Method for flaking eyeballs of primates
By cutting the line connecting the optic disc and the macula in the sampling aid, and combining the use of fixative and embedding locator, the problem of macular area loss in primate eyes during slide preparation was solved, and the optic disc center and macula center were located in the same field of vision, improving slide preparation efficiency and reducing costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-03-24
AI Technical Summary
The macula of primate eyes is easily lost during slide preparation, and the center of the optic disc and the center of the macula are difficult to locate in the same field of vision, resulting in missing pathological diagnostic data and increased human and economic costs.
A method for preparing primate eyeballs was developed. By cutting along the line connecting the optic disc and the center of the macula in a sampling aid, the center of the optic disc and the center of the macula are in the same field of vision. Combined with the use of fixative and embedding positioning device, the accuracy and efficiency of sampling are improved.
It improved the macular film yield, reduced the macular loss rate, significantly improved film production efficiency, and reduced costs.
Smart Images
Figure CN121720799A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for preparing a primate eyeball. Background Technology
[0002] For routine paraffin slide preparation of ocular tissue, the tissue is usually first fixed using Davidson's fixative, modified Davidson's fixative, 4% paraformaldehyde or Bouin fixative, and then the ocular tissue is harvested, dehydrated, embedded, sectioned, stained and prepared.
[0003] Currently, the pathological preparation of macula in primate eyes usually involves routine sampling, dehydration, and embedding of the eyeball. The macula is located by slicing the macula continuously, and then the slices containing the macula are stained. Compared to animals with smaller eyeballs, primates have larger eyeballs, requiring more manpower, time, and economic costs for slicing. Moreover, since the diameter of the fovea of the macula is only about 250 μm, and there is only one in each eyeball, it is almost impossible to identify with the naked eye when slicing paraffin tissue of the eyeball, and it is very easy to lose it, resulting in the loss of pathological diagnostic data and affecting the final macular research. In addition, in the examination of eyeball pathological sections, placing the center of the optic disc and the center of the macula in the same field of vision is an important indicator: (1) The macula and the optic disc have a fixed relative positional relationship on the retina. If the two are not in the same field of vision, it is impossible to determine the position of the macula. Whether the anatomical structure of the slice is accurately located, (2) Many eye diseases (such as macular degeneration, glaucoma, retinal detachment) involve the area between the macula and the optic disc. When the two are in the same field of vision, it is easier to locate and measure the lesion more accurately and efficiently. (3) In clinical fundus photography, OCT and other examinations, the optic disc and macula are usually displayed at the same time. The pathological slice should also be displayed accordingly so as to compare and analyze with the imaging data. Therefore, placing the center of the optic disc and the center of the macula in the same field of vision is a key step to ensure that the pathological slice is oriented correctly, structurally complete and accurately located. It is also an important basis for comparing with clinical images and judging whether the anatomy of the posterior pole of the eyeball is normal.
[0004] Therefore, there is a need in this field to study a slide preparation method that can improve the detection rate of the macular region in primate eyes, ensure the simultaneous detection of the optic disc center and the macular center, and reduce slide preparation costs, so as to meet the needs of ophthalmic drug development and the diagnosis of ophthalmic diseases. Summary of the Invention
[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing primate eyeballs.
[0006] This invention provides a method for preparing a primate eyeball, comprising: Step 1: Remove the eyeball and inject fixative into the vitreous humor to fix the eyeball in place; Step 2: Divide the fixed eyeball in half along the limbus, place the vitreous part of the eyeball in the sampling aid and cut it along the sagittal plane to collect the sample; The sampling aid contains at least two sets of parallel cutting grooves. The sampling step includes: placing the area containing the optic disc and macula between the two sets of cutting grooves, making the line connecting the center of the optic disc and the center of the macula parallel to the cutting grooves, cutting along the cutting grooves with blades, and taking out the middle part containing the optic disc and macula as the sample. Step 3: The collected samples are dehydrated, embedded, sectioned, and stained.
[0007] Preferably, in step 1, after removing the eyeball, 0.5 to 1 cm of optic nerve is preserved, and connective tissue and muscle tissue outside the eyeball are removed.
[0008] Preferably, in step 1, the step of injecting fixative into the vitreous body of the eyeball includes: injecting 0.2~0.5ml of fixative into the vitreous body of the eyeball from the limbus; And / or, the step of fixing the eyeball includes: fixing the eyeball in a fixative solution of at least 5 times the volume of the eyeball for more than 48 hours, and then fixing it in 70% ethanol for at least 12 hours; And / or, the fixative is selected from Davidson's fixative, modified Davidson's fixative, Bouin fixative, 4 w / v % paraformaldehyde solution or 10 v / v % neutral buffered formalin fixative.
[0009] Preferably, in step 2, the division into two parts specifically means dividing into front and back parts; the vitreous body of the eyeball is pre-removed of the vitreous body.
[0010] Preferably, in step 2, the sampling aid includes: a base (1), an eyeball placement groove (2) is provided in the center of the top of the base (1), the eyeball placement groove (2) is hemispherical, and an optic nerve avoidance groove (3) is provided in the center of the bottom of the eyeball placement groove (2); two sets of first grooves (4) are provided parallel to each other on the top of the base (1), the two sets of first grooves (4) are symmetrically arranged on both sides of the center of the eyeball placement groove (2), the first grooves (4) penetrate the eyeball placement groove (2) and both ends penetrate the side wall of the base (1), and the bottom of the first grooves (4) exceeds the lowest point of the eyeball placement groove (2).
[0011] Preferably, the top of the base (1) is also provided with two sets of second grooves (5) in parallel. The direction of the second grooves (5) is perpendicular to the first groove (4). The two sets of second grooves (5) are symmetrically arranged on both sides of the center of the eyeball placement groove (2). The second grooves (5) penetrate the eyeball placement groove (2) and both ends penetrate the side wall of the base (1). The bottom of the second grooves (5) exceeds the lowest point of the eyeball placement groove (2). The distance between the two sets of second grooves (5) is different from the distance between the two sets of first grooves (4).
[0012] Preferably, the distance between the two sets of the first grooves (4) is 2~3mm.
[0013] Preferably, the distance between the two sets of the second grooves (5) is 3~5mm; and / or, the width of the groove gap is 0.3~0.5mm.
[0014] Preferably, in step 3, the embedding process includes: placing the sample into an embedding dish, adding paraffin wax, placing a macular locator to locate the macular area, and when the paraffin wax is semi-solidified, removing the locator, continuing to add liquid paraffin wax to fill the embedding dish, and waiting for the tissue wax block to cool.
[0015] Preferably, in step 3, the dehydration step includes: 70% ethanol for 30-45 min, 80% ethanol for 30-60 min, 90% ethanol for 30-60 min, 95% ethanol for 30-60 min, 95% ethanol for 45-60 min, anhydrous ethanol for 45-60 min, and anhydrous ethanol for 45-60 min. And / or, the dehydration is followed by a transparentizing process, the transparentizing process comprising: immersing the dehydrated sample in xylene, repeating at least 3 times, each time for 45 to 60 minutes; And / or, the sectioning steps include: rough trimming the tissue according to the markings left by the macular locator until the macular position, fine trimming until the cut surface is smooth, and sectioning using a paraffin microtome adjusted to a section thickness of 3μm~4μm.
[0016] The "sagittal plane" refers to the vertical plane that cuts the eyeball into left and right halves through the optic nerve and the center of the pupil.
[0017] The modified Davidson's Fixative has the following formula: 30% v / v formaldehyde, 15% v / v ethanol, 5% v / v acetic acid, and 50% v / v water; the formaldehyde is formalin stock solution, i.e., 37–40 m / v formaldehyde solution.
[0018] This invention provides a method for preparing primate eyeballs by changing the cutting steps in the sampling process. This method ensures that the center of the optic disc is parallel to the center of the macula in the prepared sample, achieving the same visual field during sample examination. This improves the macular recovery rate, reduces the macular loss rate, lowers preparation costs, and significantly improves preparation efficiency.
[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description
[0021] To more clearly illustrate the technical solutions of the embodiments of this application, 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 this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 A schematic diagram of vitreous sampling of the eyeball; Figure 2 A three-dimensional schematic diagram of an eyeball harvesting aid; Figure 3 Top view of the eyeball harvesting aid; reference numerals: 1-base, 2-eyeball placement groove, 3-optic nerve avoidance groove, 4-first incision groove, 5-second incision groove, 6-marker line.
[0023] Figure 4 This is a schematic diagram of the embedding process; Figure 5 Image showing the effect of hematoxylin-eosin (H&E) staining after preparing a macular slide. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely represents selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application. In the following embodiments, reagents and materials not specifically mentioned are commercially available products.
[0025] Example 1: A method for preparing a primate eyeball This embodiment provides a method for preparing a slide of a primate's (cynomolgus monkey or rhesus monkey) eyeball: 1. Dissection: Remove the eyeball from the orbit, leaving about 0.5 cm of the optic nerve intact. Remove excess tissue such as connective tissue and muscle outside the eyeball. Using a 1 ml syringe, slowly and gently inject 0.2-0.5 ml of fixative solution from the corneal limbus into the vitreous part of the eyeball. Place the eyeball in a container containing 5 times the volume of fixative solution for fixation.
[0026] The fixative in this step is selected according to the requirements. For example: 4% paraformaldehyde fixative is selected for subsequent immunofluorescence detection; Davidson's fixative is selected for H&E detection; 10% neutral buffered formalin (NBF) fixative is selected for both H&E staining and immunohistochemistry; and Bouin's fixative is selected for special tissue staining, such as Masson staining. In this example, Davidson's fixative is specifically used.
[0027] 2. After 48 hours of fixation, transfer to 70% ethanol for 12 hours of fixation before sampling.
[0028] 3. First, use a scalpel to divide the eyeball in two along the limbus (e.g., Figure 1 The eyeball is divided into two parts, front and back. Then, ophthalmic forceps and surgical scissors are used to gently remove the vitreous humor from the vitreous portion of the eyeball. The vitreous portion is then placed in an eye sampling aid, and the position of the posterior part of the eyeball is adjusted. The area including the optic disc and macula is placed within the sampling area and then cut. The prepared eyeball sample is placed in an embedding cassette and gently rinsed with tap water.
[0029] Material collection aids, such as Figure 2-3As shown, the base 1 is made of stainless steel. An eyeball placement groove 2 is provided in the center of the top of the base 1. The eyeball placement groove 2 is hemispherical. An optic nerve avoidance groove 3 is provided in the center of the bottom of the eyeball placement groove 2. Two sets of first grooves are provided parallel to each other on the top of the base 1. The two sets of first grooves are symmetrically arranged on both sides of the center of the eyeball placement groove 2. The first groove 4 penetrates the eyeball placement groove 2 and its two ends penetrate the side wall of the base 1. The bottom of the first groove 4 exceeds the lowest point of the eyeball placement groove 2.
[0030] The base 1 is also provided with two sets of second grooves 5 in parallel on the top. The direction of the second grooves 5 is perpendicular to the first groove 4. The two sets of second grooves 5 are symmetrically arranged on both sides of the center of the eyeball placement groove 2. The second grooves 5 penetrate the eyeball placement groove 2 and both ends penetrate the side wall of the base 1. The bottom of the second grooves 5 exceeds the lowest point of the eyeball placement groove 2. The distance between the two sets of second grooves 5 is different from the distance between the two sets of first grooves 4.
[0031] The distance between the two sets of first grooves 4 is 2-3 mm. The distance between the two sets of second grooves 5 is 3-5 mm. Specifically, the distance between the two sets of first grooves 4 is 3 mm, and the distance between the two sets of second grooves 5 is 4 mm. The width of each groove is 0.3-0.5 mm, specifically 0.4 mm. Appropriate grooves can be selected for cutting based on the size of the superior optic disc and macula.
[0032] The bottom of the optic nerve avoidance groove 3, the first incision groove 4, and the second incision groove 5 are provided with drainage holes to facilitate the cleaning of residual tissue and avoid cross-contamination.
[0033] The base 1 is square or cylindrical. Preferably, the top of the base 1 has marking lines 6 at the center points of each of its four sides to facilitate positioning of the eyeball. Preferably, the bottom of the base 1 has a silicone anti-slip pad to ensure the stability of the base during operation.
[0034] The method of using the material collection aid is as follows: (1) Place the pretreated vitreous body of the eyeball in the eyeball placement groove 2, and adjust the position so that the area of the optic disc and macula is located between the two sets of first grooves 4 in the middle, and try to make the line connecting the center of the optic disc and the center of the macula parallel to the first groove 4; or make the area containing the optic disc and macula located between the two sets of second grooves 5, and try to make the line connecting the center of the optic disc and the center of the macula parallel to the second groove 5.
[0035] (2) Cut along the two sets of first grooves 4 or two sets of second grooves 5 parallel to the line connecting the center of the optic disc and the center of the macula, respectively, using a blade. After cutting, take out the middle part containing the optic disc and the macula as a sample.
[0036] 4. Dehydration is carried out according to the following procedure: 70% ethanol for 30 minutes. 80% ethanol for 50 minutes 90% ethanol for 50 minutes 95% ethanol for 50 minutes 95% ethanol for 50 minutes Anhydrous ethanol 50 min Anhydrous ethanol for 60 min.
[0037] 5. The following procedure is used for transparency: Xylene I 40 min Xylene II 40min Xylene III 40 min.
[0038] 6. Wax impregnation: Paraffin I for 60 min Paraffin II 70 min Paraffin III for 80 min.
[0039] 7. Embedding: Place the sample in an embedding dish, add an appropriate amount of paraffin wax, adjust the position, place the macular locator to locate the macular area, and when the paraffin wax is semi-solidified, remove the locator, continue to add liquid paraffin wax to fill the embedding dish, place the embedding cassette, and wait for the tissue wax block to cool ( Figure 4 ).
[0040] 8. Section the embedded eye tissue in paraffin wax. Based on the markings left by the macular locator, roughly trim the tissue down to the macula, then finely trim to make the cut surface smooth. Use a paraffin microtome to adjust the section thickness to 3μm~4μm for sectioning.
[0041] 9. Select the best section from the macular region for staining.
[0042] Comparative Example 1 This comparative example provides an existing method for preparing slides of large animal eyeballs after subretinal injection. The specific steps are as follows: 1. Before removing the eyeball, mark the nasal side of the eyeball. After removal, extract 0.4-0.6 mL of vitreous humor from the large animal's eyeball and immediately inject an equal volume of Davidson's fixative into the vitreous cavity. Then, fix the large animal's eyeball in Davidson's fixative and finally transfer it to formaldehyde for fixation. 2. The anterior corneal tissue is removed. The blood vessel distribution of the eyeball wall can be seen through the cut cornea. Combined with fundus photography, the injection site is determined by observing the location of typical blood vessels in the injection area. Then, tissue is collected.
[0043] 3. Large animal eyeballs are placed in 10% neutral formalin fixative for 1-2 hours, then immersed in deionized water for 5-10 minutes, followed by dehydration, embedding, and sectioning of the eyeballs.
[0044] Existing technologies have located the subretinal injection site, but have not solved the difficulties in preparing macular pathology slides, such as the waste of manpower and consumables, and positioning issues.
[0045] This invention, based on existing techniques for preparing large animal eye slides, improves the procedures for fixation, tissue collection, embedding, and sectioning after dissection, developing a method for preparing slides for macular pathology. This method has been applied to primate macular degeneration research projects, with excellent slide preparation results. Figure 5 As shown, the macula can be clearly detected in the tissue staining image, and the center of the optic disc and the center of the macula are in the same field of view. Compared with the method of Comparative Example 1, the method of the present invention increases the slicing efficiency of this type of research project by more than 50%, and the detection rate of the macula increases from less than 50% to more than 90%.
[0046] Sectioning efficiency equals the final number of usable sections (the number of sections actually collected, unfolded without wrinkles, and structurally intact after HE staining) / sectioning time.
[0047] The detection rate of the macula is equal to the percentage of actual sections (sample size) where the optic disc and macula are cut on the same section relative to the theoretical number of samples tested. For example, if slides are prepared from 20 eye samples, theoretically all 20 slides should be acceptable. If, in actual preparation, 18 slides are acceptable, then the detection rate of the macula is 90%.
[0048] The film preparation method of the present invention has the following advantages: 1. After dissection, directly immersing the eyeball intact in fixation solution can cause the eyeball wall to collapse due to the difference in osmotic pressure between the vitreous humor inside the eye and the inside and outside of the eye, affecting the structural stability of the retina and choroid. Injecting a certain amount of fixation solution into the eyeball before dissection and fixation can accelerate the fixation of the retina and choroid, ensuring the stability and integrity of the structure.
[0049] 2. 12 hours before collection, fix the eyeball in 70% ethanol to increase its rigidity and make it easier to collect the eyeball.
[0050] 3. The macula is located at the posterior pole of the retina and cannot be seen with the naked eye from the outside of the eyeball. The fovea centralis (about 250μm in diameter) is the only coordinate of the macula that can be identified with the naked eye. Therefore, it is necessary to divide the eyeball in half to expose the inside of the eyeball and identify the fovea centralis with the naked eye to determine the macula.
[0051] 4. Eye sampling involves taking a 2-3 mm thick sample from the sagittal plane of the eyeball for slide preparation. Slides one and two must be parallel to the line connecting the fovea centralis and the optic disc. Before this invention, the accuracy of the sampling depended entirely on the skill of the sampling personnel, making it highly reliant on their expertise. By using a sampling aid, the parallelism of slides one and two to the fovea centralis and optic disc line can be ensured, maintaining the integrity of the sampling surface and reducing reliance on the skill of the sampling personnel. This significantly improves the quality of eyeball samples from the optic disc and macula region.
[0052] 5. During embedding, a macular locator is used to locate the position of the macular spot in the paraffin tissue block, which solves the problem that the position of the macular spot cannot be identified by the naked eye during sectioning, improves the accuracy and efficiency of sectioning, and reduces the time and waste of consumables.
[0053] As can be seen from the above embodiments, this invention provides a method for preparing primate eyeballs by changing the material collection method, embedding markers, and slicing and positioning steps. This method improves the macular recovery rate, reduces the macular loss rate, lowers preparation costs, and significantly improves preparation efficiency.
Claims
1. A method for preparing a primate eyeball, characterized in that: It includes: Step 1: Remove the eyeball and inject fixative into the vitreous humor to fix the eyeball in place; Step 2: Divide the fixed eyeball in half along the limbus, place the vitreous part of the eyeball in the sampling aid and cut it along the sagittal plane to collect the sample; The sampling aid contains at least two sets of parallel cutting grooves. The sampling step includes: placing the area containing the optic disc and macula between the two sets of cutting grooves, making the line connecting the center of the optic disc and the center of the macula parallel to the cutting grooves, cutting along the cutting grooves with blades, and taking out the middle part containing the optic disc and macula as the sample. Step 3: The collected samples are dehydrated, embedded, sectioned, and stained.
2. The film preparation method according to claim 1, characterized in that: In step 1, after removing the eyeball, 0.5 to 1 cm of the optic nerve is preserved, and the connective tissue and muscle tissue outside the eyeball are removed.
3. The film preparation method according to Example 1, characterized in that, In step 1, the step of injecting fixative into the vitreous body of the eyeball includes: injecting 0.2~0.5ml of fixative into the vitreous body of the eyeball from the limbus; And / or, the step of fixing the eyeball includes: fixing the eyeball in a fixative solution of at least 5 times the volume of the eyeball for more than 48 hours, and then fixing it in 70% ethanol for at least 12 hours; And / or, the fixative is selected from Davidson's fixative, modified Davidson's fixative, Bouin fixative, 4 w / v % paraformaldehyde solution or 10 v / v % neutral buffered formalin fixative.
4. The film preparation method according to claim 1, characterized in that, In step 2, the division into two parts specifically means dividing the eyeball into two parts, front and back; the vitreous humor of the eyeball is removed beforehand.
5. The film preparation method according to claim 1, characterized in that, In step 2, the sampling aid includes: a base (1), an eyeball placement groove (2) is provided in the center of the top of the base (1), the eyeball placement groove (2) is hemispherical, and an optic nerve avoidance groove (3) is provided in the center of the bottom of the eyeball placement groove (2); two sets of first grooves (4) are provided parallel to each other on the top of the base (1), the two sets of first grooves (4) are symmetrically arranged on both sides of the center of the eyeball placement groove (2), the first grooves (4) penetrate the eyeball placement groove (2) and both ends penetrate the side wall of the base (1), and the bottom of the first grooves (4) exceeds the lowest point of the eyeball placement groove (2).
6. The film preparation method according to claim 5, characterized in that, The base (1) is also provided with two sets of second grooves (5) in parallel on the top. The direction of the second grooves (5) is perpendicular to the first groove (4). The two sets of second grooves (5) are symmetrically arranged on both sides of the center of the eyeball placement groove (2). The second grooves (5) penetrate the eyeball placement groove (2) and both ends penetrate the side wall of the base (1). The bottom of the second grooves (5) exceeds the lowest point of the eyeball placement groove (2). The distance between the two sets of second grooves (5) is different from the distance between the two sets of first grooves (4).
7. The film preparation method according to claim 6, characterized in that: The distance between the two sets of the first grooves (4) is 2~3mm.
8. The film preparation method according to claim 6, characterized in that: The distance between the two sets of the second grooves (5) is 3~5mm; and / or the width of the groove gap is 0.3~0.5mm.
9. The film preparation method according to claim 1, characterized in that, In step 3, the embedding process includes: placing the sample into an embedding dish, adding paraffin wax, placing a macular locator to locate the macular area, and when the paraffin wax is semi-solidified, removing the locator, continuing to add liquid paraffin wax to fill the embedding dish, and waiting for the tissue wax block to cool.
10. The film preparation method according to claim 1, characterized in that, In step 3, the dehydration steps include: 70% ethanol for 30-45 min, 80% ethanol for 30-60 min, 90% ethanol for 30-60 min, 95% ethanol for 30-60 min, 95% ethanol for 45-60 min, anhydrous ethanol for 45-60 min, and anhydrous ethanol for 45-60 min. And / or, the dehydration is followed by a transparentizing process, the transparentizing process comprising: immersing the dehydrated sample in xylene, repeating at least 3 times, each time for 45 to 60 minutes; And / or, the sectioning steps include: rough trimming the tissue according to the markings left by the macular locator until the macular position, fine trimming until the cut surface is smooth, and sectioning using a paraffin microtome adjusted to a section thickness of 3μm~4μm.