CN101756752A - Molding method of standardized spinal cord injury animal model - Google Patents
Molding method of standardized spinal cord injury animal model Download PDFInfo
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- CN101756752A CN101756752A CN201010033354A CN201010033354A CN101756752A CN 101756752 A CN101756752 A CN 101756752A CN 201010033354 A CN201010033354 A CN 201010033354A CN 201010033354 A CN201010033354 A CN 201010033354A CN 101756752 A CN101756752 A CN 101756752A
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Abstract
The invention relates to a molding method of a standardized spinal cord injury animal model. The molding method is characterized in that the four limbs, the head and the tail of an experimental animal are fixed, a spinal column part of the experimental animal is exposed by operation, a spacer, the shape of which corresponds to the shape of the spinal column, is arranged at a part that is ready to be injured, and a hollow impact channel is vertically fixed above the spacer; an impact bar with the standard weight which can just slide freely in the impact channel is arranged in the impact channel; the impact bar freely falls from a rated height to impact the spacer at the back side of the spinal column of the experimental animal; and the impact is moved away in time or remained for the rated time to cause back side injury of the spinal cord to a certain degree. Compared with a preparation method of the spinal cord injury model that is used frequently before, the invention has the outstanding characteristics that the standardization of the experimental method is realized, and the molded animal model has adjustable injury degree and good repeatability, can simulate situations of trauma buffer and oppression, and the like and more approaches to the human spinal cord injury situation.
Description
Technical field
The present invention relates to the modeling method of animal model, be specifically related to a kind of modeling method of animal model with spinal cord damnification.
Background technology
(spinal cord injury is because the following spinal function obstacle of level of damage after the spinal cord structure that causes of a variety of causes, functional lesion SCI) to spinal cord injury.In the U.S., annual every million people's mouth kainogenesis 30～40 examples, some the area in addition up to 60 examples.According to incompletely statistics, the spinal cord injury number of China has broken through 1,000,000, and increases severely with annual 120000 speed, and global spinal cord injury number has broken through 3,000,000.Development along with modern society, traffic and building falling accident are on the increase, and SCI population constantly increases, and SCI patient's majority is between twenty and fifty in addition, they are playing the part of important role in society and family, will cause huge economy and emotional distress to society and family after meeting accident.The Wenchuan earthquake that took place in 2008 be since the founding of New destructive the strongest, involve one earthquake disaster widest in area, that casualty loss is maximum, earthquake magnitude reaches 8.0 grades of Richter scales, maximum earthquake intensity reaches 11 degree, casualties heaviness.Current precipitate earthquake has caused tens thousand of wounded's spine and spinal cords to be subjected in various degree damage, and the wounded's functional rehabilitation problem seems particularly outstanding, therefore, the research of relevant spinal cord injury international and domestic all are hot subjects.
Carry out the research of spinal cord injury, at first want the ideal animal model with spinal cord damnification of reproducing standards.Ideal animal model with spinal cord damnification should possess following requirement: 1. clinical similarity: promptly the animal model with spinal cord damnification of Zhi Zuoing should be with clinical close, and animal model can be simulated the special pathological process that human spinal cord injury takes place, as far as possible near human situation.2. Modulatory character: promptly can adjust degree of injury according to actual needs, duplicate the animal model with spinal cord damnification of different degree of injury.3. repeatability and operability: promptly can the committed step of spinal cord injury model be objectified, quantification, the model manipulation technology is simple, is easy to grasp, and is convenient to promote.
The still spinal cord dorsal part injured animal model that present stage clinical application research use is maximum.The main method of traumatic spinal cord injury animal model modeling has: weight fall (weight drop, WD) method, clamp platen press, balloon tamponade method etc.The WD method also claims All en punch method, promptly falls from differing heights with weight, hits the animal spinal cord dorsal part, causes damage in various degree.Classical WD method has been started the beginning of making animal model with spinal cord damnification, can be by selecting weight whereabouts differing heights, or change weight quality adjustment impact force size, or limit the bump of different spinal segments, thereby copy in various degree, dissimilar animal model with spinal cord damnification.Studies show that, animal model with spinal cord damnification that the WD method is duplicated and human spinal cord injury cause edema of spinal cord, ischemia after all belonging to certain strength bump, and a series of injury responses of secondary, the character of this model and human spinal cord injury is very close, is a kind of with human spinal cord injury good relationship so far.The outstanding feature of WD method is exactly the loading process of simulating human spinal cord injury to a great extent, and the position of causing injury, scope can artificially be controlled.This method has kept spinal dura mater complete, can prevent exogenous components intrusion spinal cord injury zone effectively, and prevent that myelocele and cerebrospinal fluid from leaking outside.
But the WD method still exists defectives such as non-constant such as the external force effect, that spinal cord injury is asymmetric, scope is non-constant.In the moment that weight hits, the skew of spine and spinal cord also usually can cause the discordance of damage field and degree, and same potential energy also can cause degree of impairment to differ greatly owing to act on the area discrepancy of spinal cord.In addition, the WD method has only been simulated the initial strike state when causing injury, and ignored the squeezing action of persistence, and human acute spinal cord injury often exists the squeezing action of persistence because of spinal fracture.Therefore, need further improve the WD method.
Summary of the invention
The modeling method that the purpose of this invention is to provide a kind of standardization animal model with spinal cord damnification, adjustable with the animal model degree of injury of this method modeling, and good reproducibility.
The modeling method of standardization animal model with spinal cord damnification of the present invention is specially:
1) the fixedly extremity of laboratory animal, head and afterbody, operation appears the spine portion of laboratory animal;
2) place and the corresponding pad of spinal cord profile at the position of preparing damage;
3) the impact passage of a hollow of top vertical fixing of pad;
4) impact bar with a standard weights that be able to be free to slide in impacting passage places in the impact passage, freely falls from the height of stipulating, impinges upon on the dorsispinal pad of laboratory animal;
5) in time remove impact bar or impact bar kept somewhere the long time of regulation, cause spinal cord dorsal part injured animal model to a certain degree.
In the said method, the size of pad, the weight of impact bar, height of fall and impact bar pressing time are quantitatively, and can adjust according to the difference of laboratory animal or the degree of injury of needs.
Wherein, the impact channel outlet is 0.5～50mm apart from the distance of pad.
Height of fall is quantitative for convenience, can be provided with altitude scale on the impact passage.
Standardization animal model with spinal cord damnification modeling method of the present invention is that its advantage and innovation are to the improvement of traditional preparation process spinal cord dorsal part damage model method and perfect:
1, in fixing laboratory animal extremity, also the head and the afterbody of laboratory animal are fixed, thereby the laboratory animal activity is reduced to minimum level to the influence of model stability, guarantee the maximum concordance of each impact position;
2, pad has been used in increase on the laboratory animal spinal cord, and the quality and the area of unified pad, guarantees that impact the suffered external force of spinal cord is uniformly distributed in the onesize segments of spinal cord of causing injury at every turn, and scope is constant;
3, the pad that uses on the laboratory animal spinal cord can be selected the pad of differing texture according to the concrete condition of preparation damage model, makes its existence buffering in various degree, with the buffering of different-thickness soft tissue in the simulating reality;
4, used the vertical impact passage of hollow in experimental technique, impact bar can only fall along the direction of regulation in dropping process, departing from of impact position can not occur;
5, the weight that will impact usefulness changes the impact bar with certain-length into, owing to there is the passage of impact to fix, following backwardness can keep falling the position, can not fall to the side, causes the unnecessary damage at other positions;
6, use vertical impact passage and impact bar, can make impact bar on the spinal cord pad, keep constant motionless, stipulate that the concrete time just can finish standardization compressing process, after simulation is fractured to the compressing of spinal cord;
7, quality and the height of drop of having unified impact bar make impact velocity and potential energy obtain unification; Quality and the area of having unified pad, and the local compression time of impact bar make the external force effect unified constant, thereby make model standardization.
Therefore, compare with the spinal cord injury model preparation method of using always in the past, the most outstanding characteristics of the present invention are the standardization that realized experimental technique, and good repeatability is arranged, and can simulate situations such as wound buffering and compressing, more approach human spinal cord injury situation.
The specific embodiment
1, laboratory animal modeling
Male SD rat, body weight 263.00 ± 17.32g is divided into sham operated rats and experimental group at random, and rat is with 10% chloral hydrate intraperitoneal anesthesia, and is prostrate fixing, in fixing animal foot, also head and the afterbody of animal all fixed.Row T12 laminectomy, otch is the center with T12, is about 3cm, cuts skin and muscle, appears T11～13 spinous process and vertebral plate, stings except that T12 spinous process and vertebral plate.Sham operated rats after aforesaid operations is finished, suture muscles and skin.Experimental group is that the center appears the strike district that size is 3 * 4mm with the spinal cord, the vertical impact passage of hollow is moved to the spinal cord exposure portion that appears, utilize impact bar location in advance, and place pad at this position, impact bar can be met exposed after falling on the pad above the spinal cord by the location.Press the spinal cord position of differing heights, Different Weight strike exposure, and make impact bar fall back indwelling different time.Finish experiment back suture operation otch.
According to height, weight, pressing time, pad quality the experimental group rat is divided into some groups again, observes its motor function and myeloid tissue and learn variation.
2, result
2.1 the animal basic condition is observed
Hit two hind legs twitches, the whipping of the visible rat in back in experiment, lax fully subsequently.Behind the rat modeling recovery from anesthesia, two hind legs occurring can not autonomic activities, and obvious urine retention, and muscular tension reduces, and shows the modeling success.In the postoperative 3d, activities in rats is less, and diet is few, and body weight alleviates to some extent, and after this diet recovers normally gradually, and the body constitution amount increases, and activity increases.Through manual massage's bladder, all rats can recover automatic micturition substantially after 5～7 days.Have part rat postoperative hematuria to occur in 3 days, after in time pushing the emptying bladder, most urine colors transfer to normally, but the death of minority rat hematuria is arranged.All rat otch dryings are not seen infection.Because it is timely to change bedding and padding, the hind leg of SD rat is not seen the decubital ulcer phenomenon.In the experimentation, matched group has 1, experimental group 2 rats to be arranged because of anesthetic accident death.
2.2 motor function is measured
1) hind leg motion standards of grading (adopting the Tarlov scoring of improvement)
0 minute: paralysis fully, lower limb are reactionless during acupuncture; 1 minute: paralysis fully, lower limb respond during acupuncture, but limbs are stiff; 2 minutes: limbs were movable, but can not stand or astasia (＜5 seconds); 3 minutes: can stand, but can't walk; 4 minutes: the number of can the walking step, but unstable; 5 minutes: can slowly walk, but dumb, there is certain defective; 6 minutes: normal walking.
2) assessment extremity muscular strength standard (adopting the swash plate experiment)
Swash plate is linked to each other by hinge by two orthogonal alloy sheets and forms, and makes base plate for one, and another piece is a movable plate, the thick rubber blanket of the surface about 6mm in shop.With rat headfirst, long axis of body is placed with the swash plate longitudinal axis is vertical, and plate increases the angle between movable plate and horizonal base plate gradually from horizontal level (0 degree), when rat can just stop 5s onboard, and recording angular.Every rat is surveyed 3 times, averages.
3) motor function measurement result
1. equal quality impact bar, along with the increase of impact bar height of fall, rat hindlimb motion scoring descends gradually, and the inclined-plane critical angle descends gradually, shows that rat extremity muscular strength reduces.
When 2. the impact bar height of fall was identical, along with the increase of impact bar quality, rat hindlimb motor function and inclined-plane critical angle descended gradually.
3. increase impact bar height of fall and impact bar quality, the spinal cord injury degree of rat model increases the weight of, and its hind leg motor function and inclined-plane critical angle descend.
4. equal quality impact bar, when the impact bar height of fall was also identical, along with the prolongation that impact bar falls pressing time, rat hindlimb motion scoring was on a declining curve, and the inclined-plane critical angle also diminishes.
5. after the spinal cord injury, the spinal cord of rat has certain self-repair function, degree of injury light after the wound about 2 weeks, part motor function in rats and inclined-plane critical angle can return to level before the damage basically, the self-repair function of prompting spinal cord and the degree of injury of spinal cord have certain relation.
2.3 histological observation
1) sample preparations
Get 3 of rats for every group, after SCI was 12 hours, with 10% chloral hydrate intraperitoneal anesthesia, it was fixing to lie on the back after the anesthesia, cut off the thoracic cavity, expose heart, pour into the normal saline flushing blood vessel from left ventricle fast by venous duct, it is colourless substantially to treat that liquid is reserved in the right auricle, immediately with cold 4% paraformaldehyde fixative perfusion, first quick and back slow, take out myeloid tissue behind the 30min, get the spinal cord that damage sections and adjacent segment are about 8mm, 4 ℃ move into fixedly 6h of 4% paraformaldehyde down, move in 20% and 30% the sucrose solution until sinking to the bottom conventional then dehydration, paraffin embedding successively, the horizontal plane serial section, thick 5 μ m, HE dyeing, light microscopic is observed down.
2) spinal cord slice om observation result
1. sham operated rats: spinal cord ash, white matter clear in structure, normal, the as seen more much and polygonal neurons of grey matter, the nucleus circle and big, dyeing is shallow, kernel is clear, it is tight that white matter nerve fiber is arranged;
2. equal quality impact bar, along with the increase of impact bar height of fall, rat model spinal cord structure destructiveness increases the weight of; When the impact bar height of fall was identical, along with the increase of impact bar quality, rat model spinal cord structure destructiveness increased the weight of;
3. increase impact bar height of fall and impact bar quality, rat model spinal cord structure destructiveness increases the weight of;
4. equal quality impact bar, when the impact bar height of fall was also identical, along with the prolongation that impact bar falls pressing time, the spinal cord structure destructiveness of rat model increased the weight of;
5. under high height and greater impact bar quality situation, the rats with spinal cord injury model spinal cord structure destructiveness that the rats with spinal cord injury model that the pad on thick soft ground is made is made than hard gasket is slightly light, under the less situation of shock height and impact bar quality, both difference is not obvious;
6. spinal cord injury tissue performance (by gently to heavy)
Central canal are faintly visible, hemorrhagic focus is arranged in the grey matter, but necrosis area is less, remaining neuron is arranged in the grey matter, white matter nerve fiber edema---spinal cord ash, white matter normal configuration and central canal disappear, ash, white matter patch shape is hemorrhage, neuron swelling, necrosis, the nerve fiber edema, part-structure is smudgy---and spinal cord structure is destroyed and is disappeared, ash, the large stretch of hemorrhagic necrosis of white matter, neuron almost all disappears, glial cell necrosis, the downright bad disintegrate of white matter, the cell rareness is " a slice is bleak and desolate " scene.
Motor function and histological observation all confirm: can regulate impacting height, impact bar weight, impact bar and fall the degree that pressing time is controlled spinal cord injury according to actual needs, make it to reach the purpose of objective quantification.
We also find by experiment, and behind the use pad, the animal model with spinal cord damnification of preparation is from motor function evaluation and histological observation aspect, and its model repeats the animal model with spinal cord damnification that degree all is better than not using the pad preparation.
Therefore, the modeling method of standardization animal model with spinal cord damnification of the present invention is as the model of spinal cord classification damage, the whole operation technology is easy, repeatability better, for analyzing spinal cord classification injured tissues morphological change, pathomechanism and inquiring into the corresponding treatment measure, observing therapeutic outcome or more different experimental results all has crucial meaning.
Claims (3)
1. the modeling method of standardization animal model with spinal cord damnification may further comprise the steps:
1) the fixedly extremity of laboratory animal, head and afterbody, operation appears the spine portion of laboratory animal;
2) place and the corresponding pad of spinal cord profile at the position of preparing damage;
3) the impact passage of a hollow of top vertical fixing of pad;
4) impact bar with a standard weights that be able to be free to slide in impacting passage places in the impact passage, freely falls from the height of stipulating, impinges upon on the dorsispinal pad of laboratory animal;
5) in time remove impact bar or impact bar kept somewhere the long time of regulation, cause spinal cord dorsal part injured animal model to a certain degree.
2. the modeling method of standardization animal model with spinal cord damnification according to claim 1, it is characterized in that impacting channel outlet is 0.5～50mm apart from the distance of pad.
3. the modeling method of standardization animal model with spinal cord damnification according to claim 1 is characterized in that being provided with altitude scale on the impact passage.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101947136A (en) * | 2010-10-20 | 2011-01-19 | 中国人民解放军第三军医大学第三附属医院 | Miniature impact device for injuring experimental animals |
| RU2461071C2 (en) * | 2010-10-28 | 2012-09-10 | Государственное образовательное учреждение высшего профессионального образования "Омская государственная медицинская академия Федерального агентства по здравоохранению и социальному развитию" Росздрава | Method for simulating spinal cord injury |
| TWI493374B (en) * | 2013-12-20 | 2015-07-21 | Univ Taipei Medical | Quantitative Method for the Induction of Traumatic Brain Injury Animal Model and System thereof |
| CN107765601A (en) * | 2017-11-17 | 2018-03-06 | 中国人民解放军成都军区总医院 | Controlling cortex injured animal model producing device striker control circuit |
| CN109077825A (en) * | 2018-08-02 | 2018-12-25 | 中国人民解放军第二军医大学第二附属医院 | The tension stimulating apparatus and purposes of induced rat Thoracolumbar disk ossification of the yellow ligament |
| CN110859156A (en) * | 2019-10-23 | 2020-03-06 | 南方医科大学第三附属医院（广东省骨科研究院） | Construction method of posterior longitudinal ligament ossification animal model |
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2010
- 2010-01-08 CN CN201010033354A patent/CN101756752A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101947136A (en) * | 2010-10-20 | 2011-01-19 | 中国人民解放军第三军医大学第三附属医院 | Miniature impact device for injuring experimental animals |
| CN101947136B (en) * | 2010-10-20 | 2012-05-23 | 中国人民解放军第三军医大学第三附属医院 | Miniature impact device for injuring experimental animals |
| RU2461071C2 (en) * | 2010-10-28 | 2012-09-10 | Государственное образовательное учреждение высшего профессионального образования "Омская государственная медицинская академия Федерального агентства по здравоохранению и социальному развитию" Росздрава | Method for simulating spinal cord injury |
| TWI493374B (en) * | 2013-12-20 | 2015-07-21 | Univ Taipei Medical | Quantitative Method for the Induction of Traumatic Brain Injury Animal Model and System thereof |
| CN107765601A (en) * | 2017-11-17 | 2018-03-06 | 中国人民解放军成都军区总医院 | Controlling cortex injured animal model producing device striker control circuit |
| CN109077825A (en) * | 2018-08-02 | 2018-12-25 | 中国人民解放军第二军医大学第二附属医院 | The tension stimulating apparatus and purposes of induced rat Thoracolumbar disk ossification of the yellow ligament |
| CN109077825B (en) * | 2018-08-02 | 2023-12-05 | 中国人民解放军第二军医大学第二附属医院 | Tension stimulation device for inducing rat thoracolumbar yellow ligament ossification and application thereof |
| CN110859156A (en) * | 2019-10-23 | 2020-03-06 | 南方医科大学第三附属医院（广东省骨科研究院） | Construction method of posterior longitudinal ligament ossification animal model |
| CN110859156B (en) * | 2019-10-23 | 2021-04-30 | 南方医科大学第三附属医院（广东省骨科研究院） | Construction method of posterior longitudinal ligament ossification animal model |
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Open date: 20100630 |