set scanning parameters based on the optimal ratio of signal intensity to noise of primary antibody staining in the control slides, and apply these parameters to all scanned slides; the inter-slice spacing is 2 μm and the magnification is ×200;

[0017] 4. Calculate the cholinergic nerve fiber density:

[0018] a. Image J software was used to manually circle the sweat gland region of interest based on the merged image stained with three antibodies: DAPI, PGP 9.5 (green) (a) and VIP (red) (g);

[0019] b. Convert the original fluorescence images stained with PGP 9.5 antibody and VIP antibody into grayscale images respectively;

[0020] c. removing the background from the grayscale image to obtain a base image, blurring the base image to obtain a defocused image, and subtracting the defocused image from the base image to obtain a composite image;

[0021] d. Intersection of the two composite images labeled with PGP 9.5 and VIP antibodies;

[0022] e. Adjust the light threshold to highlight the nerve fibers and obtain the actual cholinergic nerve fibers labeled with VIP antibodies;

[0023] f. Calculate the actual area of ​​cholinergic nerve fibers labeled with VIP antibodies by dividing the area of ​​the sweat gland region of interest in step a, that is, the density of cholinergic nerve fibers labeled with VIP antibodies in the sweat glands;

[0024] g. Calculate the average cholinergic nerve fiber density of the three sweat glands of each receptor, that is, the cholinergic nerve fiber density of the sweat glands in the dermis of each receptor.

[0025] The beneficial effects of the present invention are as follows: the present invention develops a simple and feasible method for quantitatively evaluating cholinergic nerve fibers in neuronal nuclear inclusion disease; skin biopsy is performed on neuronal nuclear inclusion disease patients and control recipients without nervous system diseases, and immunofluorescence staining is performed using VIP antibody after slicing, and the density of cholinergic nerve fibers labeled by VIP antibody in the sweat glands in the dermis is quantitatively calculated using Image J software, and whether there is a difference between the two groups of receptors is analyzed, and cholinergic nerve damage and severity are evaluated; the present invention provides a new way to accurately evaluate autonomic nerve damage and severity in neuronal nuclear inclusion disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the quantitative method for cholinergic nerve fiber innervation of sweat glands of control subjects in an embodiment of the present invention; manually selecting the sweat gland region of interest based on the merged image stained with DAPI, PGP 9.5 and VIP three antibodies; converting the original images showing PGP9.5 and VIP staining into grayscale images (a, f) respectively; removing the background from the grayscale images (a, f) to obtain the base images (b, g), blurring to obtain the defocused images (c, h), subtracting the defocused images (c, h) from the base images (b, g) to obtain the composite images (d, i); adjusting the light threshold to highlight the PGP 9.5 antibody labeled nerve fibers (e); vasoactive intestinal peptide is also present in sweat gland cells, increasing nonspecific cell staining; considering that the cholinergic nerves labeled with VIP antibody are closely related to PGP 9.5 The myelinated and unmyelinated nerves stained with antibodies overlap, and the intersection of the two composite images (d, i) is taken, and the light threshold is adjusted to highlight the actual cholinergic nerve fibers (j); the density of cholinergic nerve fibers in the sweat gland VIP is 2.83%, scale bar = 100 μm, magnification: ×200;

[0027] Figure 2 It is a statistical diagram (mean ± standard deviation) of the cholinergic nerve fibers innervated by VIP antibodies of NIID and CON receptor sweat glands in the embodiment of the present invention; quantitative analysis of the density of VIP cholinergic nerve fibers revealed that NIID receptors were indeed lower than control receptors; **: p<0.01. DETAILED DESCRIPTION

[0028] The specific technical scheme of the present invention is further described in detail below with reference to specific examples.

[0029] As shown in the figure, the present invention included 20 matched subjects with neuronal intranuclear inclusion disease and 21 control subjects without neurological diseases. After local anesthesia with 2% lidocaine, skin biopsy needles were used to obtain samples at the distal end of the upper limb (10 cm above the wrist on the lateral side of the forearm), with a skin diameter of 3 mm;

[0030] The specimens were immediately immersed in 4% paraformaldehyde universal tissue fixative overnight, dehydrated in 25% sucrose solution, and then embedded in OCT embedding medium;

[0031] Skin samples were sectioned discontinuously with a thickness of 30 μm using a freezing microtome, and the sections were incubated with the corresponding primary antibodies overnight;

[0032] The primary antibodies included mouse PGP 9.5 antibody (Abcam Cat# ab72911, RRID: ab_1269733) that labels myelinated and unmyelinated nerve fibers and rabbit vasoactive intestinal peptide antibody (VIP) (ImmunoStar Cat# 20077, RRID: AB_572270) that labels sweat gland cholinergic fibers;

[0033] Sections were incubated with secondary antibodies, including anti-rabbit Alexa Fluor 594 antibody (Abcam Cat#ab150080, RRID:ab_2650602) and anti-mouse Alexa Fluor 488 antibody (Abcam Cat#ab150105, RRID:ab_2732856), and then DAPI (Abcam Cat#ab104139, RRID:no) was added to label cell nuclei; three-dimensional digital images were acquired using confocal microscopy with a slice spacing of 2 μm and a magnification of ×200;

[0034] Scanning parameters were set based on the optimal ratio of signal intensity to noise of primary antibody staining in the control slides, and these parameters were applied to all scanned slides;

[0035] Image J software was used for analysis, and sweat glands were identified based on staining with DAPI and PGP 9.5;

[0036] Image J software was used to quantitatively calculate the density of cholinergic nerve fibers labeled with VIP antibody in sweat glands, i.e., the ratio of nerve fiber area labeled with VIP antibody to sweat gland area;

[0037] Subtracting the defocused image of the sweat gland from the base image to create a composite image to highlight the nerve fibers; because vasoactive intestinal peptide is also present in sweat gland cells, it increases nonspecific cell staining;

[0038] Because vasoactive intestinal peptide is also present in sweat gland cells, increasing nonspecific cell staining, and the cholinergic nerves labeled by the VIP antibody overlap with the myelinated and unmyelinated nerves labeled by the PGP 9.5 antibody, the present invention takes the intersection of the two composite images labeled by the PGP 9.5 and VIP antibodies, and adjusts the light thresholds respectively to highlight the nerve fibers, thereby obtaining the actual cholinergic nerve fibers labeled by the VIP antibody;

[0039] The density of cholinergic nerve fibers labeled by VIP antibody in sweat glands was quantitatively calculated, i.e., the ratio of nerve fiber area labeled by VIP antibody to sweat gland area;

[0040] The cholinergic nerve fiber density of three sweat glands per receptor is usually calculated as the average value.

[0041] The experimental operation is as follows:

[0042] (1): The present invention included 20 matched patients with neuronal intranuclear inclusion disease and 21 controls without neurological diseases, and performed skin biopsies;

[0043] (2) Acquisition of specimens:

[0044] (a): Prepare the items needed for sampling: iodine, cotton swabs, sterile gloves, dressing packs, 5 ml syringes, 2% lidocaine, epinephrine hydrochloride injection, 3 mm skin biopsy needles, disposable surgical blades, EP tubes, 4% paraformaldehyde universal tissue fixative, markers, cotton balls, and tape;

[0045] (b): All receptors were sampled after disinfection with iodine-containing cotton swabs;

[0046] (c): Wear sterile gloves, draw 2% lidocaine and a small amount of epinephrine, and perform local anesthesia subcutaneously at the selected site;

[0047] (d) Press and rotate a sterile, 3 mm diameter skin biopsy needle to obtain a sample, with a sampling depth of approximately 3 mm; use a surgical blade to separate the sample;

[0048] (e): bandaging the biopsy site;

[0049] (3): Fixation of specimens:

[0050] (a): Soak the sample in 4% paraformaldehyde universal tissue fixative and store at 4°C for 18-24 hours;

[0051] (b): Wash with 0.01% PBS three times, 5 minutes each time;

[0052] (c): Dehydrated overnight in 25% sucrose solution (25 g sucrose + 100 ml 0.01% PBS) at 4°C;

[0053] (4): Preparation of slices:

[0054] (a): The tissue was embedded in OCT gel in a cryostat (-20°C). The skin epidermis was perpendicular to the mold base and the epidermis position was marked.

[0055] (b): The slice thickness was 30 μm, the epidermis was facing the blade during slicing, the slices were not continuous, and the slices were washed with 0.01% PBS for 5 minutes;

[0056] (5): Immunohistochemical staining:

[0057] (a): Wash with 0.01% PBS three times, 5 minutes each time;

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

[0059] (c): Add 200ul 5% BSA and block for 1 hour at room temperature;

[0060] (d): Primary anti-mouse PGP 9.5 (Abcam Cat#ab72911, RRID: ab_1269733) antibody and rabbit VIP (ImmunoStar Cat#20077, RRID: AB_572270) antibody were diluted with 0.1% BSA to a concentration of 1:500 and 1:200, respectively, at 4°C overnight;

[0061] (e): Wash with 0.3% PBST three times, 5 min each time;

[0062] (f): Fluorescent secondary antibodies anti-rabbit Alexa Fluor 594 (Abcam Cat#ab150080, RRID:ab_2650602) and anti-mouse Alexa Fluor 488 (Abcam Cat#ab150105, RRID:ab_2732856) were diluted in 0.1% BSA at a concentration of 1:500 and incubated at room temperature in the dark for 2 hours;

[0063] (g): Wash with 0.01% PBS three times, 5 min each time;

[0064] (h): Patching, adding 1-2 drops of DAPI (Abcam Cat#ab104139, RRID:no), sealing, and storing at 4°C;

[0065] (6): Slide scanning:

[0066] (a): Three-dimensional digital images were collected using confocal microscopy, and sweat glands were identified based on staining with DAPI and PGP 9.5.

[0067] (b): Scanning parameters were set according to the optimal ratio of signal intensity to noise of primary antibody staining in the control slides, and these parameters were applied to all scanned slides; the inter-slice spacing was 2 μm and the magnification was ×200;

[0068] (7): Calculation of cholinergic nerve fiber density:

[0069] (a): Image J software was used to manually circle the sweat gland region of interest based on the merged image stained with three antibodies: DAPI, PGP 9.5 (green) (a) and VIP (red) (g);

[0070] (b): The original fluorescence images showing myelinated and unmyelinated nerve fibers stained with PGP 9.5 antibody and cholinergic nerve fibers labeled with VIP antibody were converted into grayscale images respectively;

[0071] (c) The background of the grayscale image is removed to obtain a base image, the base image is blurred to obtain a defocused image, and the defocused image is subtracted from the base image to obtain a composite image;

[0072] (d): The two composite images labeled with PGP 9.5 and VIP antibodies were intersected, and the light thresholds were adjusted to highlight the nerve fibers, respectively, to obtain the actual cholinergic nerve fibers labeled with VIP antibodies;

[0073] (e): Calculate the actual area of ​​cholinergic nerve fibers labeled with VIP antibody and divide it by the area of ​​the sweat gland region of interest in a., i.e., the density of cholinergic nerve fibers labeled with VIP antibody in sweat glands;

[0074] (d) Calculate the average cholinergic nerve fiber density of the three sweat glands of each receptor, that is, the cholinergic nerve fiber density of the sweat glands in the dermis of each receptor.

Claims

1. A quantitative detection method for cholinergic nerves in sweat glands with neuronal intranuclear inclusion disease, characterized in that: The operation steps are as follows: (1): Skin biopsy; (2): Immunohistochemical staining; (3): Slide scanning; (4): Calculate the cholinergic nerve fiber density.

2. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step (1), the specific steps of the skin biopsy are as follows: a. Inclusion of matched control recipients with neuronal intranuclear inclusion disease and without neurological disease; b. After applying 2% lidocaine for local anesthesia, use a skin biopsy needle to obtain a sample 10 cm above the wrist on the outer side of the forearm; c. The specimens were immersed in 4% paraformaldehyde overnight, dehydrated with 25% sucrose solution, and then embedded in OCT embedding medium.

3. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 2, characterized in that: In step b, the diameter of the skin sample is 3 mm.

4. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step (2), the specific steps of the immunohistochemical staining are as follows: a. Use a freezing microtome to discontinuously slice the skin sample; b. Incubate the sections with the corresponding primary antibodies overnight; c. Incubate sections with secondary antibodies; d. Add DAPI to label cell nuclei.

5. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 4, characterized in that: In step a, the thickness of the slice is 30 μm.

6. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 4, characterized in that: In step b, the primary antibodies include mouse PGP 9.5 antibody for labeling myelinated and unmyelinated nerve fibers and rabbit VIP antibody for labeling sweat gland cholinergic fibers.

7. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 4, characterized in that: In step c, the secondary antibodies include anti-rabbit Alexa Fluor 594 antibody and anti-mouse Alexa Fluor 488 antibody.

8. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 4, characterized in that: In step (3), the specific operation steps of the slide scanning are as follows: a. Three-dimensional digital images were acquired using confocal microscopy, and sweat glands were identified based on staining with DAPI and PGP 9.

5. b. Set scanning parameters based on the optimal ratio of signal intensity to noise of primary antibody staining in the control slides, and apply the above parameters to all scanned slides.

9. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 8, characterized in that: In step b, the interlayer spacing of the scanning slide is 2 μm and the magnification is ×200.

10. The method for quantitatively detecting cholinergic nerves of sweat glands in neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step (4), the specific operation steps for calculating the cholinergic nerve fiber density are as follows: a. Use Image J software to manually circle the sweat gland region of interest based on the merged image stained with DAPI, PGP 9.5 and VIP antibodies; b. Convert the original fluorescence images stained with PGP 9.5 antibody and VIP antibody into grayscale images respectively; c. removing the background from the grayscale image to obtain a base image, blurring the base image to obtain a defocused image, and subtracting the defocused image from the base image to obtain a composite image; d. The two composite images labeled with PGP 9.5 and VIP antibodies were intersected, and the light thresholds were adjusted to highlight the nerve fibers, respectively, to obtain the actual cholinergic nerve fibers labeled with VIP antibodies; e. Calculate the actual area of ​​cholinergic nerve fibers labeled with VIP antibodies divided by the area of ​​the sweat gland region of interest in step a, that is, the density of cholinergic nerve fibers labeled with VIP antibodies in the sweat glands; d. Calculate the average cholinergic nerve fiber density of the three sweat glands of each receptor, that is, the cholinergic nerve fiber density of the sweat glands in the dermis of each receptor.

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