A method for quantitatively detecting cholinergic nerves in sweat glands of neuronal intranuclear inclusion disease

Abstract

The present invention discloses a method for quantitatively detecting cholinergic nerves in sweat glands of neuronal intranuclear inclusion disease; belonging to the field of biological science and technology, the operation steps are as follows: skin biopsy; immunohistochemical staining; slide scanning; calculating the density of cholinergic nerve fibers. The method described in the present invention is a simple and easy method for quantitatively evaluating cholinergic nerve fibers in neuronal intranuclear inclusion disease; skin biopsies are performed on control recipients with neuronal intranuclear inclusion disease and without neurological diseases. After sectioning, immunofluorescence staining is performed using VIP antibody, and Image J software is used to quantitatively calculate the density of cholinergic nerve fibers labeled with VIP antibody in the dermal sweat glands, analyze whether there are differences in the two groups of recipients, and evaluate cholinergic nerve injury and severity; the present invention provides a new way for accurately evaluating the autonomic nerve injury and severity of neuronal intranuclear inclusion disease.

Description

Technical Field

[0001] The present invention belongs to the field of biological science and technology, and relates to a method for quantitatively detecting cholinergic nerves in the sweat glands of neuronal intranuclear inclusion disease (a method for quantitatively detecting cholinergic nerve damage in the sweat glands of neuronal intranuclear inclusion disease); specifically, it relates to a technique 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 and slowly progressive neurodegenerative disease, and its clinical manifestations are highly heterogeneous. Sweating dysfunction can be the initial symptom or can appear at different stages of the disease course, severely reducing the quality of life of patients. The sympathetic nerves that innervate sweat gland secretion include cholinergic nerves and adrenergic nerves. There are not many existing related detection techniques. Skin sympathetic nerve responses are mainly used to detect postganglionic C-type nerve fibers of the sympathetic nerves, but they are affected by skin temperature, stimulation intensity, and patient adaptability, and their localization significance is not clear, and it is impossible to distinguish cholinergic nerves from adrenergic nerves. The detection of sweating nerve function is easy to operate and inexpensive, but it is not clear whether it directly or simultaneously stimulates sweating nerve fibers or sweat glands. The thermoregulatory sweating test is used to evaluate the overall sweating function, and this technique has not been widely carried out in China yet. Therefore, in order to detect sweat gland nerve damage at an early stage and intervene as early as possible, it is of great significance to find a simple and inexpensive method to quantitatively evaluate the state of sweat gland nerves. Summary of the Invention

[0003] In view of the above problems, the object of the present invention is to propose a method for quantitatively detecting cholinergic nerves in the 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; perform skin biopsies on control recipients with neuronal intranuclear inclusion disease and without nervous system diseases, after sectioning, perform immunofluorescence staining with VIP antibody, use Image J software to quantitatively calculate the density of cholinergic nerve fibers labeled with VIP antibody in the sweat glands in the dermis, analyze whether there are differences in the two groups of recipients, and evaluate cholinergic nerve damage and its severity.

[0004] The technical solution of the present invention is as follows: A method for quantitatively detecting cholinergic nerves in the sweat glands of neuronal intranuclear inclusion disease according to the present invention comprises the following operating steps:

[0005] 1. Skin biopsy:

[0006] a. Include matched control recipients with neuronal intranuclear inclusion disease and without nervous system diseases;

[0007] b. After local anesthesia with 2% lidocaine, use a skin biopsy needle to obtain a sample at 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 in 25% sucrose solution, and then embedded in OCT compound;

[0009] 2. Immunohistochemical staining:

[0010] a. The skin samples were sectioned discontinuously using a cryostat at a thickness of 30 μm;

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

[0012] c. The sections were incubated with secondary antibodies, and the secondary antibodies included 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. DAPI (Abcam Cat#ab104139, RRID: no) labeled with cell nuclei was added;

[0014] 3. Slide scanning:

[0015] a. Three-dimensional digital images were acquired using a confocal microscope, and sweat glands were identified and circled as regions of interest according to the staining of DAPI and PGP 9.5;

[0016] b. The scanning parameters were set according to the optimal ratio of the signal intensity to noise of the primary antibody staining in the control group slides, and these parameters were applied to all scanned slides; the layer spacing was 2 μm and the magnification was ×200;

[0017] 4. Calculating the density of cholinergic nerve fibers:

[0018] a. Using Image J software, regions of interest of sweat glands were manually circled according to the merged images stained with 3 kinds of antibodies, namely DAPI, PGP 9.5 (green) (a), and VIP (red) (g);

[0019] b. The original fluorescence images stained with PGP 9.5 antibody and VIP antibody were respectively converted into grayscale images;

[0020] c. Remove the background from the grayscale image to obtain a basic image. After blurring the basic image, obtain a defocused image. Subtract the defocused image from the basic image to obtain a composite image;

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

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

[0023] f. Calculate the area of the actual 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 sweat glands;

[0024] g. Calculate the average density of cholinergic nerve fibers in 3 sweat glands of each receptor, that is, the density of cholinergic nerve fibers in the intradermal sweat glands of each receptor.

[0025] The beneficial effects of the present invention are as follows: The present invention develops a simple and easy method for quantitatively evaluating cholinergic nerve fibers in neuronal intranuclear inclusion disease. Skin biopsies are performed on control subjects with neuronal intranuclear inclusion disease and those without nervous system diseases. After sectioning, immunofluorescence staining is performed using VIP antibodies. The density of cholinergic nerve fibers labeled with VIP antibodies in the intradermal sweat glands is quantitatively calculated using Image J software, and whether there are differences between the two groups of receptors is analyzed to evaluate cholinergic nerve injury and its severity. The present invention provides a new way to accurately evaluate autonomic nerve injury and its severity in neuronal intranuclear inclusion disease. Description of the Drawings

[0026] Figure 1 It is a schematic diagram of the quantitative method for cholinergic nerve fiber innervation of sweat glands in the control subjects in the embodiments of the present invention. Manually select the sweat gland region of interest according to the merged images stained with DAPI, PGP 9.5 and VIP antibodies. Convert the original images showing PGP9.5 and VIP staining into grayscale images (a, f) respectively. Remove the background from the grayscale images (a, f) to obtain basic images (b, g), obtain defocused images (c, h) through blurring processing, subtract the defocused images (c, h) from the basic images (b, g) to obtain composite images (d, i). Adjust the light threshold to highlight the nerve fibers labeled with PGP 9.5 antibodies (e). Vasoactive intestinal peptide also exists in sweat gland cells, increasing non-specific cell staining. Considering that the cholinergic nerves labeled with VIP antibodies coincide with the myelinated and unmyelinated nerves stained with PGP 9.5 antibodies, take the intersection of the two composite images (d, i), and adjust the light threshold to highlight the actual cholinergic nerve fibers (j). The density of cholinergic nerve fibers of VIP in sweat glands in the figure is 2.83%, scale bar = 100μm, magnification: ×200;

[0027] Figure 2 This is a statistical chart (mean ± standard deviation) of the innervation of cholinergic nerve fibers labeled with VIP antibody in NIID and CON receptors in the embodiments of the present invention; quantitative analysis of the density of VIP cholinergic nerve fibers found that the NIID receptor was indeed lower than the control receptor; **: p < 0.01. Detailed implementation manners

[0028] The following further elaborates on the specific technical solutions of the present invention in conjunction with specific examples.

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

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

[0031] The skin samples were sectioned discontinuously using a cryostat, with a thickness of 30 μm, 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) for labeling myelinated and unmyelinated nerve fibers and rabbit vasoactive intestinal peptide antibody (VIP) (ImmunoStar Cat#20077, RRID: AB_572270) for labeling cholinergic fibers of sweat glands.

[0033] The 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) for labeling cell nuclei was added; three-dimensional digital images were collected using a confocal microscope, with a layer spacing of 2 μm and a magnification of ×200.

[0034] The scanning parameters were set according to the best ratio of the signal intensity to noise of the primary antibody staining in the control group glass slides, and these parameters were applied to all scanned glass slides.

[0035] Analysis was performed using Image J software, and sweat glands were identified based on the staining of DAPI and PGP 9.5.

[0036] The density of cholinergic nerve fibers labeled with VIP antibody in sweat glands was quantitatively calculated using Image J software, which is the ratio of the area of nerve fibers labeled with VIP antibody to the area of sweat glands;

[0037] The defocused image of the sweat gland was subtracted from its base image to obtain a composite image to highlight the nerve fibers; since vasoactive intestinal peptide is also present in sweat gland cells, non-specific cell staining was increased;

[0038] Since vasoactive intestinal peptide is also present in sweat gland cells, non-specific cell staining was increased, and the cholinergic nerves labeled with VIP antibody overlapped with the myelinated and unmyelinated nerves labeled with PGP 9.5 antibody. Therefore, in this invention, the intersection of two composite images labeled with PGP 9.5 and VIP antibodies was taken, and the light threshold was adjusted respectively to highlight the nerve fibers, and the actual cholinergic nerve fibers labeled with VIP antibody were obtained;

[0039] The density of cholinergic nerve fibers labeled with VIP antibody in sweat glands was quantitatively calculated, which is the ratio of the area of nerve fibers labeled with VIP antibody to the area of sweat glands;

[0040] Generally, the average value of the density of cholinergic nerve fibers in 3 sweat glands of each subject was calculated.

[0041] The experimental operations are as follows:

[0042] (1): Twenty cases of neuronal intranuclear inclusion disease and 21 control subjects without neurological diseases were included in this invention for matching, and skin biopsies were performed;

[0043] (2): Acquisition of specimens:

[0044] (a): Prepare the items required for sampling: iodophor, cotton swabs, sterile gloves, dressing change kit, 5 ml syringe, 2% lidocaine, epinephrine hydrochloride injection, 3 mm skin biopsy needle, disposable surgical blade, EP tube, 4% paraformaldehyde universal tissue fixative, marker pen, cotton balls, tape;

[0045] (b): Disinfect with iodophor cotton swabs and take samples from all subjects;

[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 for sampling, and the sampling depth is about 3 mm; use a surgical blade to separate the sample;

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

[0049] (3): Fixation of specimens:

[0050] (a): Immerse the collected samples in a 4% paraformaldehyde general 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): Immerse in 25% sucrose solution (25 g sucrose + 100 ml 0.01% PBS) and dehydrate overnight at 4°C;

[0053] (4): Preparation of sections:

[0054] (a): Operate in a cryostat (-20°C), embed the tissue with OCT compound, keep the skin epidermis perpendicular to the mold base, and mark the position of the epidermis;

[0055] (b): The section thickness is 30 μm. When sectioning, the epidermis layer faces the blade. Cut discontinuous sections and wash 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 with 0.3% PBST three times, 5 minutes each time;

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

[0060] (d): Dilute the primary antibodies anti-mouse PGP 9.5 (Abcam Cat#ab72911, RRID: ab_1269733) and rabbit VIP (ImmunoStar Cat#20077, RRID: AB_572270) with 0.1% BSA at concentrations of 1:500 and 1:200 respectively, and incubate overnight at 4°C;

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

[0062] (f): Dilute the 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) with 0.1% BSA at a concentration of 1:500, and incubate in the dark at room temperature for 2 hours;

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

[0064] (h): Apply 1 - 2 drops of DAPI (Abcam Cat#ab104139, RRID:no) to the patch, cover the slide, and store at 4°C;

[0065] (6): Slide scanning:

[0066] (a): Use a confocal microscope to acquire three - dimensional digital images, and identify sweat glands based on the staining of DAPI and PGP 9.5;

[0067] (b): Set the scanning parameters according to the best ratio of the signal intensity to noise of the primary antibody staining in the control group slides, and apply these parameters to all scanned slides; the layer spacing is 2μm, and the magnification is ×200;

[0068] (7): Calculate the cholinergic nerve fiber density:

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

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

[0071] (c): Remove the background from the grayscale image to obtain a basic image, blur the basic image to obtain a defocused image, and subtract the defocused image from the basic image to obtain a composite image;

[0072] (d): Take the intersection of the two composite images labeled with PGP 9.5 and VIP antibodies, and adjust the light threshold respectively to highlight the nerve fibers, to obtain the actual cholinergic nerve fibers labeled with VIP antibody;

[0073] (e): Calculate the area of the actual cholinergic nerve fibers labeled with VIP antibody divided by the area of the region of interest of sweat glands in a., that is, the cholinergic nerve fiber density of sweat glands labeled with VIP antibody;

[0074] (d): Calculate the average value of the cholinergic nerve fiber density of 3 sweat glands of each receptor, that is, the cholinergic nerve fiber density of dermal sweat glands 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, the steps 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, a skin biopsy needle was used to obtain a sample 10 cm above the lateral forearm wrist; the diameter of the sampled skin was 3 mm; c. The specimens were immersed in 4% paraformaldehyde overnight, dehydrated with 25% sucrose solution, and then embedded in OCT embedding medium; (2): Immunohistochemical staining; The steps 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 mark the cell nucleus; (3): Slide scanning; The steps 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. Setting scanning parameters according to the optimal ratio of signal intensity to noise of primary antibody staining in the control slides, and applying the above parameters to all scanned slides; (4): Calculate the cholinergic nerve fiber density; The steps 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; Wherein, the primary antibodies include mouse PGP 9.5 antibody for marking myelinated and unmyelinated nerve fibers and rabbit VIP antibody for marking sweat gland cholinergic fibers; The secondary antibodies included anti-rabbit Alexa Fluor 594 antibody and anti-mouse Alexa Fluor 488 antibody.

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 a of step (2), the thickness of the slice is 30 μm.

3. The method for quantitatively detecting cholinergic nerves of sweat glands with neuronal intranuclear inclusion disease according to claim 1, characterized in that: In step b of step (3), the interlayer spacing of the scanning slide is 2 μm and the magnification is ×200.

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