CN116058996A - Construction method of cerebral hemorrhage animal model - Google Patents
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- CN116058996A CN116058996A CN202310075761.2A CN202310075761A CN116058996A CN 116058996 A CN116058996 A CN 116058996A CN 202310075761 A CN202310075761 A CN 202310075761A CN 116058996 A CN116058996 A CN 116058996A
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- 206010008111 Cerebral haemorrhage Diseases 0.000 title claims abstract description 35
- 238000010276 construction Methods 0.000 title claims abstract description 12
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- 241001465754 Metazoa Species 0.000 claims abstract description 39
- 102000029816 Collagenase Human genes 0.000 claims abstract description 30
- 108060005980 Collagenase Proteins 0.000 claims abstract description 30
- 229960002424 collagenase Drugs 0.000 claims abstract description 29
- 239000007924 injection Substances 0.000 claims abstract description 29
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- 239000000243 solution Substances 0.000 claims abstract description 23
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- 210000004556 brain Anatomy 0.000 claims abstract description 15
- 210000002637 putamen Anatomy 0.000 claims abstract description 13
- 206010018852 Haematoma Diseases 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 23
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- 206010002091 Anaesthesia Diseases 0.000 claims description 11
- 230000037005 anaesthesia Effects 0.000 claims description 11
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 claims description 6
- 230000006399 behavior Effects 0.000 claims description 6
- 229960003299 ketamine Drugs 0.000 claims description 6
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 claims description 5
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- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D1/00—Surgical instruments for veterinary use
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
- A61D7/04—Devices for anaesthetising animals by gases or vapours; Inhaling devices
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Surgery (AREA)
- Anesthesiology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of disease model construction, in particular to a construction method of a cerebral hemorrhage animal model, which can be used for constructing the cerebral hemorrhage animal model by injecting collagenase solution from an injection operation passage of an animal to a striatal putamen area in the skull of the animal, calculating the cerebral hematoma volume of the animal through brain images after injection, recording the behavioral changes of the animal, has high construction success rate, has quick and long duration time compared with the formation time of beagle hematoma of a successfully constructed model, is close to a clinical cerebral hemorrhage disease area, can be used for obtaining different models by designing different doses, and can be used for simulating clinical acute or chronic cerebral hemorrhage conditions through the cerebral hemorrhage and behavioral manifestations of animals with different survival times and animals with different periods, thereby providing a choice for a later-stage drug treatment window.
Description
Technical Field
The invention relates to the technical field of disease model construction, in particular to a construction method of a cerebral hemorrhage animal model.
Background
Cerebral hemorrhage is a public health problem seriously jeopardizing human health, accounting for 10% -30% of all admitted cerebral apoplexy patients, and can lead to serious disability and Gao Bingsi rate. In recent two years, data has shown that cerebral hemorrhage onset age tends to approach young people. Therefore, the prevention and treatment of cerebral hemorrhage are more and more important, and large-scale clinical experiments and observational researches show that the standard treatment and special nursing can obviously reduce the death rate of cerebral hemorrhage, and the technologies of ultra-early hemostasis, intracranial hypertension control, reasonable blood pressure reduction, cerebral indoor tPA thrombolysis, minimally invasive surgery and the like are expected to improve the clinical treatment strategy of the current cerebral hemorrhage patient, reduce the death rate and improve the long-term life quality of the patient.
There are many animal model building methods currently available for drug testing, for example: 1. autologous blood injection method refers to that autologous blood is extracted and injected into corresponding brain areas to form cerebral hemorrhage, and the cerebral hemorrhage formed by the method is not caused by rupture of cerebral blood vessels and is not suitable for mechanism research; 2. the micro-balloon inflation method belongs to a mechanical model, can only simulate the occupying effect of hematoma in brain after cerebral hemorrhage, can not simulate the damage effect of blood components on brain tissues, has a large gap with clinic, and has a limited application range; 3. collagenase injection method is to inject collagenase into brain region to dissolve small blood vessel to cause cerebral hemorrhage, but the method has the characteristics of diffuse bleeding, long hematoma formation time and incapability of simulating rapid rupture of blood vessel.
In the construction method, the most used animal model closest to clinic is induced by collagenase, and is usually injected into the brain endothelial layer area, however, the animal individual difference causes great difficulty in collagenase dosage selection, and the success rate of the model is not high.
Disclosure of Invention
The invention aims to provide a construction method of a cerebral hemorrhage animal model, which aims to solve the technical problems in the background technology.
In order to achieve the above object, the present invention discloses a construction method of an animal model of cerebral hemorrhage, comprising: injecting collagenase solution from an injection surgical pathway of an animal to a target nucleus, wherein the target nucleus is a striatal putamen region in the cranium of the animal; the brain hematoma volume of the animal was calculated from brain images after injection was completed and the behavioral changes of the animal were recorded.
Preferably, the collagenase solution is a type IV collagenase solution, and the method of preparing the collagenase solution includes dissolving collagenase in a solvent, and the solvent includes a balanced salt solution.
Alternatively, the collagenase solution is injected at a dose of 1-200U/10. Mu.L.
Preferably, the collagenase solution is injected at a dose of 2.4-2.8U/10. Mu.L.
Preferably, the collagenase solution is injected at a dose of 2.5U/10. Mu.L
Preferably, the collagenase solution is injected at a rate of 0.5-1. Mu.L/min.
Further, the method for determining the surgical path for injection comprises: and (3) carrying out three-dimensional reconstruction on the animal cranium and the target nucleus to obtain the space coordinates of the target nucleus, and confirming the operation passage for injection and the striatum shell nucleus region coordinates in the cranium by taking the animal cranium as a space zero point according to the space coordinates of the target nucleus.
Preferably, before the injection of the collagenase solution, further comprising: during the anesthesia of animals, ketamine and ranoxazine are firstly adopted to induce anesthesia, and isoflurane is then adopted to continuously perform anesthesia.
Optionally, the dosage of ketamine is 5-15mg/kg, and the dosage of the ranolazine is 0.2-2mg/kg.
Preferably, the ketamine dose is 8.75mg/kg and the ranolazine dose is 0.75mg/kg.
Preferably, ketamine and xylazine are injected intramuscularly.
Alternatively, anesthesia is continued by intubation with 1.5-3% isoflurane gas.
Preferably, 2% isoflurane gas is used for continuous anaesthesia.
Alternatively, the animal comprises a beagle dog.
Alternatively, beagle dogs have a weight of 7-15kg.
Alternatively, beagle dogs are 1.5-2 years old.
Optionally, the behaviours include: any one or a combination of voluntary movement, consciousness and sensation.
Optionally, the voluntary movement includes any one or combination of standing, walking directionality, a paralyzed state of fore and aft limbs, running ability, and a tail posture, the consciousness includes any one or combination of consciousness and behavioral consciousness, and the sensation includes vision.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the collagenase solution is injected from an injection operation passage of an animal to a striatal putamen area in the cranium of the animal, so that a cerebral hemorrhage animal model can be constructed, the cerebral hematoma volume of the animal is calculated through brain images after the injection is completed, and the behavioral change of the animal is recorded.
Drawings
FIG. 1 is a graph showing the percent cerebral hematoma detection of all modeled animals in an embodiment of the present invention;
FIG. 2 is a graph showing the overall score of the behavior of all modeled animals in one embodiment of the present invention;
FIG. 3 is a graph showing the percent cerebral hematoma detection of a modeled animal in accordance with one embodiment of the present invention;
FIG. 4 is a graph showing the overall score of the behavior of a modeled animal in accordance with one embodiment of the present invention;
FIG. 5 is a graph showing the score of autonomous exercise for a modeled animal in accordance with one embodiment of the present invention;
FIG. 6 is a graph showing the results of consciousness score recording for a modeled animal in accordance with one embodiment of the present invention;
FIG. 7 is a graph showing the sensory score of a modeled animal in accordance with one embodiment of the present invention;
fig. 8 is a diagram of brain image detection of a modeled animal in accordance with an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be explained in detail by referring to the drawings and the specific embodiments. It is to be understood that the following description is intended to be illustrative of the invention and not limiting in nature.
1. Modeling animal information
Table 1 modeling animal information
2. Experimental device and reagent information
Table 2 experimental setup and reagent information
| Instrument/reagent name | model/CAS number | Manufacturing factories |
| CT | ScintCareCT16 | Mingfeng Medical System Co.,Ltd. |
| MRI | PanionPremier1.5T | TIME MEDICAL JIANGSU Co.,Ltd. |
| Brain stereotactic instrument | 68916 | RWD LIFE SCIENCE Co.,Ltd. |
| Collagenase enzyme | IV type | Sigma |
3. Scoring criteria for behaviours
TABLE 3 behavioural scoring criteria
The above experiments were carried out with 7 beagle dogs numbered 1001, 2001, 3001, 4001, 5001, 6001, 7001 in order.
Through the co-positioning of MRI and CT, MRI is three layers of T22D FSE Ax, T22D FSE Dor and T22DFSE Sag, and the thinner the scanning layer is, the better the scanning layer is, the more thin the machine field intensity is, the modeling adopts 1.5T field intensity, the scanning layer thickness is 3mm, CT is used for conventional skull helical scanning, the scanning layer thickness is 0.58mm, and the layer spacing is 0.58mm. It should be noted that care is taken to align the beagle heads as much as possible during CT and MRI scans, so that the time can be greatly shortened in the subsequent software process.
After the scanning is finished, firstly, fusion pairing is carried out on MRI and CT images by using open source analysis software 3D sler, and then three-dimensional reconstruction is carried out on the skull and the striatal putamen area of each beagle, so that the space coordinates of the striatal putamen area can be obtained.
Prior to injection of the collagenase type IV solution, beagle dogs were anesthetized, during which anesthesia was first induced by intramuscular injection with ketamine at a dose of 8.75mg/kg and ranolazine at a dose of 0.75mg/kg, and after relaxation of the beagle dogs' limbs, there was no reaction by gently pinching the limbs, and then continued anesthesia with 2% isoflurane gas via cannula.
After anesthesia is completed, the beagle is fixed on a brain positioning instrument, the space level of the beagle is regulated, the position of the striatal putamen is confirmed by selecting the bregma of the beagle as a space zero point according to the space coordinates of the striatal putamen obtained by the three-dimensional reconstruction, namely, the bregma of the beagle is taken as the origin of three-dimensional coordinate axes (x, y, z), and therefore, the operation passage for injection of the beagle and the coordinates of the striatal putamen in the skull can be confirmed according to the space coordinates of the striatal putamen obtained by the three-dimensional reconstruction.
During injection, firstly, an operation part on the skull is selected according to an operation passage for injection, the skin is cut to expose the skull after the operation part is disinfected, secondly, a small opening is drilled by a skull drill after the exposed position is used as a puncture point of the operation passage for injection, and finally, the IV collagenase solution is injected into the striatum putamen area along the operation passage for injection by a microsyringe needle, and only one single-point injection is needed during injection.
5. Injection dose and modeling results
Referring to table 4, 7 beagle dogs were injected by dissolving collagenase in a balanced salt solution, for example, in physiological saline, and IV collagenase solutions of different activities (activities are generally expressed by injection doses) were prepared, and the volumes of injections were different, thereby obtaining different modeling results.
Table 4 injection case
In addition, after the injection was completed, the 7 beagle dogs were scanned for MRI brain images at the survival time at different times, the cerebral hematoma volume of each beagle dog was calculated from the brain images, and the behavioral changes of each beagle dog were recorded according to the scoring criteria of the behaviors.
Referring to fig. 1, 2 and 4, it is shown that:
beagle dogs numbered 1001-4001 showed cerebral hemorrhage (cerebral hematoma percentage > 0% was considered cerebral hemorrhage) and behavioural abnormality (behavioural score > 0 was considered abnormality), but survived for a short period of time, with the longest survived beagle dogs numbered 4001 survived for only 15hr (hours);
the beagle with the number of 5001 shows cerebral hemorrhage in operation to cause coma, namely, the beagle is not awake after the operation is finished, so the percentage of cerebral hematoma is not recorded, and the percentage of cerebral hematoma is defaulted to 0 in the figure;
beagle No. 7001 survived for a long period of time without behavioural abnormalities, but had a short duration of cerebral hemorrhage symptoms, in which the time of cerebral hemorrhage (labeled as time in the figure) lasted for 10 days.
Details of the beagle dogs referenced 6001 above are shown in figures 3-8:
cerebral hemorrhage is expressed in different periods, namely, the first period is an acute period, the second period is a subacute period (advanced period), the third period has a trend from the subacute period (advanced period) to the chronic period, the fourth period is a chronic period, specifically, the cerebral hemorrhage is expressed in a hyperacute period-an acute period at 4 hours (marked as-4 hr in the figure), the cerebral hemorrhage is expressed in an acute period at 24 hours, the cerebral hemorrhage is expressed in a subacute period (advanced period) at 3 days (marked as-3 d in the figure), the cerebral hemorrhage is expressed in a subacute period (advanced period) at 5 days to 16 days, the cerebral hemorrhage is expressed in a subacute period (advanced period) -chronic period at 20 days to 24 days, the cerebral hemorrhage is expressed in a chronic period at 27 days to 37 days, the behavioural can be fed by people in the first period, and vomiting can disappear in the second period;
in the behavioulogy of beagle dogs No. 6001, autonomic motor abnormalities (autonomic motor scores > 0, considered abnormal) lasted for more than 37 days, conscious abnormalities (conscious scores > 0, considered abnormal) lasted for 31 days and paresthesia (sensory scores > 0, considered abnormal) lasted for 29 days.
From the results, it is found that 7 beagle brain hemorrhage models are successfully constructed, and the beagle hematoma formation time and duration of the successfully constructed models are fast and approximate to clinical brain hemorrhage onset areas, and different models can be obtained by designing different doses, so that clinical acute or chronic brain hemorrhage conditions are simulated through brain hemorrhage and behavioral manifestations of animals with different survival times and animals with different periods, and a choice is provided for a later drug treatment window, for example, the beagle hematoma formation time and duration are applied to research on brain hemorrhage etiology, development processes, screening of therapeutic drugs and the like.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the invention, but any modifications, equivalent substitutions, improvements, etc. within the design concept of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for constructing an animal model of cerebral hemorrhage, comprising:
injecting a collagenase solution from an injection surgical pathway of an animal to a target nucleus, wherein the target nucleus is a striatal putamen region in the cranium of the animal;
the brain hematoma volume of the animal was calculated from brain images after injection was completed and the behavioral changes of the animal were recorded.
2. The method of claim 1, wherein the collagenase solution is a type IV collagenase solution, and the method of preparing the collagenase solution comprises dissolving collagenase in a solvent, and the solvent comprises a balanced salt solution.
3. The method of claim 2, wherein the collagenase solution is injected at a dose of 1-200U/10 μl.
4. The method of claim 3, wherein the collagenase solution is injected at a rate of 0.5 to 1 μl/min.
5. The method of constructing a surgical pathway for injection according to claim 1, wherein the method of determining a surgical pathway for injection comprises:
three-dimensional reconstruction is carried out on the cranium of the animal and the target nucleus to obtain the space coordinates of the target nucleus;
and according to the space coordinates of the target nucleus, confirming the operation passage for injection and the striatal shell nucleus region coordinates in the cranium by taking the cranium of the animal as a space zero point.
6. The method of claim 1, further comprising, prior to injecting the collagenase solution: animals are anesthetized, and during the anesthesia process, ketamine and cetoxazine are firstly adopted to induce anesthesia, and isoflurane is then adopted to continuously perform anesthesia.
7. The method of claim 1, wherein the animal comprises a beagle.
8. The method of claim 7 wherein the beagle weight is 7-15kg.
9. The method of construction of claim 8 wherein the beagle dog is 1.5 to 2 years old.
10. The method of construction of claim 1, wherein the behaviours comprise: any one or a combination of voluntary movement, consciousness and sensation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050234063A1 (en) * | 2002-06-28 | 2005-10-20 | Kazuhiro Terai | Therapeutic agent for brain hemorrhage |
| CN101543427A (en) * | 2008-03-26 | 2009-09-30 | 陈兆秋 | Common posture-fixing bedplate for multi-mode medical image fusion |
| CN104548078A (en) * | 2013-10-25 | 2015-04-29 | 香港医疗科技研究院有限公司 | Spontaneous intracerebral hemorrhage animal model platform and application thereof in medicament screening |
| CN110013546A (en) * | 2018-01-08 | 2019-07-16 | 郑州大学 | A kind of method for building up of CPSP animal model and application |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050234063A1 (en) * | 2002-06-28 | 2005-10-20 | Kazuhiro Terai | Therapeutic agent for brain hemorrhage |
| CN101543427A (en) * | 2008-03-26 | 2009-09-30 | 陈兆秋 | Common posture-fixing bedplate for multi-mode medical image fusion |
| CN104548078A (en) * | 2013-10-25 | 2015-04-29 | 香港医疗科技研究院有限公司 | Spontaneous intracerebral hemorrhage animal model platform and application thereof in medicament screening |
| CN110013546A (en) * | 2018-01-08 | 2019-07-16 | 郑州大学 | A kind of method for building up of CPSP animal model and application |
Non-Patent Citations (4)
| Title |
|---|
| J.K.WASSERMAN: "Ddevelopment and cahracterization of a hemorrhagic rat model of central post-stroke pain", NEUROSCIENCE, pages 173 - 183 * |
| 任福银: "α-生育酚对SD大鼠脑出血治疗作用的实验研究", 《中南大学硕士研究生学位论文》 * |
| 冯铭: "《骨髓间充质干细胞治疗食蟹猴脑出血及脑内单次给药的毒性实验研究》", 《北京协和医学院博士学位论文》 * |
| 冯铭: "《骨髓间充质干细胞治疗食蟹猴脑出血及脑内单次给药的毒性实验研究》", 《北京协和医学院博士研究生学位论文》, 31 May 2008 (2008-05-31) * |
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