Animal craniocerebral injury model construction method and system

Abstract

The invention relates to an animal craniocerebral injury model construction method and system, and the method comprises the following steps: 1, selecting a plurality of healthy male SD (Sprague Dawley) rats, breeding the rats in different cages in a 12-hour illumination environment and a 6-hour dark environment at the temperature of 25 DEG C, dividing the rats into three groups, and enabling the rats to eat and drink water; 2, the rats are divided into an X group, a Y group and a Z group through random grouping, the X group is a mild craniocerebral injury group, the Y group is a moderate craniocerebral injury group, the Z group is a severe craniocerebral injury group, and the number of the rats in each group is equal; according to the animal craniocerebral injury model, animal craniocerebral injury of different degrees can be simulated, the injury degree can be evaluated, and the model has good repeatability, reliability and stability and has good reference value in practical application.

Description

Technical Field

[0001] The present invention relates to the technical field of constructing animal brain injury models, and in particular to a method and system for constructing an animal craniocerebral injury model. Background Art

[0002] Traumatic brain injury is one of the main diseases threatening human life today. Especially, the mortality rate of patients with severe craniocerebral injury is very high, about 30%-50%. Craniocerebral trauma has become an important disease threatening people's lives. Brain injury mainly refers to primary injuries such as concussion, diffuse axonal injury, cerebral contusion, and cerebral contusion and laceration, as well as secondary changes such as intracerebral hematoma, cerebral edema, cerebral softening, and brain hernia formation. Craniocerebral injury is mainly related to the site, size, speed, and degree of compression of the violent action. According to the injury range, brain injury is generally divided into two types: focal and diffuse. The former includes cerebral contusion and laceration, intracranial hematoma, and brain damage caused by brain displacement, torsion, or increased intracranial pressure. The latter includes diffuse axonal injury, diffuse brain swelling, and hypoxic brain damage, etc. According to whether the meninges are ruptured, it can also be divided into two types: open and closed.

[0003] Chinese Patent with the publication number: CN 111820186 A discloses a method for constructing a traumatic brain injury animal model, which relates to the technical field of brain injury animal models. The construction method includes selecting normal rats or mice and performing intraperitoneal injection anesthesia; injecting a protective agent into the experimental animals and the traumatic brain injury animal model before the operation; constructing a traumatic brain injury animal model under different condition methods.

[0004] When the method in this patent is actually used, the following disadvantages exist: In practice, the occurrence of brain injury in different situations is of different degrees, so it is impossible to construct models for different degrees of brain injury in this patent. Summary of the Invention

[0005] In view of the technical problems existing in the patent mentioned in the background art, the present invention provides a method and system for constructing an animal craniocerebral injury model.

[0006] The technical solution adopted by the present invention is: A method for constructing an animal craniocerebral injury model specifically includes the following steps:

[0007] Select a number of healthy male SD rats. Under the environment of 25°C, 12h lighting and 6h darkness, through separate cage feeding, and divided into three groups. The rats can eat and drink water;

[0008] By random grouping, the rats are divided into three groups: X, Y, and Z. Group X is the mild craniocerebral injury group, Group Y is the moderate craniocerebral injury group, and Group Z is the severe craniocerebral injury group. The number of rats in each group is equal;

[0009] The rats were anesthetized by intraperitoneal injection, the hair on the rats' heads was shaved off, the rats were fixedly connected to the shelf board, an incision was made in the middle of the top of the rats' skulls to expose the dura mater;

[0010] Then blunt rods with different diameters were used, with an outward inclination of 15 - 20° with the sagittal plane and parallel to the coronal plane, abutting against the dura mater, and the rat brains were impacted according to different parameters;

[0011] 5 hours after the craniocerebral injury model was established, the rats were maintained in an anesthetized state and subjected to nuclear magnetic imaging;

[0012] 5 hours after the rats had craniocerebral injury, magnetic resonance imaging was used. The injured area was divided into several thin slices of the scanning layers. The cross-sectional areas of the injured parts of each layer were respectively obtained, and the approximate volume of the injured range of each layer was obtained by multiplying by the layer thickness. After summing, the approximate total volume of the injured range was obtained;

[0013] Before and 24 hours after the model was established, the behavioral changes of the rats were observed under different stimulation conditions to evaluate the severity of their craniocerebral injury;

[0014] One to two weeks after the craniocerebral injury model was established, several rats with different degrees of injury were taken respectively. After intraperitoneal anesthesia, they were sequentially injected with sodium chloride injection and paraformaldehyde solution. The whole brains of the rats were taken, and the rat brains were fixed in paraformaldehyde solution;

[0015] After 24 hours, the rat brains were washed with water, dehydrated, embedded, and 4-μm continuous sections were made in the coronal position. After HE staining, the histopathological changes were observed under a light microscope, and then data processing was carried out.

[0016] Furthermore, anesthetizing the rats by intraperitoneal injection, shaving the hair on the rats' heads, fixedly connecting the rats to the shelf board, and making an incision in the middle of the top of the rats' skulls to expose the dura mater specifically include the following steps:

[0017] The skin and subcutaneous tissue were incised, and the parietal muscles on the top of the skull were dissected laterally to expose the sagittal suture, coronal suture and anterior fontanelle of the skull;

[0018] At a position far from the left parietal bone, away from the sagittal suture and lambdoid suture, a manual drill with a certain diameter was used to slowly drill a hole to expose the dura mater.

[0019] Furthermore, the incision was about 1.0 cm long. At a position on the left parietal bone, 5.0 mm away from the sagittal suture and 5.0 mm away from the lambdoid suture, a manual drill with a diameter of 3.0 mm was used to slowly drill a hole to expose the dura mater.

[0020] Furthermore, the indicators for evaluating the severity of the craniocerebral injury include balance ability, reaction when falling from a height, postural reflex, reaction when being dragged, righting reflex, ear reflex, eye reflex, startle reflex, reaction when pain is stimulated on the hind feet, and reaction when pain is stimulated on the rat tail.

[0021] Furthermore, the volume of the injury range = ∑ scan layer thickness × cross-sectional area of the injury site.

[0022] Furthermore, the rats were anesthetized by intraperitoneal injection with sodium pentobarbital, and the standard was 60 mg / kg.

[0023] Furthermore, several healthy male SD rats with a body weight of 200 - 300 g were used.

[0024] Furthermore, an animal cranial injury model construction system

[0025] The magnetic resonance imaging module divides the injury site into several thin slices of the scan layer number, respectively obtains the cross-sectional area of the injury site for each layer, multiplies it by the layer thickness to obtain the approximate volume of the injury range for each layer, and sums them up to obtain the total approximate volume of the injury range;

[0026] The image processing module processes the images collected by the magnetic resonance imaging module;

[0027] The data processing module performs data processing. The numerical values are expressed in the form of x-±s. One-way ANOVA is used for inter-group comparison, and SNK test is used for pairwise comparison. P < 0.05 indicates that the difference is statistically significant.

[0028] The beneficial effects of the present invention are as follows: Compared with the prior art, the present invention can simulate models of different degrees of animal cranial injuries, and can also evaluate the degree of injury. The models have good repeatability, reliability and stability, and have good reference value in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the flowchart in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] Referring to the attached Figure 1 , in order to solve the problems existing in the background technology, the present application proposes the following technical solutions:

[0032] Example 1

[0033] A method for constructing an animal cranial injury model specifically includes the following steps:

[0034] Step 1: Select several healthy male SD rats (several healthy male SD rats with a body weight of 200 - 300 g are used). Under the conditions of a temperature of 25°C, a 12-hour lighting environment, and a 6-hour dark environment, the rats are raised in separate cages and divided into three groups. The rats can eat and drink.

[0035] Step 2: Through random grouping, the rats are divided into three groups: Group X, Group Y, and Group Z. Group X is the mild traumatic brain injury group, Group Y is the moderate traumatic brain injury group, and Group Z is the severe traumatic brain injury group. The number of rats in each group is equal.

[0036] Step 3: Anesthetize the rats by intraperitoneal injection (sodium thiopental is used for intraperitoneal injection of the rats, and the standard is 60 mg / kg). Shave the hair on the rats' heads, fix the rats and connect them to the shelf board, make an incision in the middle of the cranial vertex of the rats to expose the dura mater.

[0037] Among them, the incision is about 1.0 cm long. At a position 5.0 mm from the sagittal suture and 5.0 mm from the lambdoid suture on the left parietal bone, a manual drill with a diameter of 3.0 mm is used to slowly drill a hole to expose the dura mater.

[0038] Step 4: Then, blunt rods with different diameters are used to abut against the dura mater at an angle of 15 - 20° outward from the sagittal plane and parallel to the coronal plane, and the rat brains are impacted according to different parameters.

[0039] Step 5: 5 hours after the traumatic brain injury model is established, maintain the anesthetized state of the rats and perform nuclear magnetic imaging on the rats.

[0040] Step 6: 5 hours after the rats' traumatic brain injury, use magnetic resonance imaging to divide the damaged area into several thin slices of the scanning layers. Calculate the cross-sectional area of the damaged area of each layer respectively, multiply it by the layer thickness to obtain the approximate volume of the damaged area of each layer, and sum them to obtain the approximate total volume of the damaged area.

[0041] Specifically: The volume of the damaged area = ∑ scanning layer thickness × cross-sectional area of the damaged area.

[0042] 5 hours after the rats' traumatic brain injury, perform magnetic resonance imaging detection. Divide the damaged area into several thin slices of the scanning layers. Calculate the cross-sectional area of the damaged area of each layer respectively, multiply it by the layer thickness to obtain the approximate volume of the damaged area of each layer, and sum them to obtain the approximate total volume of the damaged area. The specific method: Use the image processing module to process the collected nuclear magnetic images. First, adjust the image gray threshold, select the damaged area, and the software automatically calculates the damaged area of each layer and sums them. After multiplying by the scanning layer thickness, the approximate volume of the damaged area is obtained.

[0043] Step 7: Observe the behavioral changes of the rats under different stimulation conditions before and 24 hours after modeling, and evaluate the severity of their traumatic brain injury.

[0044] Specifically, the indicators for evaluating the severity of craniocerebral injury include balance ability, reaction during a fall from a height, postural reflex, reaction when being dragged, righting reflex, ear reflex, eye reflex, startle reflex, reaction when pain is stimulated on the hind foot, and reaction when pain is stimulated on the rat tail.

[0045] In addition, each item is assigned a value according to the degree of reaction, with 0 points for normal, 1 point for partial reflex, and 2 points for no reflex. Evaluation is based on the total score. A score of 0 - 7 indicates mild craniocerebral injury, 8 - 14 indicates moderate craniocerebral injury, and > 15 indicates severe craniocerebral injury.

[0046] Step 8, one to two weeks after craniocerebral injury modeling, several rats with different degrees of injury are taken respectively. After intraperitoneal anesthesia, they are sequentially injected with sodium chloride injection and paraformaldehyde solution. The whole brain of the rats is taken, and the rat brain is fixed in paraformaldehyde solution.

[0047] Step 9, after 24 hours, the rat brain is washed with water, dehydrated, embedded, and 4 - μm continuous coronal sections are made. After HE staining, the histopathological changes are observed under a light microscope, and then data processing is carried out.

[0048] In a further design,

[0049] The rats are anesthetized by intraperitoneal injection, the hair on the rat's head is shaved off, and the rats are fixedly connected to the shelf board. An incision is made in the middle of the rat's cranial vertex to expose the dura mater, which specifically includes the following steps:

[0050] Step 1, the skin and subcutaneous tissue are incised, and the cranial vertex muscles are separately dissected laterally to expose the sagittal suture, coronal suture, and anterior fontanelle of the skull.

[0051] Step 2, at a position away from the left parietal bone, at a distance from the sagittal suture and lambdoid suture, a manual drill with a diameter is used to slowly drill a hole to expose the dura mater.

[0052] The following explanations are made for the above technical solutions:

[0053] Eighteen rats are randomly divided into 3 groups, corresponding to the mild, moderate, and severe craniocerebral injury groups respectively. Different groups use specific injury - causing parameters, and the injury depth of the probe: 1 - 4 mm for mild, 4 - 5 mm for moderate, and 5 - 6 mm for severe; the injury speed is 3 m / s for all, and the injury time is 0.1 s to cause different degrees of craniocerebral injury;

[0054] Then, magnetic resonance imaging and behavioral quantitative evaluation are respectively used, and qualitative analysis of cranial pathology is carried out to evaluate the degree of craniocerebral injury; as a result, the rats all showed different degrees of behavioral abnormalities after injury, and the cranial pathology of the injured rats showed a decrease in tissue neurons and infiltration of microglial cells;

[0055] In addition, there is congestion and edema to varying degrees. The injury volume of the mild craniocerebral injury group is 12 mm 3, the injury volume of the moderate craniocerebral injury group was 21 mm 3 , the injury volume of the severe craniocerebral injury group was 50 mm 3；

[0056] Therefore, the model constructed by this embodiment has good reliability and stability.

[0057] Embodiment 2

[0058] A system for constructing an animal craniocerebral injury model

[0059] A magnetic resonance imaging module that divides the injured part into a number of slices equal to the number of scanning layers, respectively calculates the cross-sectional area of each layer of the injured part, multiplies it by the layer thickness to obtain the approximate volume of the injury range of each layer, and sums them up to obtain the approximate total volume of the injury range;

[0060] An image processing module that processes the images collected by the magnetic resonance imaging module;

[0061] A data processing module that performs data processing. The numerical values are expressed in the form of x-±s. One-way analysis of variance is used for between-group comparison, and the SNK test is used for pairwise comparison. P < 0.05 indicates that the difference is statistically significant.

[0062] In summary, the model in the present invention can simulate different degrees of animal craniocerebral injuries, and can also evaluate the degree of injury. The model has good repeatability, reliability and stability, and has good reference value in practical applications.

[0063] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an animal craniocerebral injury model, characterized in that: The specific steps include: A number of healthy male SD rats were selected and housed in separate cages at a temperature of 25°C, a 12-hour lighting environment, and a 6-hour dark environment. The rats were allowed to eat and drink water. The rats were randomly divided into three groups: X, Y, and Z. Group X was a mild craniocerebral injury group, group Y was a moderate craniocerebral injury group, and group Z was a severe craniocerebral injury group. The number of rats in each group was equal. The rats were anesthetized by intraperitoneal injection, the hair on their heads was shaved, the rats were fixed on a board, an incision was made in the middle of the skull of the rats to expose the dura mater; Then, blunt rods of different diameters were used to impact the rat brain at different parameters, with the rods tilted 15-20° outward from the sagittal plane and parallel to the coronal plane and resting on the dura mater; 5 hours after the craniocerebral injury model was established, the rats were kept in anesthesia and subjected to MRI. 5 hours after craniocerebral injury in rats, magnetic resonance imaging was used to divide the injured area into thin slices of the same number of scanned layers, and the cross-sectional area of ​​the injured area in each layer was obtained, and the approximate volume of the injured range in each layer was obtained by multiplying it with the layer thickness. The total approximate volume of the injured range was obtained by summing them up. Before and 24 hours after modeling, the behavioral changes of rats were observed under different stimulation conditions to evaluate the severity of craniocerebral injury. One to two weeks after the modeling of craniocerebral injury, several rats with different injury degrees were selected, and after intraperitoneal anesthesia, sodium chloride injection and paraformaldehyde solution were sequentially injected, and the whole brain of the rat was taken and fixed in paraformaldehyde solution; After 24 hours, the mouse brain was washed, dehydrated, embedded, and 4 μm continuous sections were made in the coronal position. The tissue pathological changes were observed under a light microscope after HE staining, and then the data were processed.

2. The method for constructing an animal craniocerebral injury model according to claim 1, characterized in that: The rats were anesthetized by intraperitoneal injection, the hair on the head of the rats was shaved, the rats were fixed on a frame, an incision was made in the center of the rats' skull, and the dura mater was exposed, which specifically included the following steps: Cut the skin and subcutaneous tissue, free the muscles of the skull on both sides, and expose the sagittal suture, coronal suture and anterior fontanelle; A diameter hand drill was used to slowly drill a hole at the sagittal and lambdoid sutures away from the left parietal bone to expose the dura mater.

3. The method for constructing an animal craniocerebral injury model according to claim 2, characterized in that: The incision was about 1.0 cm long and was made at the left parietal bone 5.0 mm away from the sagittal suture and 5.0 mm away from the lambdoid suture. A 3.0 mm diameter manual drill was used to slowly drill a hole to expose the dura mater.

4. The method for constructing an animal craniocerebral injury model according to claim 3, characterized in that: Indicators for assessing the severity of craniocerebral injury include balance ability, reaction when falling from a height, homeostasis reflex, reaction when being dragged, righting reflex, ear reflex, eye reflex, startle reflex, reaction when receiving painful stimulation of the hind foot, and reaction when receiving painful stimulation of the rat tail.

5. The method for constructing an animal craniocerebral injury model according to claim 4, characterized in that: The volume of the damaged area = ∑ scanning layer thickness × cross-sectional area of ​​the damaged part.

6. The method for constructing an animal craniocerebral injury model according to claim 5, characterized in that: Rats were anesthetized by intraperitoneal injection of sodium thiopental at a standard of 60 mg / kg.

7. The method for constructing an animal craniocerebral injury model according to claim 6, characterized in that: Several healthy male SD rats weighing 200 to 300 g were used.

8. A system for constructing an animal craniocerebral injury model, characterized in that: The magnetic resonance imaging module divides the damaged part into thin slices of the same number of scanning layers, calculates the cross-sectional area of ​​the damaged part in each layer, multiplies it with the layer thickness to obtain the approximate volume of the damaged range in each layer, and then sums them up to obtain the approximate volume of the total damaged range; An image processing module, for processing images acquired by the magnetic resonance imaging module; The data processing module was used for data processing. The values ​​were expressed as x-±s. One-way analysis of variance was used for comparison between groups, and SNK test was used for pairwise comparison. P < 0.05 was considered statistically significant.

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