Method for detecting retinal ganglion cell ion channel and glaucoma rat model used by same

Abstract

The invention discloses a method for detecting a retinal ganglion cell ion channel and a glaucoma rat model used by the method. The method comprises the following steps: detecting an ion channel of a retinal ganglion cell through a patch clamp; the retinal ganglion cells are prepared by the following steps: sequentially performing incubation, digestion and cell dispersion on the retina of a glaucoma rat with a complete optic nerve papilla under an oxygen introduction condition; the digestion refers to digestion in papain digestive juice, the papain digestive juice comprises Hank's liquid, papain with the concentration of 10-50 [mu] g / mL, L-cysteine with the concentration of 0.5-1 mg / mL and BSA with the concentration of 0.5-1 mg / mL, and the specific activity of the papain is larger than or equal to 10 U / mg. According to the method disclosed by the invention, an in-vitro manual patch clamp is used for carrying out retinal ganglion cell ion channel detection on a magnetic bead induced intraocular hypertension rat, and a good foundation is laid for subsequent glaucoma mechanism research.

Description

Technical Field

[0001] The invention belongs to the field of animal models, and in particular relates to a method for detecting ion channels of retinal ganglion cells and a glaucoma rat model used therein. Background Art

[0002] The incidence of ophthalmic diseases in the clinic has been rising sharply in recent years. With the rise of new ophthalmic drug delivery technologies and new therapeutic drugs (such as gene therapy drugs), the current mechanism research on ophthalmic diseases faces huge challenges. So far, there are no guidelines / guidelines specifically for related technologies of ophthalmic drugs at home and abroad, and no recommended standard test combinations. Considering the special anatomy and physiology of the eye (blood-eye barrier) and the differences between different species, the basic ophthalmic examination evaluation methods used in conventional systemic toxicity studies can no longer meet the ophthalmic toxicity evaluation of the emerging new ophthalmic drugs or new drug delivery technologies. How to make more comprehensive evaluations safely and effectively has become a direct problem faced by scientific research institutions at home and abroad. The efficacy evaluation of drugs related to the treatment of glaucoma needs to be completed through animal models of ophthalmic diseases, but there are currently no scientific research institutions at home and abroad that can conduct a complete evaluation from glaucoma animal modeling to optic ganglion damage mechanism, which cannot meet the growing demand for preclinical evaluation of glaucoma drugs. Therefore, it is necessary to establish in vivo and in vitro test methods to fully and effectively evaluate the efficacy and ocular toxicity of drugs, meet the needs of the ophthalmic drug market for preclinical safety and efficacy evaluation, and thus safeguard the late clinical trials of new ophthalmic biological agents or new drug delivery routes. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for detecting ion channels of retinal ganglion cells and a glaucoma rat model used therein.

[0004] Specifically, the present invention provides a method for detecting ion channels of retinal ganglion cells, wherein the method detects ion channels of retinal ganglion cells by patch clamping;

[0005] The retinal ganglion cells are prepared by the following method:

[0006] The retina of glaucomatous rats with intact optic nerve head was incubated, digested and cell dispersed in sequence under oxygenated conditions.

[0007] In some embodiments, the digestion is performed in a papain digestion solution, wherein the papain digestion solution comprises Hank's solution, 10-50 ug / mL papain, 0.5-1 mg / mL L-cysteine ​​and 0.5-1 mg / mL BSA, wherein the specific activity of the papain is ≥10 U / mg.

[0008] In some embodiments, the oxygen supply temperature is 30-37°C.

[0009] In some specific embodiments, the oxygen supply temperature is 35°C.

[0010] In some embodiments, the incubation is incubation in Hank's solution for 10-15 min.

[0011] In some specific embodiments, the incubation is incubation in Hank's solution for 15 min.

[0012] In some embodiments, the digestion time is 20-25 min.

[0013] In some specific embodiments, the digestion time is 20 min.

[0014] In some embodiments, the cell dispersion is performed by blowing the cells apart in Ringer's fluid.

[0015] In some specific embodiments, the Hank's solution comprises: 137 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgSO4·7H2O, 0.5 mM NaHCO3, 1 mM Na2HPO4·12H2O, 20 mM HEPES, 16 mM Glucose, pH 7.4, and an osmotic pressure of 300-320 mOsm / kg H2O.

[0016] In some specific embodiments, the Ringer's solution comprises: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 20 mM Glucose, pH 7.4, and an osmotic pressure of 300-320 mOsm / kg H2O.

[0017] In some specific embodiments, the papain digestion solution comprises Hank's solution, 20-30 ug / mL papain, 0.5-1 mg / mL L-cysteine ​​and 0.5-1 mg / mL BSA.

[0018] In some specific embodiments, the papain digestion solution comprises Hank's solution, 25 ug / mL papain, 0.75 mg / mL L-cysteine ​​and 0.75 mg / mL BSA, wherein the specific activity of the papain is ≥10 U / mg.

[0019] In some embodiments, the detection includes electrode preparation, liquid filling, sealing and membrane breaking.

[0020] In some embodiments, the tip diameter of the electrode is 0.9-1.1 μm.

[0021] In some specific embodiments, the tip diameter of the electrode is 1 μm.

[0022] In some embodiments, the resistance of the inlet fluid is 4-6 MΩ.

[0023] In some specific embodiments, the resistance of the inlet liquid is 5 MΩ.

[0024] In some embodiments, the detecting is performed at 22-25°C.

[0025] In some embodiments, the clamping voltage is -60 mV or -70 mV.

[0026] In some embodiments, the stimulation voltage increases from -70 mV to +70 mV in 10 mV steps.

[0027] In some embodiments, the clamping time is 400-600 ms.

[0028] In some specific embodiments, the clamping time is 400 ms;

[0029] In some specific embodiments, the extracellular fluid is potassium ion extracellular fluid.

[0030] In some specific embodiments, the electrode internal solution is a potassium ion electrode internal solution.

[0031] In some embodiments, the glaucoma rat model is constructed by the following method:

[0032] Silica magnetic beads containing 10 mg / mL hyaluronic acid were injected into the anterior chamber of the rats to evenly distribute them in the anterior chamber angle.

[0033] In some embodiments, the concentration of the silica magnetic beads is 25-50 mg / ml.

[0034] In some specific embodiments, the concentration of the silica magnetic beads is 50 mg / ml.

[0035] In some embodiments, the injection point of the intracameral injection is about 1-2 mm from the cornea of ​​the rat corneoscleral limbus.

[0036] In some specific embodiments, the injection point for intracameral injection is about 2 mm from the cornea of ​​the rat corneoscleral limbus.

[0037] In some specific embodiments, the rat is a BN rat.

[0038] The present invention also provides a method for preparing rat retinal ganglion cells, the preparation method comprising:

[0039] Rat retinas with intact optic nerve heads were incubated, digested, and dispersed in sequence under oxygenated conditions;

[0040] The aeration, incubation, digestion and dispersion are as defined in the method.

[0041] In some specific embodiments, the rat is a glaucomatous rat.

[0042] The present invention also provides an application of a microelectrode in preparing a patch clamp, wherein the microelectrode has an outer diameter of 1.5 mm, an inner diameter of 0.86 mm, a length of 10 cm, and an electrode tip of 1 μm.

[0043] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0044] The reagents and raw materials used in the present invention are commercially available.

[0045] The positive and progressive effects of the present invention are:

[0046] The method of the present invention induces elevated intraocular pressure in rats by injecting silica magnetic beads containing 1% hyaluronic acid into the anterior chamber of BN rats, thereby establishing a rat glaucoma model. Compared with other magnetic bead models, the refractive medium of the anterior segment of the rat's eye is clear, and ophthalmic examinations such as slit lamp microscope anterior segment photography, OCT, fundus photography, and electroretinogram can be smoothly performed. At the same time, the ion channel detection of retinal ganglion cells in rats with high intraocular pressure induced by magnetic beads is performed using an in vitro manual patch clamp, which can lay a good foundation for subsequent research on the mechanism of glaucoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a slit lamp examination of the control group of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention, with a magnification of 16×.

[0048] Figure 2 This is a slit lamp examination of the experimental group of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention, with a magnification of 16×.

[0049] Figure 3 This is an OCT examination of the control group of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention.

[0050] Figure 4 This is an OCT examination of the experimental group of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention.

[0051] Figure 5 This is an ERG test of the control group of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention.

[0052] Figure 6 This is the ERG test of the experimental group of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention.

[0053] Figure 7 This is the potassium ion channel detection of the method for detecting ion channels of retinal ganglion cells in glaucoma according to an embodiment of the present invention.

[0054] Figure 8 This is a diagram of the HE staining status of the magnetic bead microsphere injection model.

[0055] Fig. 9 Schematic diagram of retinal ganglion cell apoptosis at different stages after modeling. DETAILED DESCRIPTION

[0056] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0057] Unless otherwise specified, the formula of Hank's solution used in the following examples is as follows (mM): NaCl 137, KCl 3, CaCl22, MgSO4·7H2O 1, NaHCO3 0.5, Na2HPO4·12H2O 1, HEPES 20, Glucose 16, adjusted to pH 7.4 with NaOH, osmotic pressure of 305mOsm / kg H2O; Ringer's solution formula is as follows (mM): NaCl 140, KCl 5, CaCl22, MgCl21, HEPES 10, Glucose 20, adjusted to pH=7.4 with NaOH, osmotic pressure of 310mOsm / kg H2O. The potassium ion extracellular solution uses Ringer's solution; the potassium ion intracellular solution formula is as follows (mM): KCl 140, NaCl 9, MgCl21, HEPES 10, EGTA 0.2, GTP-Na, ATP-Mg, adjusted to pH=7.2 with KOH, osmotic pressure of 290mOsm / kg H2O.

[0058] Example 1 Glaucoma Modeling

[0059] (1) Preparation of 1% hyaluronic acid solution (taking 5 mL as an example)

[0060] Weigh 0.05 g of hyaluronic acid powder and place it in a 15 mL centrifuge tube, then add 5 mL of sterile 1× HBSS solution. Shake it up and down several times and place it in a 2-8 ℃ refrigerator for more than 2 hours to allow the hyaluronic acid powder to fully absorb water to form a uniform hyaluronic acid solution. Filter it through a 0.22 μm sterile microporous filter membrane before use.

[0061] (2) 50 mg mL -1 Preparation of magnetic bead suspension stock solution

[0062] After vortexing the silica magnetic bead suspension prepared with 50 mg·mL-1 sterile water for injection for 30 s, take the required amount and centrifuge at 1000 g and 4 ℃ for 5 min, carefully discard the supernatant, add an equal volume of 1×HBSS solution and shake and wash. Repeat the above washing three times to wash away the impurities in the magnetic beads as much as possible. After the fourth centrifugation, carefully discard the supernatant, add an equal volume of 1% hyaluronic acid solution to resuspend, and obtain a magnetic bead stock solution with a concentration of 50 mg·mL-1.

[0063] (3) Preoperative treatment

[0064] Three days before intracameral injection, levofloxacin eye drops were administered twice a day into the conjunctival sacs of both eyes for anti-infection pretreatment.

[0065] (4) Anterior chamber injection process

[0066] Then, general anesthesia (intramuscular injection of Shutai® 50 and xylazine hydrochloride injection, doses of 5-10 mg / kg and 2-4 mg / kg, respectively, general anesthesia is used in this way unless otherwise specified) and corneal surface anesthesia (proparacaine hydrochloride eye drops are given in the eyes) are given to ensure that the toe pinch reflex and corneal reflex disappear. The conjunctival sac of the operated eye is rinsed with 0.5% povidone-iodine solution, and the operated eye is disinfected from the inside to the outside three times, and then immediately rinsed with normal saline. Placed under a stereomicroscope or surgical microscope, use an insulin syringe with a needle diameter of 30 G to carefully puncture the anterior chamber about 2 mm from the corneal limbus to the cornea in a "tunnel-like" manner, and inject 10 μL of 50 mg·mL into the anterior chamber. -1 The silica magnetic bead suspension was added to avoid damaging the iris and lens. After the injection, wait for a while and remove the needle. When removing the needle, gently press the injection site for 10 seconds with a cotton swab to prevent a large amount of magnetic beads from leaking out. Then, an ophthalmic foreign body suction was immediately used to attract the magnetic beads along the corneal margin so that they were evenly distributed in the chamber angle. After ofloxacin eye ointment was administered into the conjunctival sac of both eyes, the rat was placed on a warming pad with the operated eye facing up and nursed until it woke up.

[0067] (5) Postoperative care

[0068] Three days after the operation, levofloxacin eye drops and tobramycin dexamethasone eye drops were given three times a day for anti-infection treatment, and atropine sulfate eye gel was used twice a day to dilate the pupil and prevent iridocyclitis.

[0069] Example 2 Acute Isolation of Retinal Ganglion Cells

[0070] (1) Prepare three 15 ml centrifuge tubes, add 3 ml of Hank's solution to one of them, and place it in a 35℃ water bath to preheat and keep it aerated for more than 30 minutes; add 15 ml of Hank's solution and 15 ml of Ringer's solution to the other two, and place them in a 35℃ water bath to preheat and keep it aerated; burn a 12 mm diameter round glass slide soaked in anhydrous ethanol with an alcohol lamp, and then spread it flat in a 24-well plate for use; prepare several 3.5 cm culture dishes, pour 2 ml of 2-8℃ oxygenated Hank's solution for use;

[0071] (2) After the rat is anesthetized, use ophthalmic scissors to cut the eyelids, peel off the muscles around one side of the eye, and remove the eyeball. Place the eyeball in a 3.5 cm culture dish filled with oxygenated Hank's solution, use Venus scissors to split the eyeball in half along the corneal limbus, and the back half will be shaped like an optic cup. Then remove the lens, iris, vitreous body, and choroid in turn, remove the retina and keep the optic nerve head intact. Place the removed retina in Hank's solution pre-oxygenated at 35°C and incubate at 35°C for 10 minutes.

[0072] (3) After the incubation, discard the Hank's solution in the centrifuge tube, add 600 μl of papain digestion solution at 35°C under continuous oxygenation, and digest the retinal tissue for 20 min;

[0073] Papain digestion solution was prepared with 4 ml Hank's solution, 4 μl papain (Sigma P4762, prepared at 25 mg / mL), 3 mg L-cysteine, and 3 mg BSA;

[0074] (4) After digestion, aspirate the papain digestion solution, wash three times with preheated Hank's solution, and then place in oxygenated Ringer's solution. Use a Pasteur pipette to blow the retinal tissue apart and let it stand for 3 minutes. Then use a pipette to draw 100 μl of the supernatant and drop it onto the treated glass slide in the 24-well plate. Let it stand for 1 hour until the cells are attached. Then add 400 μl of preheated Ringer's solution to each well for electrophysiological recording.

[0075] Example 3 Potassium channel patch clamp assay

[0076] (1) Use a P-97 glass puller to pull the glass electrode to be used. Under a microscope, the tip of the pulled electrode is about 1 μm in size. Use a polisher to polish the electrode, and the liquid resistance is about 5 MΩ.

[0077] (2) Remove the cell slide after patching from the 24-well plate and fix it in a bath under a microscope. Add a certain volume of corresponding potassium ion extracellular fluid into the bath. Infuse the corresponding potassium ion electrode internal solution into the electrode. If there are bubbles, tap the electrode to expel them. Apply positive pressure to the electrode before entering the solution, and use a micromanipulator to immerse the electrode into the solution.

[0078] (3) Use a micromanipulator to control the electrode to carefully approach the cell. When the resistance suddenly increases, apply appropriate negative pressure to the electrode to make the electrode closely contact with the cell membrane of the cell to be tested, forming a high-resistance seal of the GΩ level.

[0079] (4) Select the Whole-Cell mode and set the clamp voltage to -60 mV. When recording ion channel IV, the clamp voltage was -60 mV, the stimulation voltage increased from -70 mV to +70 mV in 10 mV steps, and the clamp time was 400 ms. Then, negative pressure was slowly applied to break the cells, the signal sampling frequency was 5 kHz, and the low-pass filter frequency was 1 kHz. All experiments were performed at room temperature (22-25°C).

[0080] The results are as follows Figure 1-9 The results show that the results obtained by using the detection method of ion channels of retinal ganglion cells in glaucoma of the present invention show that the refractive medium of the anterior segment of the rat eye is clear, and the anterior segment photography can be smoothly performed under slit lamp microscope ( Figure 1-Figure 2 )、OCT( Figure 3-Figure 4 )、ERG( Figure 5-Figure 6 ) and other ophthalmological examinations. At the same time, the ion channel of retinal ganglion cells in rats with high intraocular pressure induced by magnetic beads was detected using in vitro manual patch clamp ( Figure 7 ).

[0081] Figure 8 The HE staining status of the magnetic bead microsphere injection model is shown, indicating that the magnetic beads have been injected into the corner of the eye.

[0082] Fig. 9 The results showed that after modeling, the number of ganglion cells (GCL) and inner granule layer cells (INF) gradually decreased over time, and the overall retinal thickness became thinner.

Claims

1. A method for detecting ion channels of retinal ganglion cells, characterized in that: The method detects ion channels of retinal ganglion cells by patch clamping; The retinal ganglion cells are prepared by the following method: The retina of glaucomatous rats with intact optic nerve heads was incubated, digested and cell dispersed in sequence under oxygenated conditions; The digestion is carried out in a papain digestion solution, wherein the papain digestion solution comprises Hank's solution, 10-50ug / mL papain, 0.5-1mg / mL L-cysteine ​​and 0.5-1mg / mL BSA, wherein the specific activity of the papain is ≥10U / mg.

2. The method according to claim 1, characterized in that The sequential incubation, digestion and cell dispersion under oxygen conditions meet one or more of the following conditions: (1) The oxygen flow temperature is 30-37°C, for example 35°C; (2) the incubation is incubating in the Hank's solution for 10-15 min, for example, 15 min; (3) The digestion time is 20-25 min, for example 20 min; (4) The cell dispersion is performed by blowing the cells apart in Ringer's solution.

3. The method according to claim 2, characterized in that The Hank's solution contains: 137 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgSO4·7H2O, 0.5 mM NaHCO3, 1 mM Na2HPO4·12H2O, 20 mM HEPES, 16 mM Glucose, pH 7.4, and an osmotic pressure of 300-320 mOsm / kg H2O; and / or, the Ringer's solution comprises: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 20 mM Glucose, pH 7.4, and an osmotic pressure of 300-320 mOsm / kg H2O; And / or, the papain digestion solution comprises Hank's solution, 20-30 ug / mL papain, 0.5-1 mg / mL L-cysteine ​​and 0.5-1 mg / mL BSA; preferably, the papain digestion solution comprises Hank's solution, 25 ug / mL papain, 0.75 mg / mL L-cysteine ​​and 0.75 mg / mL BSA.

4. The method according to claim 1, characterized in that The detection includes electrode preparation, liquid entry, sealing and membrane breaking; The tip diameter of the electrode is 0.9-1.1 μm, preferably 1 μm; and / or the resistance of the inlet liquid is 4-6 MΩ, for example 5 MΩ.

5. The method according to any one of claims 1 to 4, characterized in that: The detection is performed at 22-25° C.; and / or, the clamping voltage is -60 mV or -70 mV, the stimulation voltage increases from -70 mV to +70 mV in 10 mV steps, and the clamping time is 400-600 ms, for example 400 ms; Preferably, the extracellular fluid is potassium ion extracellular fluid; and / or, the electrode internal fluid is potassium ion electrode internal fluid.

6. The method according to claim 1, characterized in that The glaucoma rat model was constructed by the following method: Silica magnetic beads containing 10 mg / mL hyaluronic acid were injected into the anterior chamber of the rats to evenly distribute them in the anterior chamber angle.

7. The method according to claim 6, characterized in that The concentration of the silica magnetic beads is 25-50 mg / ml, for example 50 mg / ml; And / or, the injection point of the anterior chamber injection is about 1-2 mm, for example, 2 mm, from the cornea of ​​the rat corneoscleral limbus; And / or, the rat is a BN rat.

8. A method for preparing rat retinal ganglion cells, characterized in that: The preparation method comprises: Rat retinas with intact optic nerve heads were incubated, digested, and dispersed in sequence under oxygenated conditions; The oxygenation, incubation, digestion and dispersion are as defined in any one of claims 1-3.

9. The method according to claim 8, characterized in that The rats are glaucoma rats.

10. Application of a microelectrode in preparing a patch clamp; The microelectrode has an outer diameter of 1.5 mm, an inner diameter of 0.86 mm, a length of 10 cm, and an electrode tip of 1 μm.

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