Method for modeling SD rat brain injury by using different falling body hitting

文档序号：1896316 发布日期：2021-11-30 浏览：23次中文

阅读说明：本技术 利用不同落体打击对sd大鼠脑外损伤造模的方法 (Method for modeling SD rat brain injury by using different falling body hitting ) 是由江斌熊忠贤叶健于 2021-08-06 设计创作，主要内容包括：本发明利用不同落体打击对SD大鼠脑外损伤造模的方法,包括轻型外伤性颅脑损伤造模,神经功能缺损脑损伤造模和昏迷脑损伤造模,所述落体包括砝码,克氏针或圆柱形撞击锤。本发明通过自制自由落体撞击器,结合不同重量的不同落体自由坠落对SD大鼠脑外损伤造模,可以建立轻型外伤性颅脑损伤模型,神经功能缺损脑损伤模型和昏迷脑损伤模型。(The invention relates to a method for modeling the brain external injury of an SD rat by utilizing different falling body striking, which comprises light traumatic brain injury modeling, nerve function defect brain injury modeling and coma brain injury modeling, wherein the falling body comprises a weight, a Kirschner wire or a cylindrical striking hammer. According to the invention, a light traumatic brain injury model, a neurological function impairment brain injury model and a coma brain injury model can be established by self-making a free fall impactor and combining different fall free falls with different weights to model the external brain injury of the SD rat.)

1. A method for modeling SD rat brain external injury by using different falling body striking comprises light traumatic brain injury modeling, neurologically deficient brain injury modeling and coma brain injury modeling, wherein the falling body comprises a weight, a Kirschner wire or a cylindrical striking hammer, and the method comprises the following steps:

the modeling of the light traumatic craniocerebral injury comprises the following steps: fixing the anesthetized SD rat, placing a sponge pad of 4cm below the head of the rat, waiting for the rat to awaken, when the rat gradually awakens and the limb moves, freely dropping a weight of 120g from a position 0.34m above the rat, acting on the head of the rat, and repeating for 3 times;

the brain injury modeling of the neurological deficit comprises the following steps: fixing the anesthetized head of the SD rat after skin preparation, sterilizing the head, exposing the skull, peeling off the periosteum and keeping the dura intact, freely dropping a 20g weight/Kirschner wire from a position 15-20 cm above the head, acting on the dura of the rat to enable brain tissues to generate instant deformation to cause the apical lobe brain contusion and laceration, and quickly removing a device for fixing the rat;

the modeling of the coma brain injury comprises: fixing the anesthetized SD rat head after skin preparation, disinfecting the head, exposing the skull, padding a metal gasket on the surface of the skull, waiting for the rat to revive, and when the rat revives gradually, freely dropping a 400g cylindrical impact hammer from the upper part to impact the metal gasket on the surface of the skull, wherein the height of the impact hammer is 38-42 cm according to the weight of the rat, and the skull of the rat has depressed sclerotin after the impact.

2. The method for modeling brain injury of SD rat by different falling body strikes according to claim 1, wherein the anesthesia modes in the modeling of light traumatic brain injury and the modeling of coma brain injury both comprise induction anesthesia and maintenance anesthesia, the induction anesthesia comprises: putting the SD rat into an isoflurane respiratory anesthesia induction box, adjusting an air pump, controlling the flow of oxygen gas to be 1L/min, adjusting the output concentration of an anesthesia tank, and adjusting the output concentration to 5 percent isoflurane inhalation induction anesthesia of the SD rat; the maintaining anesthesia comprises: anesthesia was maintained by inhalation with a 2% isoflurane mask after induction of anesthesia success.

3. The method for modeling brain external injury of SD rats by different falling body strikes according to claim 1, wherein in the modeling of the light traumatic brain injury, SD rats show limited lateral limb activities, uncoordinated limbs and oral or nasal bleeding in a small number, which indicates success of modeling of the light traumatic brain injury.

4. The method for modeling the brain injury of the SD rat by using different falling body strikes as claimed in claim 1, wherein the anesthesia mode in the modeling of the brain injury with neurological deficit is as follows: SD rats were weighed and injected intraperitoneally with 10% chloral hydrate at a dose of 350 mg/kg.

5. The method for modeling the brain injury of the SD rat by using different falling body strikes according to claim 1, wherein in the modeling of the brain injury with neurological deficit and the modeling of the coma brain injury, the method for exposing the skull is as follows: the midline incision of the head is 3cm long, and the soft tissue and periosteum are separated bluntly to expose the skull.

6. The method for modeling the brain injury of the SD rat by using different falling body strikes as claimed in claim 5, wherein in the modeling of the brain injury with neurological deficit, the periosteum is peeled by a method comprising the following steps: a bone window with a diameter of 5mm was drilled in the area around the midline after the coronal suture with a dental drill, keeping the dura intact.

7. The method for modeling the brain injury of the SD rat by using different falling body strikes as claimed in claim 1, wherein the modeling of the brain injury with neurological deficit further comprises injecting 4-5 drops of 16 ten thousand IU penicillin into the incision after the collision, suturing the scalp, and judging whether the modeling is successful or not by the neurological deficit score mNSS.

8. The method for modeling extra-brain injury of SD rat by using different falling body strikes as claimed in claim 1, wherein in the modeling of coma brain injury, the coma evaluation criteria are: the consciousness state of rat postoperative observation is divided into 6 grades: level 1, moving as usual in the cage; level 2, reduced activity in the cage; 3, reducing the activity and dyskinesia in the cage; stage 4, rolling but not standing when the back is placed at the bottom of the cage; grade 5, the righting reflex disappears, but there is a limb retraction response to pain stimulation; grade 6, no response to any stimulus, of which grade 5, 6 are considered to be coma.

9. The method for modeling the brain injury of the SD rat by using different falling body hitting according to any one of claims 1 to 8, wherein the light traumatic brain injury modeling, the brain injury modeling with neurological deficit and the coma brain injury modeling are all performed by using a brain stereotaxic apparatus to fix the SD rat.

10. The method for modeling the extracerebral injury of the SD rat by using different falling body strikes as claimed in claim 9, wherein the following method is adopted for fixing: poking the incisors by using tweezers, clamping the incisors in square holes of incisor clamps of the animal adapter, pressing down the incisor clamp cross rods, and adjusting the height of the incisor clamps to keep the interaural line of the animal consistent with the connecting line of the ear rods of the positioning instrument; the animal body is padded by adopting foam on the base of the locator, so that the head and the body of the animal are kept horizontal, and the respiratory obstruction of the animal is prevented; insert the animal duct with left side ear pole, adjust left and right side ear pole and make the animal head keep in U type open-ended central point and put, lock fixed left side ear pole earlier, the fixed right side ear pole of back makes the animal head can not rock, adjusts the door tooth clamp screw simultaneously.

Technical Field

The invention belongs to the technical field of animal modeling, and relates to a method for modeling an SD rat with brain injury by using different falling body strikes.

Background

Traumatic Brain Injury (TBI) refers to injury of the brain due to external mechanical forces, which can cause transient or permanent physical dysfunction, cognitive disorders, and psychological disorders in survivors of brain trauma, and craniocerebral trauma is one of the important published factors of neurodegenerative disorders. In order to understand the physiological process of craniocerebral trauma pathology and formulation a new treatment method, the establishment of a good animal model is of great importance.

In recent years, researchers at home and abroad make extensive and intensive researches on the pathophysiological change process and the injury mechanism of craniocerebral injury. A large number of basic experimental and clinical research results show that the injury mechanism of craniocerebral trauma mainly comprises two aspects of primary injury and secondary injury.

The primary injury is closely related to the magnitude of external force, stress position, injury degree and the like, directly causes structural damage, brain tissue deformation and the like, and is one of important research directions of craniocerebral trauma prevention measures. The secondary injury is the basis of clinical treatment and experimental research of craniocerebral trauma, relates to multiple aspects of neuroinflammation reaction, oxygen free radicals, necrosis and the like, is easy to cause vicious circle of 'injury-inflammation-injury', further aggravates the neural injury and causes indirect injury of the brain. The difficult recovery is mainly embodied in two aspects: 1. the external environment hinders the regeneration and repair of neurons. 2. Factors that inherently hinder regeneration and repair.

Rodents have been used more and more extensively for brain trauma studies since 1990 due to their low cost, small footprint and easy standardized assessment of outcomes. In an experimental animal model of a rat, the impact-type injury mechanism regulates the injury degree of the brain through controllable external force, so that the pathophysiology mechanism under different injury conditions is quantitatively researched, and the method is more significant for guiding treatment and prognosis evaluation. Of these 3 models have been widely used in recent years: weight-drop models, fluid brain injury models (FPI), and controlled cortical impact models (CCI).

Disclosure of Invention

Based on the above, the invention aims to provide a method for modeling the extracerebral injury of the SD rat by using different falling body strikes.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for modeling SD rat brain external injury by using different falling body striking comprises light traumatic brain injury modeling, neurologically deficient brain injury modeling and coma brain injury modeling, wherein the falling body comprises a weight, a Kirschner wire or a cylindrical striking hammer, and the method comprises the following steps:

Preferably, the mode of anesthesia in both the modeling of light traumatic brain injury and the modeling of coma brain injury comprises induction anesthesia and maintenance anesthesia, the induction anesthesia comprising: putting the SD rat into an isoflurane respiratory anesthesia induction box, adjusting an air pump, controlling the flow of oxygen gas to be 1L/min, adjusting the output concentration of an anesthesia tank, and adjusting the output concentration to 5 percent isoflurane inhalation induction anesthesia of the SD rat; the maintaining anesthesia comprises: anesthesia was maintained by inhalation with a 2% isoflurane mask after induction of anesthesia success.

Preferably, in the modeling of the light traumatic craniocerebral injury, SD rats show limited lateral limb activity, uncoordinated limbs and oral hemorrhage or nasal hemorrhage in a few cases, which indicates that the modeling of the light traumatic craniocerebral injury is successful.

Preferably, the anesthesia mode in the modeling of the brain injury with neurological deficit is as follows: SD rats were weighed and injected intraperitoneally with 10% chloral hydrate at a dose of 350 mg/kg.

Preferably, in the modeling of the brain injury with neurological deficit and the modeling of the coma brain injury, the method for exposing the skull comprises the following steps: the midline incision of the head is 3cm long, and the soft tissue and periosteum are separated bluntly to expose the skull.

More preferably, in the modeling of the brain injury with neurological deficit, the method for stripping periosteum is as follows: a bone window with a diameter of 5mm was drilled in the area around the midline after the coronal suture with a dental drill, keeping the dura intact.

Preferably, the modeling of the brain injury with neurological deficit further comprises dripping 4-5 drops of 16 ten thousand IU penicillin into an incision after the impact, suturing the scalp, and judging whether the modeling is successful or not through a neurological deficit score (mNSS).

Preferably, in the modeling of the coma brain injury, the coma assessment criteria are: the consciousness state of rat postoperative observation is divided into 6 grades: level 1, moving as usual in the cage; level 2, reduced activity in the cage; 3, reducing the activity and dyskinesia in the cage; stage 4, rolling (presence of righting reflex) but not standing when the back is placed at the bottom of the cage; grade 5, the righting reflex disappears, but there is a limb retraction response to pain stimulation; grade 6, no response to any stimulus, of which grade 5, 6 are considered to be coma.

Preferably, the light traumatic brain injury model, the brain injury model with neurological deficit and the coma brain injury model are fixed by a brain stereotaxic apparatus.

More preferably, the following method is used for fixation: the front teeth are poked by tweezers, the front teeth are clamped in square holes of a front tooth clamp of the animal adapter, and the front tooth clamp cross rod is pressed down. Adjusting the height of the incisor clamp to keep the interaural line of the animal consistent with the ear rod connecting line of the locator; the animal body is padded by adopting foam on the base of the locator, so that the head and the body of the animal are kept horizontal, and the respiratory obstruction of the animal is prevented; insert the animal duct with left side ear pole, adjust left and right side ear pole and make the animal head keep in U type open-ended central point and put, lock fixed left side ear pole earlier, the fixed right side ear pole of back makes the animal head can not rock, adjusts the door tooth clamp screw simultaneously.

The invention has the following beneficial effects:

according to the invention, a light traumatic brain injury model, a neurological function impairment brain injury model and a coma brain injury model can be established by self-making a free fall impactor and combining different fall free falls with different weights to model the external brain injury of the SD rat.

Drawings

FIG. 1 is a schematic structural view of a home-made free-fall impactor in accordance with the invention, wherein: 1. an iron stand; 2. a crocodile clip; 3. a rubber tube; 4. a stainless steel rod; 5. a sponge cushion; 6. a small sponge.

FIG. 2 shows the SD rat being fixed on the brain stereotaxic apparatus according to the present invention.

Figure 3 shows the placement of the pre-impact rats of the present invention.

FIG. 4 shows the state of the skull of a rat after periosteum removal in example 2 of the present invention.

Fig. 5 shows a rat after suturing the scalp according to example 2 of the present invention.

FIG. 6 shows the sampling of the molded article of example 2 of the present invention.

FIG. 7 shows the fixation of the spacer to the surface of the rat skull in example 3 of the present invention.

FIG. 8 shows the appearance of depressed bone in the skull of rats after impact according to example 3 of the present invention.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

In the following examples, a self-made free-fall impactor was used, and the injury-causing device was modified with reference to the marmarmarrou model, as shown in fig. 1, specifically including:

i. a cylindrical rubber tube 3 with a length of 1.0m, an inner diameter of 19.0mm and an outer diameter of 25.0 mm; and drilling small holes with the diameter of 5mm on the pipe wall every 10cm, wherein the small holes correspond to two rows, so that the air resistance is reduced.

ii, a cubic spongy cushion 5 of 40.0cm by 30.0cm by 10.0 cm; small pieces of sponge 6, 3.0cm × 2.0cm × 1.0cm, one piece each.

A strike pad: a round stainless steel sheet with the diameter of 10mm and the thickness of 3mm is used for manufacturing a diffuse axon injury model, and another round stainless steel sheet is provided with a small hole with the diameter of 1.5mm drilled at the position of a stainless steel gasket 2.5mm away from the center, and a stainless steel cylinder is placed in the small hole so as to be 3mm higher than the plane of the gasket to manufacture a diffuse brain injury model and combine a focal brain injury model.

Stainless steel rod 4, 18.0mm diameter, 470.0g weight.

v. other: iron stand platform 1, crocodile clamp 2 etc. iron stand platform 1 includes the straight metal pole of base and perpendicular to base, is the conventional spare part in laboratory.

The two crocodile clips 2 fix the rubber tube 3 on the iron stand 1, so that the rubber tube 3 is perpendicular to the ground, the lower end of the rubber tube is 15.0cm away from the ground to place the sponge pad 5 and the rat, the small sponge 6 is placed under the skull of the rat to ensure that the stainless steel bar 4 perpendicularly strikes the metal gasket on the skull of the rat, and the gasket is tightly attached to the surface of the skull of the rat.

EXAMPLE 1 modeling of light traumatic craniocerebral injury

First, preparation of experiment

Experimental animals: male SD rats were 8 weeks old.

Laboratory instruments and surgical instruments: 120g weight, brain stereotaxic apparatus, isoflurane respiratory anesthesia machine and self-made free-fall impactor

Second, Experimental operation

1. Anesthesia: SD rats were anesthetized using an isoflurane respiratory anesthesia machine.

1.1 induction of anesthesia: SD rats were placed in an isoflurane respiratory anesthesia induction chamber. Adjusting an air pump, controlling the oxygen flow to be 1L/min, adjusting the output concentration of an anesthesia tank, and adjusting the output concentration to 5 percent isoflurane to be inhaled to induce and anesthetize SD rats.

1.2 maintenance of anesthesia: after the induction anesthesia was successful (index of success of anesthesia: rat limb no self-movement, muscle relaxation, smooth breathing) the anesthesia was maintained by inhalation with 2% isoflurane mask.

2. Fixing: SD rats were fixed in brain stereotaxic apparatus as shown in fig. 2. The front teeth are poked by tweezers, the front teeth are clamped in square holes of a front tooth clamp of the animal adapter, and the front tooth clamp cross rod is pressed down. Adjusting the height of the incisor clamp to keep the interaural line of the animal consistent with the ear rod connecting line of the locator; the animal body is padded by adopting foam on the base of the locator, so that the head and the body of the animal are kept horizontal, and the respiratory obstruction of the animal is prevented; insert the animal duct with left side ear pole, adjust left and right side ear pole and make the animal head keep in U type open-ended central point and put, lock fixed left side ear pole earlier, the fixed right side ear pole of back makes the animal head can not rock, adjusts the door tooth clamp screw simultaneously.

3. Impacting the head: after the SD rat is fixed in the brain stereotaxic apparatus, a 4cm sponge pad is placed below the head of the rat, as shown in fig. 3, the isoflurane respiratory anesthesia machine is closed, the rat is waited to revive, when the rat revives gradually and the limb moves, a 120g weight is freely dropped from a position 0.34m above the device, acts on the head of the rat, and the process is repeated for 3 times.

4. And (4) observation: SD rats showed limited lateral limb movement, uncoordinated limbs, and oral or nasal bleeding in a small number. The success of modeling the light traumatic craniocerebral injury is demonstrated.

This approach is suitable for establishing lightweight traumatic brain injury models.

EXAMPLE 2 modeling of brain injury with neurological impairment

First, preparation of experiment

Experimental animals: male SD rat of 8 weeks old

Laboratory instruments and surgical instruments: 10% chloral hydrate, brain stereotaxic apparatus, isoflurane respiratory anesthesia machine, self-made pipeline (20-30cm is different, suitable pipeline is made according to experimental conditions), 20g weight (which can be replaced by Kirschner wire, such as A162), dental drill, surgical blade, surgical knife handle, surgical scissors, ophthalmological forceps (2 handles), ruler, cotton swab, iodophor disinfectant, needle holder, hemostatic forceps, needle thread (4-0 braided wire)

Second, Experimental operation

1. Anesthesia: SD rats were weighed and injected intraperitoneally with 10% chloral hydrate at a dose of 350 mg/kg.

2. Skin preparation: the rat head was shaved.

3. Fixing: the SD rat was fixed on a brain stereotaxic apparatus. The front teeth are poked by tweezers, the front teeth are clamped in square holes of a front tooth clamp of the animal adapter, and the front tooth clamp cross rod is pressed down. Adjusting the height of the incisor clamp to keep the interaural line of the animal consistent with the ear rod connecting line of the locator; the animal body is padded by adopting foam on the base of the locator, so that the head and the body of the animal are kept horizontal, and the respiratory obstruction of the animal is prevented; insert the animal duct with left side ear pole, adjust left and right side ear pole and make the animal head keep in U type open-ended central point and put, lock fixed left side ear pole earlier, the fixed right side ear pole of back makes the animal head can not rock, adjusts the door tooth clamp screw simultaneously.

4. And (3) disinfection: the rat head was sterilized with iodophor.

5. Exposing the skull: the midline incision of the head is 3cm long, and the soft tissue and periosteum are separated bluntly to expose the skull.

6. Stripping the periosteum: a5 mm diameter window was drilled in the lateral midline region behind the coronal suture using a dental drill, leaving the dura intact, as shown in FIG. 4.

7. Impact molding: after an SD rat is fixed on a brain stereotaxic apparatus, a sponge pad with the length of 4cm is placed below the head of the rat, a weight with the length of 20g freely falls from the position 15-20 cm above the apparatus, and the weight acts on dura mater of the rat to enable brain tissues to generate instantaneous deformation to cause contusion and laceration of parietal lobe brain, and the apparatus is rapidly removed.

8. And (3) sewing: 4-5 drops of 16 ten thousand IU penicillin are dripped into the incision after the collision, and the scalp is sutured, as shown in figure 5.

9. Neurological deficit scoring: and judging whether the molding is successful or not by a neurological deficit score (mNSS). International standards currently define normal SD rats as 0 points, with an mNSS score of between 1 and 6 points for mild injury, between 7 and 12 points for moderate injury, and between 13 and 18 points for severe injury.

TABLE 1 neurological deficit scoring sheet

10. After molding, sampling was carried out, and as shown in FIG. 6, brain damage was evident.

This approach is suitable for establishing a model of moderate brain injury.

EXAMPLE 3 modeling of coma brain injury

First, preparation of experiment

Experimental animals: male SD rat of 8 weeks old

Laboratory instruments and surgical instruments: brain stereotaxic apparatus, isoflurane respiratory anesthesia machine, dental drill, self-made free fall impactor, thin aluminum pad, 400g cylindrical impact hammer, surgical blade, surgical knife handle, surgical scissors, ophthalmological tweezers (2 handles), ruler, cotton swab, iodophor, needle holder, hemostatic forceps, needle thread (4-0 braided wire)

Second, Experimental operation

1. Anesthesia: SD rats were anesthetized using an isoflurane respiratory anesthesia machine.

2. Skin preparation: the rat head was shaved.

4. And (3) disinfection: the rat head was sterilized with iodophor.

5. Exposing the skull: the midline incision of the head is 3cm long, and the soft tissue and periosteum are separated bluntly to expose the skull.

6. Aluminum padding sheets: a thin aluminum shim was placed on top of the skull as shown in fig. 7, and then the isoflurane anesthesia machine was turned off and the rat was waited for awakening.

7. Impact: as the rat gradually revived, a 400g cylindrical impact hammer was free-dropped from above the device along a straight metal rod of a homemade free-fall impactor, striking a thin aluminum pad on the surface of the skull bone. According to the weight difference of the rat, the height of the rat is 38-42 cm, and after the impact, the skull of the rat has sunken sclerotin, as shown in figure 8.

8. Coma assessment criteria: the consciousness state of rat postoperative observation is divided into 6 grades: level 1, moving as usual in the cage; level 2, reduced activity in the cage; 3, reducing the activity and dyskinesia in the cage; stage 4, rolling (presence of righting reflex) but not standing when the back is placed at the bottom of the cage; grade 5, the righting reflex disappears, but there is a limb retraction response to pain stimulation; grade 6, no response to any stimulus. With levels 5 and 6 being considered comatose.

This approach is suitable for establishing a severe brain injury model.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all the embodiments of the present invention are not exhaustive, and all the obvious variations or modifications which are introduced in the technical scheme of the present invention are within the scope of the present invention.

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