CN109549739B

CN109549739B - Experimental material for constructing magnetic particle-aggregated cerebral ischemia animal model

Info

Publication number: CN109549739B
Application number: CN201811591397.0A
Authority: CN
Inventors: 耿武军; 唐红丽
Original assignee: First Affiliated Hospital of Wenzhou Medical University
Current assignee: First Affiliated Hospital of Wenzhou Medical University
Priority date: 2017-12-25
Filing date: 2018-12-25
Publication date: 2021-04-02
Anticipated expiration: 2038-12-25
Also published as: CN109549739A; CN107898524A

Abstract

The invention discloses an experimental material for constructing a magnetic particle aggregation cerebral ischemia animal model, which comprises an injection for simulating thrombus formation, wherein the injection is a buffer solution containing magnetic nanoparticles, and the magnetic nanoparticles are enriched in a specific section of a blood vessel to simulate thrombus formation; the cerebral ischemia animal model constructed by using the kit disclosed by the invention can highly coincide with the symptoms of cerebral ischemia, has small damage to the animal model, and has low mortality rate constructed by the animal model.

Description

Experimental material for constructing magnetic particle-aggregated cerebral ischemia animal model

Technical Field

The invention relates to the technical field of animal pharmacology experiments, in particular to an experimental material for constructing a magnetic particle-aggregated cerebral ischemia animal model.

Background

Transient Ischemic Attack (TIA) is a transient blood supply insufficiency in the carotid or vertebrobasilar system, resulting in focal cerebral ischemia leading to sudden, transient, reversible neurological dysfunction. The onset lasts for several minutes, usually recovers completely within 30 minutes, often leaving a manifestation of mild neurological deficit over 2 hours, or CT and MRI show signs of ischemia in brain tissue. TIA occurs in 34-65 years old, 25.3% more than 65 years old, and more men than women. Sudden onset of disease is usually caused by posture change, excessive movement, sudden neck rotation or flexion and extension. The onset of the disease is not marked by aura, and the nervous system with hypersexuality locates the physical sign. Usually, involuntary disorder can occur repeatedly within 5-20 minutes, but usually, the involuntary disorder can be recovered completely within 24 hours without sequelae, the most common symptoms of TIA of the internal carotid artery system include monoplegia, hemiplegia, hemiparesis, aphasia, monocular vision disorder and the like, and syntropism hemianopsia can also occur. The main performance is as follows: sudden appearance of hyperkeratosis, or visual loss, or white blinking, or visual field defect, or diplopia in a single eye can recover after a few minutes. The contralateral limb is slightly hemiplegic or dysesthesia. Impairment of the dominant hemisphere may lead to transient aphasia or disuse or loss of reading or writing, or simultaneous weakness of the facial and tongue muscles. Even lateral hemianopsia is caused. Sudden appearance of hyperkeratosis in a single eye is a characteristic symptom of arterial ischemia in the branch of the internal carotid artery. Transient mental symptoms and disturbance of consciousness are also visible.

In clinical experiments, the influence of cerebral ischemia on an organism is researched by establishing a cerebral ischemia animal model, but in the prior art, the cerebral ischemia animal model is manufactured by compressing or cutting off through a surgical means, the simulation of thrombosis is not consistent with the reality, meanwhile, the cerebral ischemia animal model can cause direct injury to blood vessels, the death rate is high, the operation is complex, the individual variation is large, the operation is not reversible, and a sustainable cerebral ischemia animal model cannot be established because transient cerebral ischemia has the possibility of multiple relapse.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an experimental material for constructing a magnetic particle-aggregated cerebral ischemia animal model, which can be used for preparing an animal model highly matching with symptoms of cerebral ischemia, has small damage to the animal model and has low mortality rate of the animal model construction.

In order to achieve the purpose, the invention provides the following technical scheme: an experimental material for constructing a magnetic particle-aggregated cerebral ischemia animal model,

comprises an injection for simulating the formation of thrombus, wherein the injection is a buffer solution containing magnetic nanoparticles, and the magnetic nanoparticles are enriched to simulate the formation of thrombus at a specific section of a blood vessel;

the magnetic nano particle guiding device also comprises a guiding implant body used for guiding and enriching the magnetic nano particles, wherein the guiding implant body is a soft linear magnetic strip used for surrounding blood vessels.

As a further improvement of the invention, the magnetic nanoparticles are coated with an inert material.

As a further improvement of the invention, the magnetic nanoparticles are metal magnetic nanoparticles of less than 150 nm.

As a further improvement of the invention, the inert material is polyethylene glycol, glucose or dextran.

As a further improvement of the present invention, the molecular weight of polyethylene glycol is 1000-2000.

As a further improvement of the invention, the particle size of the magnetic nanoparticles after being wrapped by the inert material is 100-200 nm.

As a further improvement of the invention, the concentration of the magnetic nanoparticles in the buffer is 5-20 mg/ml.

As a further improvement of the invention, the soft linear magnetic strip is a soft magnetic strip with the diameter of 0.28 mm.

As a further improvement of the invention, the head end of the soft linear magnetic strip is dipped with hot-melt paraffin, and the paraffin is smooth and spherical at the head end of the soft linear magnetic strip.

As a further improvement of the invention, the heparin-containing composition also comprises 1% of heparin.

The invention has the beneficial effects that: by providing an experimental material, it contains the buffer solution and the soft threadlike magnetic stripe of magnetism nanoparticle, when using, through wrapping up soft linear magnet on the carotid artery of animal model, and pour into the buffer solution that contains the magnetism nanoparticle, the buffer solution that contains the magnetism nanoparticle circulates in the internal, form the embolism when the carotid artery position of placing soft threadlike magnetic stripe, and can set for the degree of embolism through the magnetism of adjusting soft threadlike magnetic stripe, the whole modeling time is short, avoid traditional cerebral ischemia model to the direct loss of blood vessel, the degree of injury to animal model is low, the modeling success rate of animal model has been improved, and the physical principle that its embolism formed is close with the physical principle that actually forms the embolism, can better simulation living body cerebral vascular embolism's influence to the organism.

Detailed Description

The present invention will be described in further detail with reference to examples and comparative examples.

Example 1 an experimental material for constructing an animal model of magnetic microparticle-induced cerebral ischemia comprises 10mg/ml magnetic nanoparticle buffer solution, soft linear magnetic stripe, sterile paraffin, and sterile 1% heparin solution.

(1) Preparing a magnetic nanoparticle buffer solution:

selecting Fe, Co, Ni magnetic nanoparticles or ferroferric oxide magnetic nanoparticles or iron sesquioxide magnetic nanoparticles, preferably selecting metal magnetic nanoparticles with the diameter of less than 150, wrapping inert materials on the outer layer of the metal magnetic nanoparticles, adopting polyethylene glycol, glucose or glucan, preferably adopting polyethylene glycol with the molecular weight of 1000-plus-2000, and wrapping the inert materials on the metal magnetic nanoparticles with the particle size of 100-plus-200 nm; after sterilization, the magnetic nano-particles are prepared into a buffer solution by mixing with physiological saline to 5-20mg/ml, preferably 10mg/ml, and the particles of the magnetic nano-particles are uniform and well dispersed in the buffer solution.

(2) Production of soft linear magnetic stripe

Cutting a soft magnetic stripe with a diameter of 0.28 mm to 6cm accurately, marking the broken end of the soft magnetic stripe with a flat and blunt circle under a mirror every 2cm, sterilizing, and dipping the end part of the prepared soft linear magnetic stripe in hot-melt sterile paraffin to make the end part of the soft linear magnetic stripe in a smooth spherical shape.

Example 2 application method of Experimental Material for constructing novel magnetic particle-aggregated cerebral ischemia animal model

1. Experimental materials:

1.1 animal Material

SD rats, male, weaning rats (day 21 of birth), were selected and purchased from the animal center of the medical college of the university of counterdenier. After the animals are bought, the animals are randomly grouped and raised in cages. During the raising period, a quiet and comfortable environment is kept, and water and food are freely drunk and eaten. The experimenter touches and grips the rat once every morning and afternoon two days before the experiment begins, and the rat adapts to the experimental device and the used device, so that the stress stimulation of the experimental environment to the rat is eliminated as much as possible, and the accuracy of the experimental result is ensured.

When the mice are raised to 120 + -10 days old, the rats weighing 230 + -30 g are selected.

1.2 Primary reagents

75% alcohol, iodophor, 10% chloral hydrate (or sodium pentobarbital), triphenyltetrazolium chloride (TTC), PBS;

1.3 Main instruments

Rat board, binding thread (thread rope), rough scissors, scalpel, hemostatic forceps, ophthalmic scissors, ophthalmic curved forceps, surgical scissors, surgical forceps, glass needle, medical dry cotton ball or gauze piece, 5ml syringe, alcohol lamp, beaker, plate, capillary dropper, razor blade, horn needle, curved plate, self-made drag hook, latex glove, working glove and long leather hand

1.4 Experimental instruments

Slicing machine: LEICA CM 1800.

And (3) constant temperature blanket: ALC-HTP

Electric heating constant temperature drying oven: JKDP-type 1.

An electronic balance: TIANFU DT-200A.

Microscope and digital camera matched with the microscope: OLMPUS IX 71.

2. Preparation of animal model for cerebral ischemia Using the test Material of example 1

(1) Soaking the soft linear magnetic stripe prepared in the example 1 in a sterile 1% heparin solution for standby;

(2) weighing the weight of the rat, injecting 80-150ug/g of the buffer solution containing the magnetic nanoparticles in example 1 into the carotid artery of the rat according to the weight of the rat, and if necessary, injecting the rat into the jugular vein or the tail vein;

(3) calculating the required dose (3 ml/kg) of anesthesia according to the body weight, preparing 10% chloral hydrate anesthetic, adopting an intraperitoneal injection mode for anesthesia in a drug administration mode, fixing the rat on a rat board in a supine posture after corneal reflection disappears and anesthesia is sufficient, and using medical adhesive tapes to fix the limbs of the rat board gently. Wiping oral cavity with sterile cotton ball and removing airway secretion;

(4) the tongue of the rat was pulled out with tissue forceps and placed on the buccal horn side to prevent glossoptosis from affecting rat respiration. Shaving and preparing skin in an operation area, sterilizing by iodophor, taking a 3cm neck median incision, performing blunt separation on neck subcutaneous tissues in sequence, exposing left Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA) according to an anatomical sequence, and fixing the operation incision by using a draw hook. When the target blood vessel is exposed, the manipulation is gentle and cautious, and the damage to other tissues, blood vessels, nerves, thyroid gland and parathyroid gland around the target blood vessel is avoided.

(5) The soft linear magnetic strip is annularly wrapped around the internal carotid artery ICA to be wrapped close to the blood vessel without causing compression. The molding time is controlled to be finished within 20 minutes.

(6) Ischemia for 2h, followed by another intraperitoneal injection of 10% chloral hydrate to anesthetize the animals, gentle unrolling of the soft linear magnetic strip, slow and gentle removal, re-suturing of the tissue incision, iodophor disinfection, restoration of blood perfusion for 24h, and monitoring of the cerebral circulation.

During the ischemia period and within 24h of restoring blood perfusion, all animals are monitored in real time for anal temperature, the rat anal temperature is kept between 36.5 and 37.5 ℃, and the survival rate of the rat is improved by controlling through a constant temperature blanket or a constant temperature box.

Comparative example 1 preparation of blank control

The other operations were the same as in example 2, and the same amount of buffer containing magnetic nanoparticles was poured in, and the soft linear magnetic strip was replaced with a soft rubber strip.

Comparative example 2 preparation of model of cerebral ischemia in rat by wire-embolus method

(1) Preparation of the wire plug: a commercially available single-stranded fishing line with the diameter of 0.28 mm is accurately cut into 6cm, after the broken end is flush and blunt under a mirror, a mark is made every 2cm, the fishing line is soaked in 75% alcohol for 10 min, the head end of the fishing line is dipped in hot-melt paraffin to make the fishing line smooth and spherical, and the fishing line is soaked in 1% heparin for later use.

(2) Surgical operation

Injecting 10% chloral hydrate (3 ml/kg) into abdominal cavity of a rat, performing cervical shearing disinfection after dorsal fixation, taking a median incision of neck, performing blunt dissection on right Common Carotid Artery (CCA), Internal Carotid Artery (ICA) and External Carotid Artery (ECA), hanging threads for standby, ligating ECA and CCA, clamping and closing the ICA distal end by using an artery clamp, quickly making an incision on CCA, inserting the prepared fish thread from the incision, opening the artery clamp for clamping and closing ICA after inserting the thread into ICA, slightly pulling the fish thread at a position close to 18mm (the dissection from the bifurcation to the middle cerebral artery), stopping when resistance exists, fixing the thread, slightly pulling the fish thread after 2h, fastening ICA, and suturing skin.

And (4) analyzing results:

(1) survival rate

The animal model prepared in example 2 and the animal models prepared in comparative examples 1 and 2, 12 rats were selected, and the mortality rate of example 2 was 91.7%, that of comparative example 2 was 50%, and that of comparative example 1 was 91.7%

(2) Neurological deficit scoring

Reference to Longa and Bederson's 5-score method scores 24h after the animals were anesthetized for arousal.

0 minute: no symptoms of nerve damage;

1 minute: the contralateral anterior paw cannot be fully extended;

and 2, dividing: turning to the opposite side;

and 3, dividing: pouring towards the opposite side;

and 4, dividing: the patient can not walk spontaneously and the consciousness is lost.

Higher scores indicate more severe animal behavior disorders.

The criteria for model success were: the body bends to the left side, the extension of the front paw at the right side is stronger than that at the left side, and the tail bends to the left side; the tail is in a compulsive posture when suspended.

The death rate of 2-3 minutes of symptoms is high, the death rate of 1-2 minutes is low, and some rats have no obvious symptoms after the next day or a plurality of days, but have infarcted foci after taking brains.

(3) Results of the experiment

Refer to Longa and Bederson' 5 point division. Score 0 (no symptoms of nerve damage) was considered as a molding failure, dead rats were also considered as a molding failure, both types of scored rats were rejected, and the remaining scored animals were included in the experiment.

The animal model prepared in example 2, except dead rats, the score of the remaining 11 rats according to 5-point method of Longa and Bederson were: 1.1, 2, 1, 3, 2, 1, the success rate of the model is 83.3 percent,

the animal model prepared in comparative example 2, except dead rats, the remaining 6 rats were scored according to 5-point method of Longa and Bederson, respectively: 2. 4, 2, 3, 2 and 4, the success rate of the model is 25 percent,

the animal model prepared in comparative example 1, except dead rats, the remaining 11 rats had scores of 0, 0 with a model success rate of 0% according to the 5-point method of Longa and Bederson.

Through the experimental data, the buffer solution containing the magnetic particles does not influence blood circulation, the modeling success rate of the example 2 is high, the scores of the animal model which is successfully modeled have no significant difference, the modeling success rate of the comparative example 2 is low, and the scores of the animal model which is successfully modeled have large significant difference.

5. Application of experimental material for constructing novel magnetic particle-aggregated cerebral ischemia animal model

The brain ischemia animal model prepared by the experimental material provided by the invention is used for observing the body damage degree of the brain ischemia animal model through the following experiments:

TTC (2, 3, 5-triphenyltetrazolium chloride) dyeing, namely, immediately cutting off the head and taking out the brain after rat ischemia is performed for 2h and reperfusion for 24h, quickly freezing, cutting 5 brain slices from front to back together, wherein the thickness is about 2mm, placing the slices in 2% TTC solution for incubation at 37 ℃ for 30 min, strictly keeping out of the sun, shaking once every 5min, finally placing the slices in 10% paraformaldehyde solution for fixation, observing the cerebral infarction volume of an animal model after embolism by microscope observation, further accurately and visually knowing the injury of cerebral ischemia to organisms, and being capable of being better applied to teaching experiments or new drug research and development experiments.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

1. An experimental material for constructing a magnetic particle-aggregated cerebral ischemia animal model is characterized by comprising an injection for simulating thrombus formation, wherein the injection is a buffer solution containing magnetic nanoparticles, and the magnetic nanoparticles are enriched to simulate thrombus formation in a specific section of a blood vessel;

the magnetic nano particle guiding and enriching device also comprises a guiding implant body used for guiding and enriching magnetic nano particles, wherein the guiding implant body is a soft linear magnetic strip used for surrounding blood vessels;

the magnetic nano particles are wrapped with inert materials, the inert materials are polyethylene glycol, glucose or glucan, the concentration of the magnetic nano particles in the buffer solution is 5-20mg/ml,

the magnetic nanoparticles are metal magnetic nanoparticles smaller than 150nm, the particle size of the magnetic nanoparticles after being wrapped by inert materials is 100-200nm, and the flexible linear magnetic strip is a flexible magnetic strip with the diameter of 0.28 mm.

2. The experimental material for constructing an animal model of magnetic particle-mediated cerebral ischemia as claimed in claim 1, wherein: the molecular weight of the polyethylene glycol is 1000-2000.

3. The experimental material for constructing an animal model of magnetic particle-mediated cerebral ischemia as claimed in claim 2, wherein: the head end of the soft linear magnetic strip is dipped with hot-melt paraffin, and the paraffin is smooth and spherical at the head end of the soft linear magnetic strip.

4. The experimental material for constructing an animal model of magnetic particle-mediated cerebral ischemia as claimed in claim 1, wherein: also included is 1% heparin.