CN111617313B

CN111617313B - Application of ophthalmic linear gel in aspect of being used as clinical hole-induced retinal detachment medicine

Info

Publication number: CN111617313B
Application number: CN202010356587.5A
Authority: CN
Inventors: 李筱荣; 任新军; 刘巨平; 柯屹峰; 郑传珍; 温德佳
Original assignee: TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
Current assignee: TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
Priority date: 2020-04-29
Filing date: 2020-04-29
Publication date: 2022-09-06
Anticipated expiration: 2040-04-29
Also published as: CN111617313A

Abstract

The invention relates to application of linear gel for eyes in serving as a clinical hole-induced retinal detachment medicine. The invention proves that the eye gel has stronger cohesion than the eye linear gel excessive crosslinking through a safety experiment. The gel is safe to fill in vitreous cavities, has no toxic or side effect, develops a new material for clinical vitreous cavity substitutes, provides data for expanding the application of the linear gel for eyes in clinical ophthalmology and accumulates experience.

Description

Application of ophthalmic linear gel in aspect of being used as clinical hole-induced retinal detachment medicine

Technical Field

The invention belongs to the technical field of ophthalmology, and particularly relates to an application of an ophthalmic linear gel in the aspect of being used as a clinical hole-induced retinal detachment medicine.

Background

Retinal Detachment (RD) refers to the separation of Retinal nerve epithelial layer and Retinal pigment epithelial layer, including three types of porogenesis, proliferation and exudation, and is a common disease and frequently encountered ophthalmology, and if the condition of disease is delayed or the treatment is improper, it is also one of the common blindness-causing fundus diseases. Retinal Detachment (RRD) is the most common type of Retinal Detachment, and occurs on the basic principle that Retinal tears form after Retinal degeneration or degenerative changes, and liquefied vitreous enters the retina through the Retinal tears and accumulates under the retina, separating the Retinal neuroepithelial layer from the pigment epithelial layer. At present, the basic treatment principle of pore-derived retinal detachment is to search and seal a fissure hole, and relieving or removing the vitreous retinal interface tension is a key step for sealing the fissure hole, so that the detached retinal neuroepithelial layer and the retinal pigment epithelial layer are closely adhered to achieve anatomical reduction as much as possible. The main treatments for rhegmatogenous retinal detachment are surgical treatments, including intravitreal dissection of the internal pathway (Pars planar vitrrectomyces, PPV) and episcleral pressor (Scleral Buckling, SB). The inner path vitrectomy is combined with gas filling to treat porogenic retinal detachment, and the main purpose is to relieve the traction of vitreous bodies and proliferation membranes on the retina and prevent liquid from continuously entering the retina through retinal holes. The external surgery mainly comprises the steps of pressing down sclera and choroid around a retinal fissure through sclera external pressurization to form a pad pressing ridge, pressing against the retinal fissure, relieving or eliminating vitreous traction, and then assisting with freezing or retinal photocoagulation to enable the retina and the choroid to be in contact with each other, adhered and finally reset.

With advances in technology and increasingly mature surgical techniques, vitrectomy (PPV) has become the most common surgical procedure for the treatment of primary poriferous retinal detachments (RRDs) in most developed and partially developing countries.

Although the retinal detachment with porogenesis is a common disease and frequently occurs, the treatment effect is not satisfactory. Studies report that the rate of first phase reduction of RRD treated using PPV technology varies from 72% to 88%. In order to increase the success rate of vitrectomy for the treatment of rhegmatogenous retinal detachment, various surgical techniques and concepts have been reported, such as combined vitrectomy and cerclage, long-acting inert gas vitreous cavity filling, use of heavy water, application of silicone oil and heavy silicone oil, and the like. Apart from the use of heavy silicone oil filling, these surgical procedures have in common that the postoperative patient is to be kept in a strictly prone position. Furthermore, the use of intraocular gas filling has the disadvantage that when the vitreous cavity is filled with gas, especially with long-lasting inert gas, there is a short visual disturbance, two weeks in the short term, and two months or more in the long term may be required for absorption (e.g. C) ₃ F ₈ ) Meanwhile, the patient needs to be in a strict prone position, the time required by different doctors is different in different conditions, and the reason is also one of the reasons for preventing the patient from selecting the vitrectomy. In addition, intraocular gas tamponade and surgical disturbance can lead to a series of complications including cataract formation, secondary glaucoma and even retinal artery occlusion. Generally, for patients with perforated retinal detachment Proliferative Vitreoretinopathy (PVR) A, B grade, although the retina can be effectively restored by combined gas filling after vitrectomy, part of ophthalmologists still select silicone oil filling, especially the shortage of long-acting gas production in China at present, and the usage rate of silicone oil is further increased. After silicone oil is filled, eye complications are more, for example, refractive state of eyes is changed, visual function of a patient is influenced, secondary ocular hypertension after silicone oil emulsification, sudden and sharp vision loss without clear inducement (most of the ocular hypertension occurs 3 months after silicone oil filling), and the like. Moreover, to prevent these complications, silicone oil in the eye is usually taken out by a secondary operation, which increases the burden on the patient.

Another classic surgical treatment for rhegmatogenous retinal detachment is external scleral cingulate surgery. Although the materials, modes and positions of the pressing have been improved greatly for years, the principle of scleral depression is the basis of the external scleral pressing operation treatment. The currently common cushion pressing materials in clinic comprise silicon sponge and silica gel belt, and scholars report that the subcutaneous connective tissue of the self limbs is taken as a cushion pressing object. There have also been experimental reports of the use of absorbable padding materials such as polycaprolactone. The concept of pad pressing mode and position selection is also changed, the minimal quantitative operation concept is provided, local pad pressing is performed as far as possible instead of cerclage, on the premise of continuously relieving or relieving various traction forces of the retina, the fissure is sealed, and the retina is reset. Although clinical practice has proved that the external scleral compression surgery has a high success rate and a good post-operative effect in treating the poriferous retinal detachment, however, the postoperative visual function is affected by the complications of scleral cerclage and external tamponade surgery, which often cause deformation of the eyeball, change of the axis of the eye, blood supply disorder of the eyeball, exudative retinal detachment, change of refractive state, damage and imbalance of the extraocular muscles, secondary glaucoma, and the like.

Its victory eye linearity

(QST, Shanghai Sheng biologics Co., Ltd., Shanghai, China) is a sterilized, biodegradable, viscoelastic, transparent and colorless homogeneous gel implant (pH 7.3, osmotic pressure 300mOsm/kg), which is prepared from non-animal Hyaluronic Acid (HA) produced by Shanghai Sheng biologics Co., Ltd., by chemical crosslinking and formulating in physiological buffer solution at a concentration of 16mg/mL, wherein the solvent system comprises phosphate buffer and NaCl salt, HAs a specific gravity of about 1.01, is close to water, HAs a refractive index i of 1.341, and HAs a kinetic viscosity of 186,733 mPas, and HAs strong cohesive force. The cross-linking agent is DVS, and the cross-linked HA dry powder is obtained by fractional precipitation of an organic solvent, so that the residual DVS can be removed to the maximum extent; the non-granular gel is uniform non-granular gel which is obtained by dissolving the prepared cross-linked HA dry powder in a buffer solution and through the swelling property of the cross-linked HA. In the cross-linking process, a low-concentration sodium hyaluronate solution is used as an initial reaction solutionThe sodium hyaluronate molecules in the reaction solution below present a linear tropism arrangement. The gel obtained by organic solvent precipitation and redissolution has a microstructure which is changed greatly, so that the microstructure generates a molecular rearrangement phenomenon, and the crosslinked sodium hyaluronate gel for intraocular filling shows obvious linear arrangement. Meanwhile, the particle-free property of the product is closer to the property of natural sodium hyaluronate gel, so that the product can reduce adverse symptoms such as delayed granuloma, red swelling, pain and the like caused by different particle sizes and surface morphologies, and the biocompatibility of the product is improved. The victory eye linear gel is a degradable viscoelastic agent, and is subjected to in vitro enzymolysis for 4 hours, 24 hours and 48 hours, wherein the degradation percentages are respectively 54%, 94% and 94%. After being implanted subcutaneously for 12 months, the medicine has good biocompatibility, and the filling effective rate reaches over 80 percent. The ophthalmic linear gel is biologically fermented and non-animal-derived, so that the allergenicity is extremely low, the sterility, the purity and the toxicity are well controlled, and the ophthalmic linear gel is a push-injection type gel, can be conveniently covered on the surface of a retinal hole, and can be applied to more ophthalmic operations besides the retinal detachment with hole source.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art caused by the stress of environment, provides the application of the eye linear gel as the clinical hole-derived retinal detachment medicine, firstly proves that the eye linear gel is safe and effective in a rabbit hole-derived retinal detachment model, and provides a new choice for the treatment of the clinical hole-derived retinal detachment.

The technical scheme adopted by the invention for solving the technical problem is as follows:

an application of the linear gel in treating clinical hole-induced retinal detachment.

Moreover, the composition of the said linear gel for eye is as follows:

each 1mL of gel contains 16mg of cross-linked sodium hyaluronate and is prepared from physiological equilibrium liquid; the physiological balanced liquid consists of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection.

An application of the linear gel for the peptide eye in rabbit rhegmatogenous retinal detachment model.

An application of the linear gel in treating macular hole, diabetic retinopathy, high-myopia retinopathy or intractable low intraocular pressure is disclosed.

The invention has the advantages and positive effects that:

1. the invention proves that the eye gel has stronger cohesion than the eye linear gel excessive crosslinking through a safety experiment. The gel is safe to fill in vitreous cavities, has no toxic or side effect, develops a new material for clinical vitreous cavity substitutes, provides data for expanding the application of the linear gel for eyes in clinical ophthalmology and accumulates experience.

2. The invention proves that the linear gel for the eye is safe and effective in a rabbit rhegmatogenous retinal detachment model for the first time, and provides a new choice for treating clinical rhegmatogenous retinal detachment.

3. Based on the characteristics of high crosslinking and high viscosity of the ophthalmic linear gel, the invention can be developed to be applied to other ophthalmic diseases, such as macular hole, diabetic retinopathy, high myopia fundus lesion, refractory low intraocular pressure and the like.

4. Based on the high crosslinking, high dynamic viscosity and degradable characteristics of the linear gel for the victory eye, the linear gel for the victory eye is adopted to cover retinal holes, and is combined with a 25G minimally invasive vitrectomy and air filling auxiliary treatment rabbit hole-originated retinal detachment model, the body position does not need to be limited after the operation, and the curative effect of the linear gel for the victory eye is observed. In addition, with the method as a trigger, based on the characteristic of the linear gel for eyes, a new viscous material which is more in line with clinical requirements, especially a vitreous cavity filler is developed, a theoretical basis and accumulated experience are provided for treating other ophthalmic diseases, and the ophthalmic application of the material is expanded.

Drawings

FIG. 1 is a technical route chart for the safety and efficacy studies of rabbit rhegmatogenous retinal detachment models by covering retinal holes with linear gel for eyes according to the present invention;

FIG. 2 is a QST validation graph in accordance with the present invention; wherein, A: anaesthetizing experimental rabbit pictures before operation; b: the vitreous cutting system cuts away the vitreous map; c: making a retinostoma pattern of about 1/2PD size, injecting BSS subretinally to about 2PD size RD; d: gas-liquid exchange reduction retina graph; e: the experimental group covered QST on the fissure hole, the control group did not (note: QST linear gel for the victory eye; PD optic disc diameter; BSS balanced salt solution; RD retinal detachment);

FIG. 3 is a graph showing a comparison of the cohesion of three gels QST, DVS and BDDE in the present invention (note: QST's linear gel for eye; DVS divinyl sulfone; BDDE 1, 4-butanediol diglycidyl ether);

FIG. 4 is a graph showing the intraocular pressure changes at various time points before and after the linear gel safety test for eye therapy in the present invention;

FIG. 5 is a graph showing the effect of the invention on retinal morphology on linear gel safety test for eye success; wherein, A: observing the non-operative eye under a 10-fold optical microscope; b: the observation picture under the optical lens of 10 times of the operation eye; c: observation under 20 times of light microscope of non-operative eyes; d: observing the operation eye under 20 times of light microscope;

FIG. 6 is a graph of retinal replacement in a typical experimental group and a control group of the present invention; wherein, A: fundus phase map after 1 week of operation in experimental group; b: eye ground phase diagram of 1 month after the operation of the experimental group; c: fundus phase map after 3 months of operation in experimental group; d: b ultrasonic image of 1 week after operation of control group; e: b-ultrasonography for 1 month after operation in the control group; f: the control group was subjected to ultrasonography B3 months after the operation.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Preferably, the composition of the said linear gel for eye is as follows:

An application of the linear gel for eye-victory in rabbit rhegmatogenous retinal detachment model.

An application of the linear gel in treating macular hole, diabetic retinopathy, high myopia, etc is disclosed.

Specifically, the preparation and verification related to the present invention are as follows (as shown in fig. 1):

firstly, the method comprises the following steps: cohesion of its linear gel for eye

1. Compare their cohesion with other commercial cross-linked sodium hyaluronate gels, which are linear gels for ophthalmic use.

II, secondly: the safety of the linear gel in rabbit eyes

1. Animal preparation

About 8 gray rabbits (2 Kg) for experiment with the qualification certificate.

2. Preoperative ERG examination: binocular ERG examination was performed 1 day before surgery.

(1) General anesthesia and mydriasis.

(2) Dark adaptation was carried out for 30 min.

(3) Surface anesthesia: oxybuprocaine hydrochloride eye drops.

(4) Connecting the cornea contact electrode slice as a positive electrode; the double-ended needle electrode is used as a common negative electrode of the two channels; the needle electrode serves as a ground electrode.

(5) An ERG check was performed.

3. And (3) operation: the left eye was selected for the experiment.

(1) General anesthesia and mydriasis.

(2) Sterilizing the operation eye.

(3)25G standard three channel vitrectomy, the vitreous cavity is filled with sterile air and 0.1ml of its ocular linear gel after the vitrectomy. After the operation, the cannula is removed and the incision is massaged with a cotton swab.

(4) Tobramycin dexamethasone eye ointment (Dianbishu ointment) is applied on eyes and bound with gauze.

4. Post-operative observation

(1) Eye dropping: compound tropicamide 4 times/day; dianshu ointment for 3 times a day. The administration time is 1 week.

(2) Intraocular pressure: intraocular pressure measurements were performed and recorded at 1d, 3d, 5d, 7d, 14d, 1 month post-operatively with an ICARE hand-held rebound tonometer. The average was taken 5 times in succession.

(3) And (3) slit lamp inspection: after 1d, 3d, 5d, 7d, 14d and 1 month of operation, the eye slit lamp is examined, and the cornea transparency, the existence of inflammation in the anterior chamber, the cataract development and the like are observed and recorded.

(4) Fundus examination: observing the vitreous cavity and the retinal condition of the eyeground of the operation under a front lens at 1d, 3d, 5d, 7d, 14d and 1 month after the operation and recording.

5. And (3) postoperative examination: ERG, HE staining of retinal sections

(1) After one month, the eyes were again examined for ERG in the descending eyes of general anesthesia. Comparing the ERG change before and after the operation of the eye. The influence of the eye-friendly linear gel on the function of the retina is observed.

(2) Air was injected into the ear vein and air embolisms were used to immobilize gray rabbits.

(3) The eyes are removed, HE staining is carried out on retinal sections, the operated eyes and the non-operated eyes are observed in a contrast mode, and the influence of the eye-surpassing linear gel on the form of each layer of the retina is judged.

Thirdly, the method comprises the following steps: the effectiveness of the linear gel in rabbit rhegmatogenous retinal detachment model

1. Molding machine

(1) About 16 experimental gray rabbits (2 Kg) with the qualification certificate. 8 experimental groups and 8 control groups were randomly assigned. The left eye was selected for the experiment.

(2) General anesthesia and mydriasis.

(3) Sterilizing the operation eye.

(4) The 25G standard three-channel vitrectomy is to cut the vitreous body, perform retinal cutting at about 2PD below the optic disc to make a retinal fissure with the size of about 1/2PD, inject BSS to the retina through the retinal fissure to cause retinal detachment with the range of about 2PD, exchange gas and liquid, reset the retina and fill the vitreous cavity with sterile air. The experimental group had the linear gel for eye use covered the retinal cleavage hole surface by cutting incision, and the control group had no such operation as the standard of completely covering the cleavage hole. After the operation, the cannula is removed and the incision is massaged with a cotton swab.

(5) Tobramycin dexamethasone eye ointment (Dianbishu ointment) is applied on eyes and bound with gauze.

2. Post-operative observation

(4) Fundus examination: observing the vitreous cavity of the operation eye and the resetting condition of the retina of the eye fundus under the front microscope and recording the conditions at 1d, 3d, 5d, 7d, 14d and 1 month after the operation.

3. And (3) postoperative examination: fundus phase, B-ultrasound

(1) Auxiliary inspection: performing eye fundus examination and B-ultrasonic examination after 1 week, 1 month and 3 months of operation. The retinal replacement rates of the experimental group and the control group were compared.

(2) Air was injected into the ear vein 3 months after surgery and the experimental grey rabbits were sacrificed by air embolism.

More specifically, the operation is as follows:

cohesion of linear gel for eye

The cohesiveness of the gel for eye use and other cross-linked sodium hyaluronate gel on the market is compared by a dispersion method, and two types of Divinyl sulfone (DVS) and 1, 4-Butanediol diglycidyl ether (BDDE) are selected for comparison. Staining was completed by first adding 30 μ L of a 1% toluidine blue solution by mass to 1mL of the gel and mixing between 2 syringes for 3 minutes. The gel was then filled into a 1ml BD glass syringe (Becton, Dickinson and Company, Franklin Lakes, N.J.) and air was removed from the gel by centrifugation. A 1000 ml beaker 14.2 cm high and 11.7 cm diameter was filled with 700 ml deionized water and placed on a magnetic stirrer. Stirring was carried out at 170rpm using a magnetic stirrer 25mm long and 0.8mm wide. The syringe containing the stained gel was placed in a well 2cm above the water surface. The gel was pushed out of the syringe at a rate of 400mm/min using a Zwick materials tester Zwick BTC-FR 2.5TH.D09(Zwick Roell, Ulm, Germany). And photographs were taken 15s, 75s, 90s, 5min, 10min after the gel impacted the surface.

Second, the safety of the eye-servicing linear gel in rabbit eyes

1. Preoperative ERG examination: binocular ERG examination was performed 1 day before surgery.

(1) General anesthesia: the rabbit was fixed in a homemade animal clip, 1.5ml of fast-sleep nova II (1.5 ml/log, Shengda animal drugs Co., Ltd., Town. city) was injected intraperitoneally, 1mg/ml of midazolam (5mg/ml, Jiangsu Enhua Co., Ltd.) was mixed with 0.9% physiological saline (Tsukawa pharmaceutical Co., Ltd., China) in mass concentration, and 5ml of auricular vein was taken for slow bolus injection. The effect of anesthesia was probed by touching the cornea with a cotton swab.

(2) Binocular mydriasis: compound tropicamide eye drops (maduoli, shentian pharmaceutical (china) limited).

(3) The gray rabbits were fixed on the electrophysiological examination instrument laboratory bench by adhesive tape for dark adaptation for 30 minutes.

(4) After dark adaptation for 30min, oxybuprocaine hydrochloride eye drops (binomi, Shentian pharmaceutical (China) Co., Ltd.) were dropped on both eyes under infrared light illumination for surface anesthesia.

(5) Under the illumination of infrared light, an electrode plate (Chongqing Aiersi medical equipment Co., Ltd.) was connected. 0.5% carboxymethyl cellulose (Allergan, Inc. USA) lubricates eyes, then a cornea contact electrode plate is placed as a positive electrode, and a connecting line is fixed by an eye adhesive tape; a double-ended needle electrode is subcutaneously inserted at the middle point of the root connecting line of the two ears, and is used as a common negative electrode of the two channels; and inserting a needle electrode under the tail skin as a ground electrode.

(6) The gray rabbit follower experimental platform was pushed into a Ganzfeld stimulation ball (chongqing exercz medical devices ltd) together, and the eyes were fully exposed to the stimulation ball.

(7) ERG recording is carried out by adopting RetimINER 4.0 software (Chongqing Airlxi medical equipment Co., Ltd.), and a grey rabbit number is recorded in a calculator to create a file. And carrying out dark adaptation of 0.01, dark adaptation of 3.0, dark adaptation of 30.0, dark adaptation of 3.0 oscillating potential, bright adaptation of 3.0 flicker, FVEP transient state and FVEP steady state inspection and recording in sequence. The ERG electrophysiological examination instrument (Chongqing Airlxi medical equipment Co., Ltd.) consists of a Ganzfeld stimulating ball, a signal amplifier and a recorder. The filter passband of the signal amplifier is 3Hz to 300 Hz. The distance between the corneal plane and the white laser is 30cm, and the light intensity range is 19200-24000cd/m ² . The flashes were spaced 30s 3 times per eye each and averaged.

(8) Eye drops of zubi levofloxacin (clonbital, shentian pharmaceutical (china) limited) are applied to eyes.

(9) The experimental rabbits were examined for respiration and heartbeat to confirm survival. Animals were removed from rabbit houses and observed every 10min until awakened.

2. And (3) operation: the left eye was selected for the experiment. As shown in fig. 2.

(1) The mydriasis of the eye is performed by general anesthesia and surgery as described above.

(2) Disinfection of surgical eyes: and (3) expanding an eyelid exposed operation area by using an eyelid opener, dropping the eye by using povidone iodine, staying for 30 seconds, and then washing by using the lactic acid ringer's solution.

(3)25G sclerotomy: standard three channels passing through pars plana are passed, an ruler measures 1.5mm behind the limbus, an Alcon 25G puncture cannula makes a perfusion incision below temples, the puncture cannula is used as an optical fiber port at 2 points, and the puncture cannula is used as a vitreous cutting head incision at 10 points.

(4) Vitrectomy: 25G vitrectomy was performed using a contact wide-angle viewing lens (Volk Mini Quad XL; Volk Optical, Inc, Mentor, Ohio), a Constellation vitreous cutting system (Alcon Laboratories, Inc., Fort Worth, TX).

(5) Gas-liquid exchange: after the vitreous was excised, gas-liquid exchange was performed to fill the vitreous cavity with sterile air, and 0.1ml of the ophthalmic linear gel was injected to the 2PD site below the optic disc through the incision using a blunt needle to adhere it to the retina. After the operation, the cannula was removed and the incision was massaged with a cotton swab.

(6) Tobramycin dexamethasone eye ointment (Dianbishu ointment, Alcon-couvreur, Belgium) is applied to eyes and bandaged with gauze.

3. Post-operative observation

(1) Eye dropping: beautiful 4 times/day; dianshu ointment for 3 times a day. The administration time is 1 week.

(2) Intraocular pressure: binocular tonometery was performed at 1d, 3d, 5d, 7d, 14d, 1 month post-operatively using an ICARE hand-held rebound tonometer (Tonolab, Icare Finland Oy, Vantaa, Finland) and recorded. The average was taken 5 times in succession.

(3) And (3) slit lamp inspection: after the operation, the eye slit lamps (SLM-7E, Chongqing Conghuaming science and technology Co., Ltd.) were examined at 1d, 3d, 5d, 7d, 14d and 1 month, and the cornea transparency, the inflammation in the anterior chamber, the cataract development and the like were observed and recorded.

(4) Fundus examination: the vitreous cavity and retinal condition of the fundus of the eye were observed and recorded under a front lens (Bl 10011, VOLK, USA) at 1d, 3d, 5d, 7d, 14d, 1 month after surgery.

4. And (3) postoperative examination: ERG, HE staining of retinal sections

(1) The binocular ERG examination was performed again after one month as before.

(2) After completion of the ERG examination, the experimental rabbits were sacrificed by air embolism with 100ml of air injected intravenously at the ear margin. The eye was removed immediately and the retinal histopathology was examined.

a. Fixing: placing into mixed fixing solution (containing 10% neutral formalin solution, glacial acetic acid, distilled water, and 95% alcohol).

b. Taking a specimen: after overnight at room temperature, the liquid in the cornea, lens and vitreous body was removed and transferred to 10% neutral formalin for sectioning.

c. Gradient dehydration: 50%, 60%, 70%, 80%, 90%, 95% alcohol for 30min each, and 100% alcohol for 30min × 2 each.

d. And (3) transparency: the xylene was clear for 2 h.

e. Wax dipping: pre-mixing xylene and 52 deg.C paraffin for 30min, mixing 65-75 deg.C beeswax and 56 deg.C paraffin for 3 hr.

f. Embedding the section: the above mixed wax is embedded, and 10 pieces of slices are continuously sliced, and the thickness is 3 um.

g. Surface mounting: baking at 60 deg.C for 30 min.

h. Dewaxing: xylene was dewaxed for 5min × 2 times.

i. Rehydration: dewatering with 100% alcohol for each lmin × 2 times, and washing with 95%, 90%, 80%, and 70% alcohol for each 3min with tap water for 5 min.

He staining: hematoxylin staining is carried out for 2min, tap water washing is carried out for 1min, 1% hydrochloric acid alcohol is differentiated for 20s, tap water washing is carried out for 2min, differentiation is stopped and the mixture returns to blue, eosin staining is carried out for 30s, tap water washing is carried out for 30s, 70%, 80%, 90% and 95% alcohol are respectively dehydrated for 1min, 100% alcohol is dehydrated for 2min multiplied by 2 times, and dimethylbenzene is transparent for 2min multiplied by 2 times.

k. Sealing: and (5) sealing the neutral gum.

l, observing with a light lens and shooting.

Third, the effectiveness of the linear gel for eyes in rabbit hole-originated retinal detachment model

1. Molding die

(1) General anesthesia and operation of mydriasis of eye, disinfection, 25G standard pars plana triple channel vitrectomy as described above.

(2) Making retinal tears and retinal detachments: after removal of the vitreous, the vitreous cutting head cuts the retina at about 2PD below the disc, creating a retinal tear of about 1/2PD size, and injecting balanced salt solution under the retina through the tear to detach the retina in the range of about 2PD size.

(3) Gas-liquid exchange: performing gas-liquid exchange to reposition retina, and filling vitreous cavity with sterile air.

(4) The experimental group completely covered the surface of the cleavage hole with the blunt needle by cutting the incision, and the injection amount was determined as the standard that the cleavage hole was completely covered with the blunt needle (see fig. 2 for the specific procedure). The control group did not have this operation.

(5) After the operation, the cannula is removed and the incision is massaged with a cotton swab. Dianshu ointment is applied to eyes and wrapped with gauze.

2. Post-operative observation

(2) Intraocular pressure: intraocular pressure measurements were performed on both eyes at 1d, 3d, 5d, 7d, 14d, 1 month post-operatively using an ICARE handheld rebound tonometer and recorded. The average was taken 5 times in succession.

3. And (3) postoperative examination: fundic phase, B-ultrasound, retinal HE staining and immunohistochemistry

(1) Fundus phase: eye fundus examination was performed at 1 week, 1 month, and 3 months after surgery (CR-2, Canon Inc., Tokyo, Japan). After the operative eye is beautiful and magnificent, the rabbit is fixed in the homemade animal clamp, the rabbit is moved to the fundus phase, the operative eye is adjusted to align with the lens, the angle is adjusted, and the retinal hole is shot.

(2) B, ultrasonic wave: the eye B ultrasonic examination (MD-2300S, Tianjin Mida medical science and technology, Inc.) was performed at 1 week, 1 month and 3 months after the operation. After the ultrasonic couplant is coated on the operative eyes, the probes are adjusted to shoot the retina and the fissure below the optic disc in each direction.

As a result:

first, ocular linear gel cohesiveness

The detection results of QST, DVS and BDDE in water dispersion method are shown in figure 3 at 15s, 75s, 90s, 5min and 10 min. The staining at each time point gave a lower degree of linear gel dispersion than the other two products.

Safety of ophthalmic linear gel

1. General condition of the eye after operation

All experimental animals survived healthily after one month after operation, no infection symptoms of the eyes, transparent cornea inspected by a slit lamp and a front lens, no anterior chamber or vitreous chamber inflammation, no cataract, clear eyeground, clear optic disc boundary, proper color and in-place retina.

2. Intraocular pressure

The results of repeated tonometry and non-parametric tests for multiple sample pairings are shown in table 1 and fig. 4. The intraocular pressure fluctuation is large within 3 days after operation, and the range is 22.41 +/-6.00 mmHg-13.95 +/-4.59 mmHg. The level recovered to preoperative level 1 week after surgery, and there was no statistical difference in intraocular pressure change after preoperative surgery (P ═ 0.086).

TABLE 1 eye pressure changes at various time points before and after eye linear gel safety test

3. Electrophysiological function

The results of the incubation period and amplitude of each examination of the operative eye ERG are shown in table 2 and table 3, respectively, and there was no statistical difference in the incubation period and amplitude change before and after the operation (P > 0.05).

TABLE 2 latent period (ms) change of retina ERG before and after eye linear gel safety experiment

TABLE 3 amplitude (uv) Change in retinal ERG before and after QST safety experiments

4. Histopathological examination of the retina

The results of HE staining of retinal sections of non-operative (right) and operative (left) eyes are shown in fig. 5, the two groups of retinas were stained uniformly, the structure was complete, the layers were arranged in order, and no significant difference was observed.

Effectiveness of first, ophthalmic Linear gels

1. General condition of the eye after operation

All experimental animals survived healthily for 3 months after operation, no infection signs in the eyes, clear cornea and examination by slit lamp, no inflammation in the anterior chamber, and mild to moderate cataract development in the experimental group and the control group.

2. Rate of retinal replacement in experimental and control groups

By the end of the observation period, the experimental and control groups were repositioned as shown in table 4 and fig. 6, with the retinas in place in the 8 eyes of the experimental group (100% retinal repositioning rate); retinas from control eyes 3 and 4 were in place and retinas from the remaining 6 eyes were detached (retinal return rate 25%). Two groups of retinas were statistically significantly different compared (P0.007).

TABLE 4 comparison of retinal replacement rates between experimental and control groups

V: the retina is in place; x: retinal detachment

Discussion:

1. it has better cohesion than eye linear gel: compared with other cross-linked sodium hyaluronate gels, the cross-linked sodium hyaluronate gel has the advantages that the gel presents obvious linear arrangement through chemical cross-linking, has obviously improved cohesiveness, can be effectively adhered to the surface of retina for a long time due to high dynamic viscosity, is not easy to move, covers retinal fissures, prevents liquid from entering subretina, and also prevents retinal pigment epithelial cells from entering a vitreous cavity and generating series cascade reactions such as endothelial mesenchymal transition, hyperplasia and the like.

2. It has better safety than eye linear gel: the safety experiment result shows that the retina ERG before and after the operation has no obvious change; compared with non-operative eyes, the HE staining of the retinal section of the operative eye has no obvious change, and the HE staining of the retinal section of the operative eye has no influence on the form and the function of the retina compared with linear gel for eyes, and is safe and non-toxic. Compared with the intraocular pressure before operation, the intraocular pressure is not obviously changed after 1 day of rabbit eye operation, the intraocular pressure is reduced after 3 days of rabbit eye operation, the intraocular pressure is recovered to the preoperative level after 1 week, and the intraocular pressure fluctuation is considered to be related to the non-suture of the incision after 25G PPV operation and sterile air filling. Meanwhile, the tunnel incision also reduces the leakage after 25G PPV operation to a certain extent. Although there is fluctuation in intraocular pressure after operation, it can recover by itself, and the change has no statistical significance.

3. It is superior to linear ophthalmic gel effectiveness: the result of the effectiveness experiment shows that the ophthalmic linear gel can effectively treat rabbit RRD model by covering retinal fissure holes with 25G PPV and filling sterile air. In the research, the retinas of the experimental group are all in place, the retinas of the control group are detached, the retinas of 2 eyes are in place, the wound repair function of rabbit eyes is presumed to be strong, in addition, after the retina is cut by a vitreous body cutting head to manufacture a fissure hole in the operation, a small amount of balanced salt solution enters the subretinas in a short time, then, gas-liquid exchange is carried out to reset the retinas, the RPE exposure time is short, no pigment or only a small amount of pigment exists in a vitreous body cavity, and animal experiments show that eyeballs have good tolerance to limited RPE damage and no liquid enters subretinal gaps, so the pathophysiological process of human RRD cannot be completely simulated.

4. To summarize: the eye-friendly anti-tumor gel can effectively treat experimental RRD by combining eye linear gel with 25G PPV and sterile air filling, and is high in safety.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Claims

1. The application of the eye linear gel as a clinical hole-induced retinal detachment medicine comprises the following components:

each 1mL of gel contains 16mg of cross-linked sodium hyaluronate and is prepared from physiological equilibrium liquid; the physiological equilibrium liquid consists of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection;

the preparation method of the gel comprises the following steps:

the method comprises the steps of using low-concentration hyaluronic acid of non-animal origin produced by Shanghai shongsheng biological preparation company Limited as a raw material, performing chemical crosslinking by using a DVS crosslinking agent, performing fractional precipitation by using an organic solvent to obtain dry powder of crosslinked HA, and performing redissolution to form gel, wherein the gel HAs the specific gravity of 1.01, the refractive index i of 1.341 and the dynamic viscosity of 186,733 mPa.

2. The application of an ophthalmic linear gel in a rabbit rhegmatogenous retinal detachment model, wherein the composition of the ophthalmic linear gel is as follows:

the preparation method of the gel comprises the following steps:

3. The application of the eye linear gel in the aspect of serving as a medicine for macular hole or diabetic retinopathy or high-myopia fundus lesion or refractory low intraocular pressure is disclosed, wherein the eye linear gel consists of the following components in percentage by weight:

each 1mL of gel contains 16mg of cross-linked sodium hyaluronate and is prepared from physiological equilibrium liquid; the physiological equilibrium solution consists of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection;

the preparation method of the gel comprises the following steps: