A method for preparing a complete retinal flat mount

Abstract

This invention discloses a method for fabricating a complete retinal patch, which can greatly improve the fabrication efficiency of retinal patches, while reducing tissue damage during the fabrication process, facilitating the positioning of various retinal tissues (such as the macula), and reducing curling and folding phenomena that occur during the fabrication process, thus facilitating the implementation of various subsequent experimental techniques.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for fabricating a retinal patch. Background Technology

[0002] Retinal planing can be applied to a variety of scientific research, including quantitative staining of optic ganglion cells, targeted injection into the retina, and electrophysiological and immunohistochemical studies of various retinal tissues. It is an indispensable part of in vivo ophthalmic research. The traditional method of retinal planing is as follows: After removing the mouse eyeball, it is cut 0.5 mm behind the corneal limbus, the cornea and lens are removed, and the retinal tissue is peeled off. At this time, the retina is naturally curled into a spindle shape. The curled retina is placed on a glass slide, and the retina is cut in four directions and gradually flattened.

[0003] Traditional retinal slide preparation methods have several drawbacks: After creating a cloverleaf section of the retina, the step of flipping it so that the nerve fiber layer faces upwards may cause varying degrees of curling or stacking of the four fan-shaped retinal lobes; retinal incision damages blood vessels in the retinal tissue, making it difficult to locate different parts of the retina, requiring researchers to spend considerable time and effort to distinguish the positions of tissues within the retinal slide, which significantly reduces the efficiency of subsequent observation and research; retinal incision also hinders histopathological and molecular biological experiments. For example, when antibody mixtures and washing solutions are applied to the slide, surface tension and other factors often cause the four retinal lobes to curl or flip, frequently resulting in the failure of immunostaining experiments.

[0004] Therefore, in order to ensure the smooth progress of subsequent research, there is a need in this field for a highly efficient method for fabricating retinal patches that can maintain the integrity of the retina. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for fabricating a complete retinal patch, which can greatly improve the fabrication efficiency of retinal patches, while reducing tissue damage during the fabrication process, facilitating the localization of various retinal tissues (such as the macula), and reducing phenomena such as curling and folding that occur during the fabrication process, thus facilitating the implementation of various subsequent experimental techniques.

[0006] Specifically, the present invention provides a method for preparing a complete retinal smear, comprising the following steps: (1) dissecting the eyeball and immersing the retinal tissue in a culture medium with the nerve fiber layer facing upward; (2) placing the glass slide directly along the edge of the culture dish away from the retinal tissue and adjusting its position; (3) removing the culture medium; (4) attaching the flattened retinal smear to the glass slide and maintaining this state for a period of time.

[0007] In some embodiments, the culture medium level in step (1) is deeper than the thickness of the glass slide.

[0008] In some embodiments, the culture medium contains insulin, progesterone solution, sodium selenite, thyroxine T3, forskolin, acetylcysteine, BDNF, CNTF, FGF, GNTF, etc., and the composition can be adjusted according to experimental requirements.

[0009] In some implementations, step (2) involves moving the slide directly beneath the retinal tissue.

[0010] In some embodiments, the culture medium is aspirated in step (3) in small, multiple steps. Further, after each aspiration, an iris restorer is used to slowly adjust the position of the retinal tissue and unroll the curled edges of the retina, ensuring that the rolled-up periphery of the retinal tissue is flattened each time the liquid level is lowered.

[0011] In some implementations, the state is maintained for more than 30 seconds in step (4). Further, the state can be maintained for approximately 2 minutes.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1) Higher Efficiency in Retinal Patch Fabrication: Traditional retinal patch fabrication requires the optic nerve fiber layer of the patch to be placed downwards against the bottom of the culture dish to prevent retinal flap tissue curling. After cutting, the patch usually needs to be flipped so that the optic nerve fiber layer is upwards for observation of RGCs. During this process, upward curling and stacking of the retinal patch often occur. In contrast, this invention uses a "small amount, multiple times" method to remove the culture medium, gradually adjusting the position of the retinal tissue and the retinal edge after each removal, eliminating the need for cutting and flipping. Furthermore, because the retinal patch fabricated using this invention is easier for the operator to control and allows for faster operation, the fabrication efficiency is greatly improved.

[0014] 2) Easier to locate: After traditional retinal smears are cut, the four-lobed retinal tissue damages the original vascular tissue of the retina, making it difficult to locate the macula and other parts. However, this invention does not cut the retinal tissue after dissecting the eyeball. Instead, it uses the liquid tension of the culture medium to gradually flatten the retina, preserving the complete retinal structure. Based on the course of blood vessels, it greatly reduces the time and difficulty of locating the macula and other parts, and shortens the time for subsequent research on retinal smears.

[0015] 3) Facilitates other experiments: In histopathology and molecular biology experiments, traditional retinal lamellae, due to their structure of four retinal tissue flaps, are more susceptible to curling and deformation by various liquid reagents, which can negatively impact the conduct of these experiments. In contrast, the intact retinal structure in the lamellae of this invention, once shaped, is less prone to curling and deformation, laying a foundation for the smooth conduct of subsequent experiments. Attached Figure Description

[0016] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0017] Figure 1 Schematic diagrams of a conventional retinal patch and the retinal patch of the present invention.

[0018] Figure 2 The fabrication process of this invention

[0019] Figure 3 Schematic diagram of the retinal patch of this invention (X10, confocal microscope)

[0020] Figure 4 Traditional retinal patch (X4)

[0021] Figure 5 Number of RGCs per unit area in a retinal patch 7 days after optic nerve clamp injury in mice. Detailed Implementation

[0022] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Improved method for preparing mouse whole retinal slices

[0025] The improved mouse whole retinal slide preparation method provided by this invention mainly consists of four steps:

[0026] (1) After dissecting the eyeball, the retinal tissue is adjusted so that the nerve fiber layer faces upward and immersed in the culture medium. The culture medium level should be deeper than the thickness of the glass slide. Figure 2 A).

[0027] (2) Then place the slide directly along the edge of the culture dish away from the retinal tissue, adjust its position, and move the slide directly under the retinal tissue. Figure 2 B).

[0028] (3) Aspirate the culture medium in small amounts multiple times. After each aspiration, use an iris restorer to adjust the position of the retinal tissue and unfold the rolled-up edges of the retina. This step must be done slowly, ensuring that the rolled-up edges of the retinal tissue are flattened with each adjustment of the liquid level. Figure 2 C).

[0029] (4) Remove all culture medium from the culture dish, flatten the retinal graft, place it on the glass slide, and maintain this state for 2 minutes. Figure 2 D).

[0030] The experimental mice were C57BL / 6 mice. The age of the experimental mice could be any age range.

[0031] In step (1), the culture medium contains insulin, progesterone solution, sodium selenite, thyroxine (T3), forskolin, acetylcysteine, BDNF, CNTF, FGF, GNTF, etc., and the composition can be adjusted according to experimental requirements. After soaking the retinal tissue in the culture medium, the position should be gently adjusted with forceps, placing the dissected retinal tissue with the optic nerve fiber layer facing upwards.

[0032] In step (2), when placing the slide, the depth of the culture dish must exceed the thickness of the slide. This is to adjust the position of the slide without damaging the retinal tissue. Simultaneously, the slide should be slowly inserted from the section furthest from the retinal tissue to avoid retinal tissue flipping. Each retina is typically obtained from a cup-shaped section of retinal tissue cut 1 mm posterior to the limbus of a mouse cornea. However, this can be adjusted slightly according to experimental needs.

[0033] In step (3), small amounts of culture medium need to be aspirated multiple times, and the retina needs to be gently unfolded and repositioned using an iris restorer after each aspiration. This is to ensure that the distance between the liquid surface and the bottom of the cup-shaped retinal tissue gradually decreases, and the surface tension of the liquid gradually flattens the retinal tissue, allowing the bottom of the retinal tissue to adhere more tightly to the slide surface. Note that the retina needs to be gently adjusted with an iris restorer after each aspiration, as the flow of the culture medium after each aspiration may cause retinal displacement, and each drop in the liquid level may cause the retinal tissue to curl and fold.

[0034] In step (4), all the culture medium must be aspirated to ensure that the retinal plank adheres tightly to the surface of the slide, which can prevent the retinal plank from curling upward. It is best to let the retinal plank stand for 2 minutes after aspirating the culture medium to shape it before fixation, in order to prevent the retinal plank from flipping and folding in the fixative.

[0035] The following describes the method for fabricating the sheet according to the present invention and its effects in conjunction with specific embodiments.

[0036] Example 1: Fabrication of a complete retinal patch

[0037] 1.1 Preparation and observation of complete retinal patch

[0038] C57 / BL6 mice weighing 20 to 40g were used as experimental animals. Animals with normal ocular examination were euthanized by intraperitoneal injection of an excessive amount of 6% chloral hydrate. The eyeballs were removed using microscissors and microforceps.

[0039] (1) Cut the eyeball 0.5 mm behind the limbus, remove the cornea and lens, and expose the eye cup. Use micro forceps to grasp the sclera with your left hand, and use microscissors to dissect the retina on the opposite side with your right hand. Continue until you reach the optic disc, then cut the retina and the tissue that is closely attached to it below, separating the spindle-shaped retina. Prepare a sterile glass slide and a 10 cm diameter culture dish. Add 15 ml of culture medium to the culture dish. Place the curled retina in the culture dish, and gently unfold the curled retinal tissue into a cup shape in the culture medium liquid. Use toothless forceps to gently adjust the cup-shaped retinal tissue so that the tip of the cup is directly below the ( Figure 2 A).

[0040] (2) Gently place the slide along the edge of the culture dish away from the retinal tissue, and gently adjust its position with tweezers, moving the slide directly under the retinal tissue. Figure 2 B).

[0041] (3) Each time, use a pipette to aspirate 1 ml of culture medium. After each aspiration, use an iris restorer to adjust the position of the retinal tissue and keep the tip of the retinal tissue pointing directly downwards until the tip of the retinal tissue begins to flatten against the surface of the slide when another 1 ml of culture medium is aspirated. The entire retinal tissue begins to resemble a bowl. Each time 1 ml of culture medium is aspirated, use the iris restorer to unfold the curled edges of the retina. This step must be done slowly and gently. Continue until all the culture medium in the culture dish is aspirated and the fully flattened retinal graft rests on the slide. Figure 2 C), maintain this state for 2 minutes for fixation, wipe the area around the retinal patch dry with a cotton swab under a microscope, and draw a circle with an immunohistochemistry pen to isolate water stains and facilitate various staining processes. Add 100ul of 4% paraformaldehyde to the immunohistochemistry circle and fix for at least 24 hours before staining and further observation. Figure 2D). It can be used for radiography; the retina is flat, meeting the needs of taking photographs. An example of a complete retinal planus observation can be seen... Figure 4 .

[0042] 1.2 Preparation and observation of traditional retinal slides

[0043] Example of traditional retinal smear preparation: C57 / BL6 mice weighing 20 to 40g were selected as experimental animals. Animals with normal ocular examination were euthanized by intraperitoneal injection of an excessive amount of 6% chloral hydrate. The eyeballs were removed using microscissors and microforceps.

[0044] (1) Cut the eyeball 0.5 mm behind the limbus, remove the cornea and lens, and expose the eye cup. Use micro forceps to grasp the sclera with your left hand, and use microscissors to separate the retina on the opposite side with your right hand. Continue until you reach the optic disc, then cut the retina and the tissue that is closely attached to it below, separating the spindle-shaped retina.

[0045] (2) Place the curled retinal tissue on a glass slide, cut the retina along four directions: above the nose, below the nose, above the temple, and below the temple, and gently adjust its position with forceps to gradually flatten it.

[0046] (3) Place the fully flattened retinal graft onto a glass slide and maintain this position for 2 minutes to set the shape. Wipe the area around the retinal graft dry with a cotton swab under a microscope, and draw a circle with an immunohistochemistry pen to isolate water stains and facilitate various staining processes. Add 100 μL of 4% paraformaldehyde to the immunohistochemistry circle and fix for at least 24 hours before staining and further observation. An example of traditional retinal graft observation can be seen below. Figure 3 .

[0047] Depend on Figure 3 and Figure 4 The comparison shows that the retinal patch fabrication method of the present invention preserves the complete retinal structure compared with the traditional method, which can greatly reduce the time and difficulty of locating the macula and other parts, and shorten the time for subsequent research on the retinal patch.

[0048] Example 2: Observation of retinal ganglion cells in a complete retinal patch

[0049] This invention provides a more complete retinal planus than traditional retinal planus preparations, allowing for easy localization based on vascular morphology and minimizing the risk of warping. This invention has already been applied in our research group's experimental studies observing retinal ganglion cells (RGCs). Figure 5 ).

[0050] 2.1 Grouping

[0051] To observe the protective effect of SCGF-β (recombinant human stem cell growth factor-β) on mouse optic nerve follicle cells (RGCs) in vivo under a mouse optic nerve clamp model, 15 C57BL / 6 mice weighing approximately 20g were randomly divided into three groups of five each. All mice underwent retinal slide preparation (all right eyes were operated on): the sham injury group served as the control group; the optic nerve clamp injury group was designated as the injury group; and the mice with optic nerve clamp injury underwent intravitreal injection of 2μL SCGF-β were designated as the SCGF-β treatment group. Seven days after treatment, all three groups underwent enucleation to prepare whole-retinal slides for RBPMS and Hochest immunofluorescence staining. The number of RBPMS-positive cells per unit area was calculated under a confocal microscope. Mice were euthanized under anesthesia, and the right eyeball was removed. After dissection, the retina was positioned, and the retinal tissue was spread out with the nerve fiber layer facing upwards and immersed in culture medium. A slide was then placed directly along the edge of the culture dish, and its position was adjusted until it was directly beneath the retinal tissue. Remove the culture medium and use an iris restorer to adjust the position of the retinal tissue and unfold the curled edges of the retina (perform this in multiple steps). Remove the culture medium to allow the retinal slide to sink, promoting the flattened retinal implant to adhere to the glass slide, and wait 2 minutes for it to set.

[0052] 2.2 Immunostaining and RGC counting observation

[0053] Immunofluorescence staining of RGCs in retinal slides was performed using rabbit anti-mouse primary antibody against RBPMS (1:100). The effect of the drug was evaluated by observing the number of RBPMS-positive cells per 400 μm2 under a fluorescence microscope. The number of cells was calculated and statistically analyzed.

[0054] 2.3 Results

[0055] The number of RBPMS-positive cells per unit area in each group was calculated using a confocal microscope. RBPMS immunofluorescence staining results showed that on day 7 after intravitreal administration, the coverage of RBPMS-positive cells increased 7 days after nerve clamp injury following SCGF-β treatment.

[0056] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0057] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for fabricating a complete retinal mount, characterized in that, The procedure includes the following steps: (1) Dissecting the eyeball and immersing the retinal tissue in the culture medium with the nerve fiber layer facing upwards; (2) Placing the slide directly along the edge of the culture dish away from the retinal tissue and adjusting its position; (3) Absorbing the culture medium; (4) Placing the flattened retinal graft on the slide and maintaining this state for a period of time; wherein, in step (1), the culture medium level should be deeper than the thickness of the slide; in step (2), the slide is moved directly under the retinal tissue; in step (3), the culture medium is aspirated in multiple small amounts, and after each aspiration, the position of the retinal tissue and the rolled-up edge of the retina are slowly adjusted using an iris restorer to ensure that the rolled-up periphery of the retinal tissue is flattened every time the liquid level is lowered. In step (4), the state is maintained for more than 30 seconds.

2. The method according to claim 1, characterized in that, This state is maintained for approximately 2 minutes.

Images