CN110584822B - Bolt line for preparing animal cerebral ischemia model and production method and application thereof - Google Patents

Abstract

The invention relates to a bolt line for preparing an animal cerebral ischemia model, a production method and application thereof. In particular, the invention relates to a suppository for preparing a model of middle cerebral artery occlusion (sMCAO) by a wire-suppository method, a production method thereof and application thereof in preparing the model of middle cerebral artery occlusion (sMCAO) by an animal wire-suppository method.

Description

Bolt line for preparing animal cerebral ischemia model and production method and application thereof

Technical Field

The present invention relates generally to a tether for use in preparing an animal brain ischemia model, and a method of producing and using the same. In particular, the invention relates to a suppository for preparing a model of middle cerebral artery occlusion (sMCAO) by a wire-suppository method, a production method thereof and application thereof in preparing the model of middle cerebral artery occlusion (sMCAO) by an animal wire-suppository method.

Background

Ischemic stroke is the leading fatal, disabling disease worldwide. Basic research and drug development for the prevention, treatment and rehabilitation of ischemic stroke are global urgent medical problems, and cerebral ischemia animal models are an indispensable important link for researching the medical problems.

Currently, the most commonly used cerebral ischemia animal model is a rat or mouse middle cerebral artery occlusion (Suture Middle Cerebral Artery Occlusion, sMCAO) model (hereinafter referred to as sMCAO model), i.e. a small incision is cut on the common carotid artery or the external carotid artery, so that the incision is inserted into a processed monofilament nylon wire, the nylon wire is pushed toward the internal carotid artery until the front end of the nylon wire enters the cranium along the internal carotid artery, reaches the initial part of the middle cerebral artery (Middle Cerebral Artery, MCA), blocks the blood flow of MCA, and causes ischemia necrosis of brain tissue in the MCA blood supply area.

The sMCAO model has small operation trauma to experimental animals, can simulate human ischemic cerebral apoplexy, especially can realize MCA blood circulation recanalization by pulling out nylon wires from the cranium after blocking MCA blood flow for a period of time, thereby conveniently controlling ischemia time or ischemia degree, and being widely applied at home and abroad. In the literature report (Str foster et al, BMC Neuroscience,2013; 14:41), 78.3% of the papers published with the rat middle cerebral artery occlusion model used the sMCAO rat model. Early sMCAO models were mainly performed in rats, and mice are now being used as sMCAO models in more and more, especially in transgenic mice. The monofilament nylon wire used for preparing the sMCAO model is called sMCAO bolt wire for short. The sMCAO tether is one of the most critical links in determining success and failure of the sMCAO model. The preparation method of rat or mouse sMCAO suppository is similar. Since the blood vessels of the mice are finer, the mice sMCAO plug wire requires the use of finer nylon wire. There are a number of disadvantages to the currently commercially available or literature-published sMCAO plugs produced using prior art techniques.

The prior art for preparing rat sMCAO tethers mainly comprises three methods: (1) Enrique Zea Longa et al (Stroke, 1989; 20:84-91), i.e., a monofilament nylon wire having a length of 5cm, a wire number of 4-0 or 5-0, is heated to a rounded shape at one end adjacent to the flame. We refer to such an sMCAO tether simply as a "single ball tether". The disadvantages of single ball tethers are: because only the tip has a single point expansion into a sphere, when the tip pellet is inserted into the anterior cerebral artery, although blood flow from the distal end of the anterior cerebral artery can be well blocked, part of blood from the internal carotid artery or the posterior transport artery can enter the MCA along the gap between the thinner nylon wire and the inner wall of the blood vessel, so that blood flow entering the MCA is not completely blocked, and the inter-animal ischemia range has large variation (Laing et al, strokes, 1993; 24:294-297;Norihito Shimamura et al, journal of Neuroscience Methods, 2006, 156:161-165); (2) The technical method reported by Koizumi J et al (Jpn J Stroke, 1986; 8:1-8), i.e., a section of one end 5mm of a No. 4-0 monofilament nylon wire was coated with liquid silicone rubber. The diameter of the solidified silicone rubber is thickened, and the thickness of the silicone rubber coating can be adjusted according to the weight of animals. We refer to such mcao bolts simply as "silicone rubber bolts". The disadvantages of silicone rubber ties are: the silicone rubber is easy to fall off, the surface of the silicone rubber is stagnant, the running resistance in blood vessels and the damage to the blood vessels are increased, and the flexibility of the silicone rubber part is reduced, so that the difficulty is increased when the silicone rubber part enters the cranium; (3) Single ball tethers prepared by the Enrique Zea Longa method were coated with 0.1% (w/v) poly-L-lysine (Belayev et al, stroke, 1996; 27:1616-1623). After coating, the diameter of the bolt is not increased, and we will refer to the bolt as lysine bolt hereinafter. Compared with a single-balloon tether, the lysine tether can improve the success rate and stability of the sMCAO model, but still can not achieve the blocking effect of the silicone rubber tether on MCA, and increase the incidence of cerebral hemorrhage and thrombosis of the sMCAO model, so that failure is caused, and therefore, the method has lower acceptance in the industry. The literature reports that the occupancy of single ball, silicone and lysine tethers in the rat sMCAO model market is 40.7%, 36.9% and 10.7%, respectively (Strm et al, BMC Neuroscience,2013; 14:41). The three bolt line physical photographs are shown in figure 1.

The prior art method for preparing mouse sMCAO suppository wire is the same as the above three methods, and generally uses 6-0, 7-0 or 8-0 monofilament nylon wire, and the diameter of heated ball end or silicone rubber coated end is smaller than that of rat. The disadvantages of the mouse sMCAO tether are similar to those of the rat sMCAO tether.

Disclosure of Invention

In order to solve the defects of the prior sMCAO wire, the invention provides a novel sMCAO wire and a production method thereof.

The invention aims to provide a novel sMCAO tether with two bulbs. We refer to such an sMCAO tether simply as a "double ball tether". The double ball tether has one ball at the top of the tether and a second ball spaced a distance apart (see figures 1-3 for photographs).

In one embodiment, the invention provides a tether for use in preparing a model of a middle cerebral artery occlusion (sMCAO) by the tether method, characterized by two adjacent smooth surfaced bulbs at one end of the tether, wherein a first bulb is at the tip and a second bulb is spaced a distance from the first bulb, the diameter of the bulb spacing portion is less than the diameter of the first bulb, and the maximum diameter of the first bulb is less than or equal to the maximum diameter of the second bulb. In another embodiment, the minimum diameter of the bulb spacer portion is equal to the diameter of the feed line. In another embodiment, the two spheres are spaced apart a distance of 0.5mm to 10mm, preferably 1mm to 5mm. In another embodiment, the maximum diameter of the bulb ranges from 0.10mm to 1mm, preferably from 0.15mm to 0.5mm.

In one embodiment, the raw wire used in the double ball tether of the present invention has good biocompatibility and has certain flexibility, elasticity and hardness. In another embodiment, the feed line is made from a high molecular polymer material, a biological material, or a metallic material. In a preferred embodiment, the high molecular polymer material is selected from the group consisting of polyamide, polypropylene, polyurethane, polyethylene, polytetrafluoroethylene, and the like. In another embodiment, the feed line is a medical monofilament nylon line.

In one embodiment, the diameter of the feed line used in the dual ball tether of the present invention is in the range of 0.001mm to 0.5mm. In another embodiment, the stock line is commonly used in specification number 0, 2-0, 3-0, 4-0, 5-0, 6-0, 7-0, 8-0, 9-0, 10-0 or 11-0, preferably 3-0, 4-0, 5-0, 6-0, 7-0 or 8-0 according to the United states national formulary (USP) standard.

In one embodiment, both spheres of the dual ball tether of the present invention are made by heat treatment (fig. 2). In one embodiment, both spheres of the dual ball tether of the present invention are made by a glue process. In one embodiment, one ball of the dual ball tether of the present invention is made by a heat treatment and the other ball is made by a glue treatment. In another embodiment, the top ball of the double ball tether of the present invention is made by a heat treatment and the other ball is made by a glue treatment (fig. 3). Preferably, both spheres of the dual ball tether of the present invention are made by heat treatment.

In one embodiment, the length and diameter of the feed line used in the double ball tether of the present invention is dependent upon the type of experimental animal used. In one embodiment, the double ball tether of the present invention is used in rats having a length of 2.5-8cm, preferably 2.5-5cm, and a diameter of preferably 3-0, 4-0 or 5-0. In one embodiment, the double ball tether of the present invention is used in mice having a length of 1.5-5cm, preferably 1.5-3cm, and a diameter of preferably 5-0, 6-0, 7-0 or 8-0. In one embodiment, the double ball tether of the present invention is used in rabbits, having a length of 6-15cm, preferably 6-10cm, and a diameter of preferably No. 2-0 or No. 3-0.

In one embodiment, the present invention provides a method of producing a double ball bond comprising the steps of:

(1) Cutting the raw material line into line segments with proper lengths;

(2) Forming a first ball by heat treatment or glue coating at one end of the wire segment;

(3) Forming a second ball by heat treatment or glue coating at a location spaced from the first ball; and

(4) The bolt is cleaned, marked and sterilized.

In one embodiment, the invention provides the use of a double balloon catheter for the preparation of a model of arterial occlusion (sMCAO) in the brain by animal wire-embolization. In one embodiment, the animal is selected from the group consisting of rat, mouse, and rabbit.

The dual ball tether of the present invention avoids or greatly reduces the disadvantages of the three tethers described above while still retaining the advantages of each.

Drawings

FIG. 1 is a photograph of four sMCAO studs (single ball studs, lysine studs, double ball studs and silicone rubber studs, respectively, from left to right).

Fig. 2 is a double balloon tether made by a heat treatment process using medical monofilament nylon suture.

Fig. 3 is a double bulb tether made by heat treatment and glue coating process using medical monofilament nylon suture (top bulb made by heat treatment and rear bulb made by glue coating process).

Detailed Description

The invention relates to a bolt line for preparing an animal cerebral ischemia model, a production method and application thereof. In particular, the invention relates to a suppository for preparing a model of middle cerebral artery occlusion (sMCAO) by a wire-suppository method, a production method thereof and application thereof in preparing the model of middle cerebral artery occlusion (sMCAO) by an animal wire-suppository method.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Generally, the nomenclature used in connection with the animal brain ischemia models, anatomies, and neurobiology described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Laboratory procedures and techniques described herein are those well known and commonly used in the art.

In order that the invention may be more readily understood, selected terms are defined below.

As used herein, the term "single ball bolt" refers to a bolt made according to the technical method reported by Enrique Zea Longa et al (Stroke, 1989; 20:84-91), i.e., a monofilament nylon wire having a length of 5cm, a wire number of 4-0 or 5-0 is heated to a rounded shape at one end thereof adjacent to a flame.

As used herein, the term "silicone rubber cord" refers to a cord made according to the technical method reported by Koizumi J et al (Jpn J Stroke, 1986; 8:1-8), i.e., a length of one end 5mm of a 4-0 monofilament nylon cord is coated with liquid silicone rubber.

As used herein, the term "lysine tether" refers to a tether made by coating a single ball tether prepared by the Enrique Zea Longa method with 0.1% (weight/volume) poly-L-lysine (Belayev et al, stroke, 1996; 27:1616-1623).

As used herein, the term "double ball tether" refers to an mcao tether having two balls according to the present invention.

As used herein, the term "bulb spacer portion" refers to the portion between the cross sections of the two bulbs where the largest diameter is measured perpendicular to the feed line.

As used herein, the term "maximum diameter of a sphere" refers to the maximum diameter of a sphere measured perpendicular to the feed line.

As used herein, the term "two-ball separation distance" refers to the distance between the cross-sections at which the maximum diameters of the two balls lie.

As used herein, the term "heat treatment" refers to the process of burning a feedstock line with heat to form spheres.

As used herein, the term "gumming treatment" refers to the application of an appropriate amount of a settable liquid or colloidal substance, such as liquid silicone rubber, epoxy, dental cement, etc., to a raw material line. The smeared droplets solidify to form spheres.

As used herein, "comprising" means "including but not limited to" in the description and claims of the present invention.

I. Production of double ball bolt line

The invention relates to a suppository for preparing a model of middle cerebral artery occlusion (sMCAO) by a wire-suppository method, which is characterized in that two adjacent balls with smooth surfaces are arranged at one end of a raw wire, wherein a first ball is arranged at the top end, a second ball is arranged at a certain distance from the first ball, the diameter of a ball spacing part is smaller than that of the first ball, and the maximum diameter of the first ball is smaller than or equal to that of the second ball. Such a tether for preparing the sMCAO model is simply referred to as a "double ball tether".

The double ball bolt of the present invention is prepared by processing one end of a stock line into two adjacent smooth surfaced balls, one ball on top and the other ball spaced a distance from the top ball. The two spheres are spaced apart by a distance of 0.5mm to 10mm, for example 0.5mm, 1mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm or 10mm. Preferably, the two spheres are spaced apart by a distance of 1mm to 5mm, such as 1mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm or 5mm. The method for manufacturing the ball of the double ball bolt wire comprises heat treatment and glue coating treatment. The two balls of the double ball bolt line can be manufactured through heat treatment, can be manufactured through glue coating treatment, and can be mixed for use. For example, the top ball is made by a heat treatment and the other ball is made by a glue treatment.

In one embodiment, the maximum diameter of the bulb of the double bulb bolt of the present invention ranges from 0.10mm to 1mm, such as 0.10mm, 0.15mm, 0.20 mm, 0.25 mm, 0.30 mm, 0.35 mm, 0.40 mm, 0.45 mm, 0.50 mm, 0.55 mm, 0.60 mm, 0.65 mm, 0.70 mm, 0.75 mm, 0.80 mm, 0.85 mm, 0.90 mm, 0.95 mm, or 1mm. Preferably, the maximum diameter of the ball ranges from 0.15mm to 0.50 mm, such as 0.15mm, 0.16 mm, 0.17 mm, 0.18 mm, 0.19 mm, 0.20 mm, 0.21 mm, 0.22 mm, 0.23 mm, 0.24 mm, 0.25 mm, 0.26 mm, 0.27 mm, 0.28 mm, 0.29 mm, 0.30 mm, 0.31 mm, 0.32 mm, 0.33 mm, 0.34 mm, 0.35 mm, 0.36 mm, 0.37 mm, 0.38 mm, 0.39 mm, 0.40 mm, 0.41 mm, 0.42 mm, 0.43 mm, 0.44 mm, 0.45 mm, 0.46 mm, 0.5237, 0.52 37, 0.48 mm, 0.49 or 0.50. In one embodiment, the maximum diameter of the bulb of the double bulb bolt of the present invention is dependent on the species and weight of the experimental animal. The preferred maximum diameters of the tip and rear spheres for the double bulb tethers for rabbits, rats and mice of different body weights are shown in the following table.

Table 1. Maximum diameter range of spheres for double-sphere tethers of scao models for experimental animals of different body weights.

Remarks: for each double ball tether, the maximum diameter of the top ball should be less than or equal to the maximum diameter of the rear ball.

In certain cases, the size of the spheroids can be determined by routine mcao animal experiments.

In one embodiment, the diameter of the feed line used in the twin-ball tether of the present invention is in the range of 0.001mm to 0.5mm, such as 0.001mm, 0.005 mm, 0.01 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.10mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, 0.15mm, 0.16 mm, 0.17 mm, 0.18 mm, 0.19 mm, 0.20 mm, 0.21 mm, 0.22 mm, 0.23 mm, 0.24 mm, 0.25 mm, 0.26 mm, 0.27 mm, 0.28 mm, 0.29 mm, 0.30 mm, 0.35 mm, 0.40 mm, 0.5237 or 0.5237, 0.17 or 0.50, preferably, is in accordance with the U.S. Pat. No. 4-5, 0.7-0.5-6, 0.7, 0.8-5-7, 0.5-7, 0.8-6-2, or 0.8-7.

II, application of double ball bolt line

The present disclosure also provides methods of preparing an animal brain ischemia model, particularly a method of preparing an animal lineembolization middle cerebral artery occlusion (sMCAO) model, using the double balloon embolization described herein. In the practice of the present disclosure, methods well known in the art for performing anesthesia, surgery, and assessment of neurological deficit conditions in experimental animals are used.

When the double ball tether of the present invention is used in rats, it has a length of 2.5-8cm, such as 2.5 cm, 3cm, 3.5 cm, 4 cm, 4.5 cm, 5cm, 5.5 cm, 6 cm, 6.5 cm, 7 cm, 7.5 cm and 8cm, preferably a length of 2.5-5cm, such as 2.5 cm, 3cm, 3.5 cm, 4 cm, 4.5 cm and 5cm. When the double ball tether of the present invention is used in mice, it has a length of 1.5-5cm, such as 1.5 cm, 2 cm, 2.5 cm, 3cm, 3.5 cm, 4 cm, 4.5 cm and 5cm, preferably a length of 1.5-3cm, such as 1.5 cm, 2 cm, 2.5 cm and 3cm. When the double ball tether of the present invention is used in rabbits, it is 6-15cm, e.g., 6 cm, 6.5 cm, 7 cm, 7.5 cm, 8cm, 8.5 cm, 9 cm, 9.5 cm, 10cm, 10.5 cm, 11 cm, 11.5 cm, 12 cm, 12.5 cm, 13 cm, 13.5 cm, 14 cm, 14.5 cm, and 15cm, preferably 6-10cm, e.g., 6 cm, 6.5 cm, 7 cm, 7.5 cm, 8cm, 8.5 cm, 9 cm, 9.5 cm, and 10cm.

While not being bound by a particular theory, the anterior and posterior bulbs of the dual bulb tether of the present invention block blood flow from the distal (e.g., anterior cerebral artery) and proximal (e.g., internal carotid artery or posterior cerebral artery), respectively, into the MCA blood supply region, thereby increasing the success rate, stability, repeatability of the cerebral ischemia model. The double balloon tether does not need to be coated with polylysine, thereby avoiding adverse effects of polylysine on the model (e.g., possible cerebral hemorrhage, cerebral thrombosis, or effects of lysine itself on blood vessels or blood components). The double-ball bolt wire does not need to be coated with silicon rubber or only needs to be coated in a punctiform manner (such as coating 1 or 2 points to form a silicon rubber ball), so that compared with the silicon rubber bolt wire prepared by the prior art, the hardness of the double-ball bolt wire is little increased, the flexibility is good, the double-ball bolt wire can more easily pass through a skull hole to enter the cranium, the friction force or the resistance is smaller when the double-ball bolt wire runs in a blood vessel, and the damage to the blood vessel is reduced.

The balloon at the tip of the double balloon tether is very smooth and has little mechanical damage to vascular endothelial cells and is used for guiding the blood flow to the front cerebral artery initiation part in the blood vessel and blocking the blood flow from the distal front traffic artery or other blood vessels. The posterior balloon blocks blood flow from the posterior transport artery or the internal carotid artery. The two spheres block blood flow in both the anterior and posterior directions of the MCA initiation, resulting in a complete, stable mcao. In contrast, a tether with only one bulb at the top, while blocking blood flow from the distal end, does not block blood flow from the posterior or internal carotid artery well due to the thin raw wire at the posterior segment of the bulb, resulting in relatively poor stability in the infarct zone. In contrast, the front end of the tether uniformly coated with silicone rubber, although blocking the blood flow of MCA well, has reduced flexibility of the silicone rubber portion, large contact area with the inner surface of the blood vessel and increased friction, with the result that the resistance of the tether entering the cranium inside the blood vessel is increased and damage to the vascular endothelium is increased. In addition, silicone rubber is easily detached. Once the silicone is detached, the silicone tether cannot be reused. If the silicone rubber falls off in the blood vessel, the sMCAO model fails. The double-ball bolt line only has front and back smooth balls, and still keeps better flexibility, smaller contact area with the blood vessel and reduced friction resistance with the blood vessel. In addition, if two balls of the double-ball bolt line are formed by melting by heating treatment of medical nylon wires, the line and the balls are integrated, and the balls cannot fall off, so that the double-ball bolt line can be repeatedly used for a plurality of times, and the cost is greatly reduced. As the ball-shaped objects can not fall off, the utility model is convenient for cleaning and disinfection when being repeatedly used. The pure nylon double-ball bolt wire does not need to be coated with other materials, so that the interference of coating material components on experimental results is avoided.

Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Abbreviations have the following meanings: "mm" means millimeter, "cm" means centimeter, "g" means gram, "mg" means milligrams, "kg" means kilograms, "ml" means milliliters, "min" means minutes, "ip" means intraperitoneally.

Example 1: manufacturing of double-ball bolt line

(1) Manufacturing a double ball bolt line by a heat treatment method: a medical monofilament nylon thread (or monofilament nylon Long Yu thread) of 4-0 is cut into 5-cm line segments. One end was slowly burned under a microscope near a heat source (e.g., an electric iron) to form a smooth ball with a diameter of 0.34.+ -. 0.01 mm, and heated near the heat source at a position 5mm from the top ball to form a ball with a diameter of 0.36.+ -. 0.01 mm. And cleaning the bolt line. Marking is done at a distance from the tip 19-20 mm. And (5) disinfecting for later use. The model of the bolt line is suitable for a rat sMCAO model with the weight of 240-270 g.

(2) The double ball bolt line is manufactured by a gluing treatment method: a medical monofilament nylon thread (or monofilament nylon Long Yu thread) of 4-0 is cut into 5-cm line segments. One end was coated with an appropriate amount of liquid silicone rubber (or epoxy) under a microscope to form a sphere with a diameter of 0.34±0.01 mm, and a position 5mm spaced from the top sphere was coated with an appropriate amount of liquid silicone rubber (or epoxy) to form a sphere with a diameter of 0.36±0.01 mm. After the silicone rubber or epoxy resin is sufficiently solidified, the tether is cleaned. Marking is done at a distance from the tip 19-20 mm. And (5) disinfecting for later use. The model of the bolt line is suitable for a rat sMCAO model with the weight of 240-270 g.

(3) The double ball bolt line is manufactured by a heat treatment method and a gluing treatment method: a medical monofilament nylon thread (or monofilament nylon Long Yu thread) of 4-0 is cut into 5-cm line segments. One end of the glass is slowly burned and melted into a smooth ball with the diameter of 0.34+/-0.01 and mm by a heat source (such as an electric iron) under a microscope, and a proper amount of liquid silicone rubber (or epoxy resin) is smeared at a position which is 5mm away from the ball at the top end to form a ball with the diameter of 0.36+/-0.01 and mm. After the silicone rubber (or epoxy) is sufficiently cured, the tether is cleaned. Marking is done at a distance from the tip 19-20 mm. And (5) disinfecting for later use. See fig. 3. The model of the bolt line is suitable for a rat sMCAO model with the weight of 240-270 g.

(4) Mouse scao model double sphere bolt line production: a medical monofilament nylon wire (or a monofilament nylon Long Yu wire) of 6-0 is cut into 3-cm line segments. One end of the ball was made into a smooth ball with a diameter of 0.20.+ -. 0.01. 0.01 mm under a microscope by one of the above 3 methods, and a ball with a diameter of 0.22.+ -. 0.01 mm was made at a position 3 mm from the top ball. And cleaning the bolt line. Marking is done at a distance of 9-10 a mm from the tip. And (5) disinfecting for later use. The model of the bolt line is suitable for a mouse sMCAO model with the weight of 25-30 g.

(5) Preparation of other body weight rats or mice sMCAO model double ball bolt: referring to table 1 and the above-mentioned method of manufacturing a double ball tether, a double ball tether suitable for rats of different body weights, mice or other animals of similar body weights may be manufactured.

(6) Manufacturing a rabbit sMCAO model double-ball bolt line: a No. 2-0 monofilament nylon wire is cut into 10cm line segments. One of the 3 methods (1), (2) and (3) above was used to form a smooth sphere with a diameter of 0.50.+ -. 0.01 mm at one end and a sphere with a diameter of 0.53.+ -. 0.01 mm at a distance of 8 mm from the top sphere under a microscope. And cleaning the bolt line. Marking is done at a distance from the tip 50 mm. And (5) disinfecting for later use. The model of the bolt line is suitable for rabbit sMCAO model with the weight of 2400-2800 g.

Example 2: comparing the effects of different suppositories to prepare rat sMCAO model

Preparation of double ball bolt wire: prepared according to the method of the invention. Namely, a medical monofilament nylon thread of 4-0 is cut into a line segment of 5cm. One end was slowly burned under a microscope near a heat source (e.g., an electric iron) to form a smooth ball with a diameter of 0.34.+ -. 0.01 mm, and heated near the heat source at a position 5mm from the top ball to form a ball with a diameter of 0.36.+ -. 0.01 mm. And cleaning the bolt line. Marking is done at a distance from the tip 19-20 mm. And (5) disinfecting for later use.

Preparation of single ball bolt wire: prepared according to the technical method reported by Enrique Zea Longa et al (Stroke, 1989; 20:84-91). That is, one end of a monofilament nylon wire (the wire diameter is the same as that of the double ball plug wire) having a length of 5cm and a wire number of 4 to 0 was heated to a smooth bulb having a diameter of 0.34.+ -. 0.01 mm by flame. And cleaning the bolt line. Marking is done at a distance from the tip 19-20 mm. And (5) disinfecting for later use.

Lysine bolt preparation: the single-balloon strands were coated with 0.1% (w/v) poly-L-lysine (Sigma P1274, shanghai, china) (see Belayev et al, stroke, 1996; 27:1616-1623), without increasing the diameter of the strands after coating.

Preparation of a silicone rubber bolt: referring to the technical method reported by Koizumi J et al (Jpn J Stroke, 1986; 8:1-8), a section of one end 5mm of a No. 4-0 monofilament nylon wire was coated with a liquid silicone rubber (DOW CORNING 748RTV sealer). The diameter of the coated silicone rubber is 0.34+/-0.01 and mm. After the silicone rubber solidifies, the tether is cleaned. Marking is done at a distance from the tip 19-20 mm. And (5) disinfecting for later use.

Experimental grouping: 45 Sprague Dawley (SD) male rats weighing 240 g-270 g were randomly divided into five groups. (1) double ball tether group (n=10): blocking MCA by adopting a double ball bolt; (2) single ball tether group (n=10): blocking MCA by adopting a single ball bolt; (3) lysine-tether group (n=10): blocking MCA by using a bolt coated by poly-L-lysine; (4) silicone rubber tether group (n=9): blocking the MCA by adopting a silicone rubber bolt; (5) sham surgery control group (n=6): the double ball bolt is used for simulating the plug wire action, and the MCA is blocked for no more than 5 seconds. The first four groups of MCA blocks for 90 min, then the thrombus line is pulled out, and the MCA blood flow is restored to recharge. After a further 24 hours of filling, neurological deficit scoring was performed. The other experimental procedure for the sham surgery group was identical to the other four groups, except for the MCA blocking time. Sham rats do not show symptoms of cerebral ischemia after anesthesia and wakefulness.

The experimental process comprises the following steps: after inhalation anesthesia of rats with isoflurane, the rats were supine on a temperature-controlled operating table, and the anal temperature was maintained at 36.5-37.5 ℃. Blood pressure, heart rate and respiration are monitored and maintained in normal ranges. Erythromycin eye ointment is smeared on eyeballs, so that the dry and damaged corneas of the eyes are avoided. The procedure is described in the following in reference to the technical method reported by Enrique Zea Longa et al (Stroke, 1989; 20:84-91):

(1) Shaving off the skin in the middle of the neck, conventionally disinfecting, cutting the skin, separating the left Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) in sequence, blowing off the branch arteries of the ECA with a microvascular electrocoagulator, ligating the distal end of the ECA and blowing off.

(2) The CCA and ICA were temporarily clamped with an arterial clamp.

(3) A small opening was cut into the ECA using Vannas micro-scissors, and the tip of the tether was inserted into the ECA and advanced into the ICA. After the rear bulb or silicone rubber portion has completely entered the ECA, the ECA is gently ligated with surgical threads on the rear side of the rear bulb or silicone rubber portion.

(4) The ICA arterial clip was released and the tether was inserted intracranially along ICA. When the tip balloon is inserted into the Anterior Cerebral Artery (ACA) starter, a feeling of resistance is felt, stopping the wire, but typically no more than 20 mm. The surgical wires on the ECA were tightened, the CCA arterial clamp was loosened, and the MCA blocking was maintained for 90 min.

(5) After blocking for 90 min, the MCA again clamps the CCA, unclamping the ligature of the ECA. Immediately after the tether is completely pulled out of the cranium with great care, the ECA is ligated, and the arterial clamp of the CCA is released. The bupivacaine hydrochloride of about 0.2 ml is evenly dripped into the operation position, thereby relieving postoperative pain. Suturing the skin. The double ball bolt line and the single ball bolt line can be reused later. The poly-L-lysine coating is lost or non-uniform after use of the lysine plug wire and is therefore not reusable. The silicone cord is not reusable if silicone spalling or deformation occurs.

(6) The thrombus line of the rats in the control group is immediately pulled out after being inserted into the brain to a similar depth, and the time from blocking MCA to pulling out the thrombus line is not more than 5 seconds, which is regarded as sMCAO pseudo operation (the transient ischemia does not cause cerebral ischemia injury). Immediately after the bolt wire is pulled out, the ECA is tightly tied, and the CCA arterial clamp is loosened. The bupivacaine hydrochloride of about 0.2 ml is evenly dripped into the operation position, thereby relieving postoperative pain. Suturing the skin.

(7) Rats from each group were scored for neurological deficit 24 hours after sMCAO or sham surgery as reported by Rogers DC et al (Stroke, 1997, 28:2060-2066). The higher the score, the more severe the neurological deficit is, from a score of 0 to 4. No neurological deficit is 0 points; the ischemic contralateral forelimb was not fully extended 1 minute; rotating to one side for 2 minutes; dumping to the opposite side of ischemia for 3 minutes; the patients cannot walk spontaneously, and the consciousness is lost, which is 4 minutes.

(8) After scoring, all rats were subjected to deep anesthesia and then were cephalized and observed for subarachnoid hemorrhage and intravascular thrombosis.

Calculating success rates of various thrombus wire preparation sMCAO models: rats without intracranial subarachnoid hemorrhage and scored for neurological deficit of at least 1 score were considered model successful. Success rate (%) = number of rats successful in the model of the group/total number of rats of the group.

Experimental results:

the neurological scores and success rates of the individual groups of rats were found in table 2 after 90 min reperfusion for 24 hours or 24 hours after sham surgery. No manifestation of neurological deficit was observed in sham control rats, and scores were all 0 points, indicating that the surgical procedure and simulated patch procedure did not cause neurological deficit. In the other four groups, all rats in the double balloon-line group exhibited 1-3 minutes of neurological deficit symptoms, and had no subarachnoid hemorrhage. The success rate of the group of models is 100%; the single ball bolt line group, the lysine bolt line group and the silicone rubber bolt line group respectively have the manifestation of no nerve function defect of 3, 1 and 1 rats, and the single ball bolt line group and the lysine bolt line group respectively have the manifestation of intracranial subarachnoid hemorrhage of 1 and 2 rats. The model success rates for these three groups were 60%, 70% and 88.9%, respectively.

The double ball bolt line group and the single ball bolt line group have no intravascular thrombosis caused by the plug wire, and the lysine bolt line group and the silicone rubber bolt line group have one rat respectively to observe intravascular thrombosis caused by the plug wire. This suggests that a thrombus formation may be caused by a cord coated with poly-L-lysine or silicone rubber.

Statistical analysis is carried out on the scoring result, and the result shows that the nerve defect symptoms of the double-balloon-wire group are obviously more severe than those of the single-balloon-wire group or the lysine-wire groupp<0.05 orp<0.005 Suggesting that the double balloon tether may better block blood flow of MCA than either the single balloon tether or the lysine tether. Although the nerve defect symptoms of the double-ball bolt line group and the silicon rubber bolt line group have no statistical differencep>0.05 But the wire plugging operation of the double-ball bolt wire is more convenient and time-saving than that of a silicon rubber bolt wire, and the smooth ball end has little damage to the vascular endothelium (the double-ball bolt wire group rat does not find intravascular thrombus). In addition, the silicone rubber bolt can not be used due to the falling off of the silicone rubber after being used for 1-2 times; the double-ball bolt is coated with no silicone rubber, so that the problem of silicone rubber falling off is avoided, the cleaning and the disinfection are very convenient, and the double-ball bolt can be reused for more times.

In conclusion, the double-ball suppository is obviously superior to the silicone rubber suppository and other suppositories in terms of the success rate of preparing the sMCAO model, thrombosis related to plug wires, blocking effect on MCA blood flow, operation convenience, suppository durability, cerebral ischemia stability and the like.

TABLE 2 scoring and model success rates of various thrombus line preparations for rat sMCAO model neurological deficit

*p<0.005 (as compared to a single ball set of pins), #p<0.05 (as compared to lysine wiring sets).

Example 3: comparing the effect of different suppositories to prepare mouse sMCAO model

Preparation of double ball bolt wire: prepared according to the method of the invention. Namely, a medical monofilament nylon wire of 6-0 is cut into a line segment of 3cm. One end was slowly burned under a microscope near a heat source to form a smooth bulb with a diameter of 0.20.+ -. 0.01 mm and heated at a position 3 mm from the top bulb to form a bulb with a diameter of 0.22.+ -. 0.01 mm. And cleaning the bolt line. Marking is done at a distance of 9-10 a mm from the tip. And (5) disinfecting for later use.

Preparation of single ball bolt wire: reference Enrique Zea Longa et al (Stroke, 1989; 20:84-91). I.e. one end of a monofilament nylon wire with the length of 3cm and the wire number of 6-0 is heated to a smooth ball with the diameter of 0.22+/-0.01 mm near the flame. And cleaning the bolt line. Marking is done at a distance of 9-10 a mm from the tip. And (5) disinfecting for later use.

Lysine bolt preparation: the single-balloon strands were coated with 0.1% (w/v) poly-L-lysine (Sigma P1274, shanghai, china) (see Belayev et al, stroke, 1996; 27:1616-1623), without increasing the diameter of the strands after coating.

Preparation of a silicone rubber bolt: a section of one end 3 mm of a 6-0 monofilament nylon wire was coated with liquid silicone rubber (DOW CORNING 748RTV sealer) according to the technique reported by Koizumi J et al (Jpn J Stroke, 1986; 8:1-8). The diameter of the coated silicone rubber is 0.22+/-0.01 and mm. After the silicone rubber solidifies, the tether is cleaned. Marking is done at a distance of 9-10 a mm from the tip. And (5) disinfecting for later use.

Experimental grouping: 45C 57 male mice weighing 25-g-30 g were randomly divided into five groups. The sham surgery group was 5, and the other four groups were 10. (1) double ball bolt wire group: blocking MCA by adopting a double ball bolt; (2) Single ball bolt wire set: blocking MCA by adopting a single ball bolt; (3) lysine-tether group: blocking MCA by using a bolt coated by poly-L-lysine; (4) silicone rubber tether group: blocking the MCA by adopting a silicone rubber bolt; (5) sham surgery group: the other surgical procedures were the same without the plug wire. Each group of sMCAO time is 45 min, and then the thrombus line is pulled out to recover the MCA blood flow and recharge. Reperfusion was performed for 24 hours.

Experimental procedure the procedure of example 2 is referenced briefly as follows:

(1) Mice were anesthetized (350 mg/kg, ip) with 5% chloral hydrate physiological saline solution and placed supine on a temperature-controlled operating table. The skin is conventionally disinfected, the middle of the neck is incised, the left Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) are separated in sequence, the branch arteries of the ECA are cauterized off, the distal end of the ECA is ligated and cauterized off.

(2) The CCA and ICA were temporarily clamped with an arterial clamp.

(3) A small incision was cut into the ECA, and a tether was inserted into the ECA and gently ligated with surgical threads.

(4) The ICA arterial clip was released and the tether was inserted intracranially along ICA. A tether is inserted into the Anterior Cerebral Artery (ACA) starter until there is a resistance, typically about 10mm (from CCA bifurcation). Blocking the blood supply to the Middle Cerebral Artery (MCA).

(5) The surgical threads on the ECA were moderately tightened.

(6) After blocking the MCA for 45 min, the thrombus line was completely pulled out from the cranium, the blood supply of MCA was restored, and ECA was fastened to prevent bleeding. Suturing the skin.

(7) Each group of mice was subjected to deep anesthesia after 24 hours after MCA recovery of blood flow, and brains were taken at intervals, and whether subarachnoid hemorrhage occurred or whether intravascular thrombosis occurred or not was observed, and then brain tissues were cut into coronary brain slices 1mm thick using a brain slice die. The brain slice is stained with 2% 2,3, 5-triphenyltetrazolium chloride (TTC) (Sigma T8877, shanghai, china) by incubation at 37deg.C in dark for about 15 min, and the normal brain tissue is stained with bright red, and ischemic necrotic brain tissue is not stained and is pale. The brain pieces were then soaked in 4% paraformaldehyde phosphate buffer (pH 7.4), photographed after fixation for 24 hours, and the percentage of ischemic side cerebral infarct volume to the side hemispheric volume was calculated with reference to the method of Chang-Mu Chen et al (Sci Rep.2018; 8:5568). Animals with subarachnoid hemorrhage or TTC staining without infarction were considered model failure, whereas were successful, model success was calculated.

Experimental results:

as can be seen from Table 3, the success rate of the sMCAO model for the double ball bolt line group was significantly better than that of the other groups. 1 example of the silicone rubber tether group showed thrombosis. Thrombus was not seen in the other groups. TTC (time to live) dyeing shows that the infarct areas of the double-ball bolt line group and the silicon rubber bolt line group are obviously larger than those of the other two groups, and the cerebral infarction volume percentage of the double-ball bolt line group or the silicon rubber bolt line group isThe ratio is obviously larger than that of a single ball bolt line groupp<0.05 And lysine bolt line groupp<0.05）。

The results show that the mouse sMCAO model prepared by the double-ball bolt line is superior to other bolts.

TABLE 3 preparation of mouse sMCAO model cerebral infarction volumes and model success rates by various thrombus lines

＊p<0.05 (as compared to single ball bolt sets); # typep<0.05 (as compared to lysine wiring sets).

The present invention incorporates by reference in its entirety techniques well known in the art of animal brain ischemia models, anatomies and neurobiology. These techniques include, but are not limited to, the techniques described in the following publications:

1. str m et al, BMC Neuroscience,2013;14:41

2. Enrique Zea Longa et al, stroke,1989;20:84-91

3. Laing et al, stroke, 1993; 24:294-297

4. Koizumi J et al, jpn J Stroke, 1986; 8:1-8

5. Belayev et al, stroke, 1996; 27:1616-1623

6. Rogers DC et al, stroke, 1997, 28:2060-2066

7. Chang-Mu Chen et al Sci Rep.2018; 8:5568.

Incorporated by reference

The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated herein by reference in their entirety for any purpose, as are the references cited therein.

Equivalent solution

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (27)

1. A tether for use in the preparation of a model of middle cerebral artery occlusion (mcao) by the tether process, characterized by two adjacent smooth surfaced bulbs at one end of the feed line, wherein a first bulb is at the tip and a second bulb is spaced from the first bulb by a distance, the diameter of the bulb spacing portion being less than the diameter of the first bulb and the maximum diameter of the first bulb being less than or equal to the maximum diameter of the second bulb.

2. The plug wire according to claim 1, wherein the minimum diameter of the bulb spacer portion is equal to the diameter of the raw wire.

3. A tether according to claim 1 or 2, wherein the two bulbs are spaced apart a distance of 0.5mm to 10mm.

4. A tether according to claim 3, wherein the two bulbs are spaced apart a distance of 1mm to 5mm.

5. The tether according to any of claims 1, 2 and 4, wherein the maximum diameter of the bulb ranges from 0.10mm to 1mm.

6. The tether according to claim 5, wherein said bulb has a maximum diameter in the range of 0.15mm to 0.5mm.

7. The tether according to any of claims 1, 2, 4 and 6, wherein said raw material wire has good biocompatibility and has a certain flexibility, elasticity and hardness.

8. The plug wire according to claim 7, wherein the raw material wire is made of a high molecular polymer material, a biological material or a metal material.

9. The tether according to claim 8, wherein said polymeric material is selected from the group consisting of polyamides, polypropylene, polyurethane, polyethylene, polytetrafluoroethylene, and the like.

10. The tether line according to claim 8, wherein said raw stock line is a medical monofilament nylon line.

11. The bolt according to any of claims 1, 2, 4, 6 and 8-10, wherein the diameter of the raw material wire ranges from 0.001mm to 0.5mm.

12. The suppository according to any one of claims 1, 2, 4, 6 and 8-10, wherein said stock line is of the general specification type number 0, 2-0, 3-0, 4-0, 5-0, 6-0, 7-0, 8-0, 9-0, 10-0 or 11-0 according to the united states national formulary (USP) standard.

13. The suppository wire according to any one of claims 1, 2, 4, 6 and 8-10, wherein said stock wire is of a common gauge type according to the united states national formulary (USP) standard No. 3-0, no. 4-0, no. 5-0, no. 6-0, no. 7-0 or No. 8-0.

14. A tether according to any of claims 1, 2, 4, 6 and 8-10, wherein both spheres are made by heat treatment.

15. A tether according to any of claims 1, 2, 4, 6 and 8-10, wherein both spheres are made by a glue-coating process.

16. A tether according to any of claims 1, 2, 4, 6 and 8-10, wherein one of the spheres is made by a heat treatment and the other sphere is made by a glue treatment.

17. A tether according to any of claims 1, 2, 4, 6 and 8-10, wherein the top bulb is made by a heat treatment and the other bulb is made by a glue treatment.

18. The tether according to any of claims 1, 2, 4, 6 and 8-10, wherein the tether is for rats and has a length of 2.5-8cm.

19. The tether according to claim 18, wherein said tether has a length of 2.5-5cm.

20. The tether according to claim 18, wherein said tether has a diameter of 3-0, 4-0 or 5-0.

21. The tether according to any of claims 1, 2, 4, 6 and 8-10, wherein the tether is for a mouse and has a length of 1.5-5cm.

22. The tether according to claim 21, wherein said tether has a length of 1.5-3cm.

23. The tether according to claim 21, wherein said tether has a diameter of 5-0, 6-0, 7-0 or 8-0.

24. The tether according to any of claims 1, 2, 4, 6 and 8-10, wherein the tether is for rabbits and has a length of 6-15cm.

25. The tether according to claim 24, wherein said tether has a length of 6-10cm.

26. The tether according to claim 24, wherein said tether has a diameter of 2-0 or 3-0.

27. A method of producing a tether according to any one of claims 1 to 26, comprising the steps of:

(1) Cutting the raw material line into line segments with proper lengths;

(2) Forming a first ball by heat treatment or glue coating at one end of the wire segment;

(3) Forming a second ball by heat treatment or glue coating at a location spaced from the first ball; and

(4) The bolt is cleaned, marked and sterilized.

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