CN104116572A - Method for building rat spinal cord injury model - Google Patents

Abstract

The invention relates to a new method for making a rat spinal cord injury model; it mainly solves many problems in the existing models such as complex operation, poor repeatability, large amount of bleeding in rats, high infection death rate, many complications, short survival time, etc. ; The technical scheme adopted is: using a self-made needle to cause accurately positioned lumbar cord dissection injury on one side of the rat spinal cord without opening the medullary cavity, which is a spinal cord injury model made by a new method, and at the same time Drugs and preparations can be injected to intervene in the injury and the regenerative repair process after the injury at the same time as the injury.

Description

Method for Establishing Rat Spinal Cord Injury Model

technical field

The invention relates to a method for establishing a spinal cord injury model, in particular to a method for establishing a rat spinal cord injury model.

Background technique

Spinal cord injury is a disease with a high morbidity rate. It is an unresolved problem in clinical practice and can cause irreversible motor sensory dysfunction. With the rapid development of various industries in society, the incidence of spinal cord injury has also increased. Therefore, the research and attention to spinal cord injury is still a subject of joint exploration at home and abroad. The spinal cord injury model provides a scientific basis for the study of spinal cord injury. An ideal spinal cord injury model should meet the following conditions. 1. It should be able to reflect the neurophysiology and motor behavior of spinal cord injury in experimental animals. 2. An animal model consistent with or similar to clinical spinal cord injury can be provided, that is, it has good clinical relevance. Third, the model must be reproducible. The study of spinal cord injury requires a large number of experimental animals, which requires the standardization of the production model.

Common spinal cord injury models in the prior art include the following:

1. Contusive spinal cord injury model

The dorsal spinal cord blow model designed by Allen for the first time uses weights of different weights to drop vertically along the cannula from different heights to hit the dorsal spinal cord of rats to cause different degrees of damage. The disadvantages of this model are that the repeatability of the operation is poor, and there are many influencing factors. For example, the damage caused by the same potential energy often varies greatly, and the shape and area of contact between the impact hammer and the spinal cord at the moment of impact are different.

2. Pincer spinal cord injury

This model meets the research needs of various injury types such as acute spinal cord compression. The specific operation process: the lamina of SD rats was opened surgically, and the spinal cord segment corresponding to T10 was clamped with an aneurysm clamp. Disadvantages of this model: The damage to the rats during the establishment of this model is relatively large, and the T10 lamina needs to be bitten off to fully expose the spinal cord segment corresponding to T10. Rats have a high chance of infection and are prone to infection and death. Moreover, the strength of the clamp on the spinal cord injury is different, and it is easy to cause total paralysis of the lower limbs of the rat. It causes a lot of inconvenience to the postoperative care of rats.

3. Incisional spinal cord injury model

Procedure: Open the lamina, expose the corresponding spinal cord, and use a sharp iris blade or microscissors to transect the spinal cord completely or semi-transected, or to resect a segment of the spinal cord. The disadvantages of this method are that the operation steps are more complicated, the damage to the rats is greater, and the cross-section of the spinal cord with a razor blade is relatively neat, which is somewhat different from the actual clinical cases.

4. Spinal cord ischemia and reperfusion model

The model is made by catheterizing the femoral artery or directly closing the descending aorta. Insufficiency of this method: while blocking the artery to cause ischemia, the operation also caused damage to other tissues and organs in the perfusion area, which affected the behavioral observation.

Contents of the invention

The present invention overcomes the deficiencies of the prior art, and the technical problems to be solved are many problems such as complex operation and poor repeatability in the prior art spinal cord injury model, large amount of bleeding in rats, high infection death rate, many complications, short survival time, etc. Furthermore, a method for establishing a rat spinal cord injury model with simple operation, good repeatability and significant dysfunction is provided.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a rat spinal cord injury model establishment method, carried out according to the following steps:

(1) Weigh the rats, inject 1% pentobarbital sodium injection into the intraperitoneal cavity at 0.4ml per 100g body weight;

(2) After the rat is anesthetized, prepare the skin on the back and disinfect the skin;

(3) Determine the position of T ₉ -T _12. The lumbar spinous process of SD rats is prominent, and the thoracic spinous process is tilted backward, and the lumbar spinous process is tilted forward. According to this mark, it is easy to locate accurately. Make an incision just above T ₉ -T ₁₂ , make a 2~3cm skin incision, separate the muscle and fascia, and expose the spinous process and lamina of T ₉ ~T ₁₂ ;

(4) Use tissue scissors and mosquito forceps to peel off the muscles and ligaments on the left side of the spinous process of T ₉ -T ₁₀ to thoroughly expose the spinous process and transverse process of T ₉ -T ₁₀ , and determine the gap of T ₉ -T ₁₀ ;

(5) Avoiding the center of the spinal cord and slightly to the left, insert the grounded 1ml syringe needle vertically into the medullary cavity from the T ₉ -T ₁₀ gap and into the corresponding segment of the spinal cord. Due to the negative pressure in the medullary cavity and the soft tissue in the intervertebral space, it is not necessary to use too much force when inserting the needle, and the needle can easily enter the medullary cavity and spinal cord. When the needle enters the spinal cord, the rat will shake violently and pull the needle vertically up and down, feeling that there is a large space for movement, indicating that the needle is in the medullary cavity;

Withdraw the needle and insert the curved needle perpendicularly from the point of entry with the curved tip pointing to the left. After the needle of the breaking needle enters the medullary cavity, swing the needle slightly from side to side, and pay attention to absolutely avoiding that the bent needle goes beyond the midline of the spinous process and enters the right half of the spinal cord, thereby only damaging the left spinal cord. When performing this operation, the left (injury side) hind limb twitched, and the needle was pulled out gently and still at a vertical angle. At this time, the establishment of the spinal cord semi-severe injury model was basically completed;

(6) After the operation, the muscles and skin are sutured. After waking up, the rats were fed with common feed under dry, quiet and natural light conditions, and were regularly cleaned and disinfected with iodophor.

The above-mentioned method for establishing a spinal cord injury model can also be operated on the right spinal cord, and the operating conditions are corresponding to the above-mentioned operation on the left spinal cord.

The needle head of the straight needle is a flat needle without a point; it can be used to determine the needle entry point of the spinal cord.

The straight needle is preferably made by grinding the needle of the syringe, and the obtained straight needle can be used to inject neurotrophic factors or neural stem cells into the spinal cord injury site to intervene in injury, and in addition to determining the insertion point of the myelotomy needle. Agents that promote regeneration after injury.

The needle of the broken marrow needle is a right-angle needle, and the free end of the right-angle needle is horizontal when the broken marrow needle is inserted vertically.

The marrow-breaking needle is preferably made by bending the beveled surface of the needle of the syringe backward by 90°.

Required material and apparatus of the present invention are as follows:

experimental animals

Healthy male rats, weighing 180-200 grams;

Experimental equipment

No. 11 surgical blade, needle forceps, mosquito forceps, vascular forceps, surgical forceps, tissue scissors, sewing needles, straight needles (self-made 1ml syringe needles: one made by grinding the 1ml syringe needles flat, as shown in Figure 1 shown), and the broken needle (made by bending the needle of a 1ml syringe to the back side of the bevel at 90 degrees, as shown in Figure 2).

Compared with the prior art, the present invention has the following beneficial effects.

The spinal cord injury model caused by a needle is easy to operate, with less damage, high repeatability, and significant dysfunction, and provides good conditions and environment for studying drugs or preparations that stimulate or promote spinal cord regeneration; for exploring the molecular mechanism of clinical drugs Provide an experimental basis. At the same time, due to the low postoperative infection rate, few complications, high survival rate and good repeatability of model animals, it has a good prospect for popularization and use.

Description of drawings

Fig. 1 is a schematic structural view of the straight needle of the present invention.

Fig. 2 is a schematic structural view of the marrow breaking needle of the present invention.

Figure 3 is a schematic diagram of the site of spinal cord injury in rats.

Figure 4 is a schematic diagram of a longitudinal section of the spinal cord at the site of spinal cord injury in a rat.

Fig. 5 is a schematic diagram of a cross section of the spinal cord at the site of spinal cord injury in a rat.

Detailed ways

The present invention will be further described below in conjunction with specific examples.

Modeling method:

(1) Weigh the rats, and inject 1% pentobarbital sodium injection into the intraperitoneal cavity at a rate of 0.4ml per 100g body weight.

(2) After the rats were anesthetized, the back skin was prepared and the skin was disinfected.

(3) Determine the position of T9-T12. The lumbar spinous process of SD rats is prominent, and the thoracic spinous process is tilted backward, and the lumbar spinous process is tilted forward. According to this mark, it is easy to locate accurately. Make an incision just above T9-T12, make a 2-3cm skin incision, separate muscle and fascia, and expose T9-T12 spinous process and lamina.

(4) As shown in Figure 3, use tissue scissors and mosquito forceps to peel off the muscles and ligaments on the left side of the T9-T10 spinous process to thoroughly expose the T9-T10 spinous process and transverse process, and determine the T9-T10 gap.

(5) As shown in Figure 4, avoiding the center of the spinal cord and slightly to the left, insert the grounded 1ml syringe needle vertically from the T9T10 gap into the medullary cavity and into the corresponding segment of the spinal cord. Due to the negative pressure in the medullary cavity and the soft tissue in the intervertebral space, it is not necessary to use too much force when inserting the needle, and the needle can easily enter the medullary cavity and spinal cord. When the needle enters the spinal cord, the rat will vibrate violently, pull and insert the needle vertically, and feel that there is a large space for movement, indicating that the needle is in the medullary cavity.

Pull out the needle, insert the curved needle into the needle vertically from the insertion point as shown in Figure 5, with the curved needle pointing to the left when the needle is inserted. After the needle enters the medullary cavity, swing the needle slightly from side to side, but absolutely avoid the curved needle going beyond the midline of the spinous process and entering the right half of the spinal cord, thus only damaging the left spinal cord. When performing this operation, the left (injury side) hind limb twitched, and the needle was pulled out gently and still at a vertical angle. At this time, the establishment of the spinal cord semi-severe injury model was basically completed.

(6) After the operation, the muscles and skin are sutured. After waking up, the rats were fed with common feed under dry, quiet and natural light conditions, and were regularly cleaned and disinfected with iodophor.

The experimental results are as follows.

Behavioral Observation of Rats

Normal rats have four feet touching the ground and can stand upright. After waking up, the spinal cord hemisection model rats had redness and swelling at the surgical incision 3-5 days after operation, and returned to normal after a few days. Compared with normal rats, the left hind limb of the model animals was paralyzed, walked on the floor, and could not stand upright. But the rats can carry out normal daily activities, diet and excretion are normal, postoperative complications are few, and the survival time is longer. Movement of the contralateral hindlimb was normal.

Grasping force (motor function) and thermal pain (sensory function) responses of rat hindlimbs at different times

We can see from the gripping force and heat pain response test of rats after spinal cord hemisection, (as shown in Table 1 and Table 2) the sensory and motor abilities of both hind limbs of rats before operation are normal. After the operation, with the passage of time, the motor sensation and reflex function of the affected hindlimb did not recover, while the motor function of the healthy hindlimb was normal, and gradually recovered after the reaction to heat pain disappeared. This shows that the half amputation has caused neurological impairment in rats.

Histomorphological observation of spinal cord

Visual observation: 24 hours after the operation, the rat's wound was swollen. After the medullary cavity was opened, the left spinal cord at the lumbar enlargement was obviously broken, and the spinal cord was congested and swollen. After 1 week, the skin wound healed and the swelling disappeared. Observation under the microscope: the longitudinal spinal cord fibers were broken in the injury area of the rat spinal cord after operation.

Above result can find out that the advantage of the present invention is:

(1) The 1ml syringe needle used in the present invention is easier to operate, the location of the injury is accurate, and the half-cut is complete, and the broken end of the spinal cord injury is closer to the actual clinical situation.

(2) The present invention uses needles to injure rats, which causes less damage to rats, shorter wound self-healing time, less chance of infection to the spinal cord, less bleeding, less requirements for experimental equipment, relatively easy operation, and causes Motor sensory impairment is clear.

(3) The biggest feature of the present invention is that it can inject drugs, neurotrophic factors or neural stem cells and other preparations that intervene in injury and promote post-injury regeneration at the injured site through the syringe connected to the injection needle. At the same time, the effects of neurotransmitters, neurotrophic factors, drugs, and neural stem cells on the spinal cord injury and regeneration process and their molecular mechanisms can be observed and explored through spinal cord hemisection. Because the model of the present invention can be compared with the healthy side and can utilize the needle to inject medicine in the spinal cord cavity, it has unique advantages.

The spinal cord injury model caused by a needle is easy to operate, with less damage, high repeatability, and significant dysfunction, and provides good conditions and environment for studying drugs or preparations that stimulate or promote spinal cord regeneration; for exploring the molecular mechanism of clinical drugs Provide an experimental basis. At the same time, due to the low postoperative infection rate, few complications, high survival rate and good repeatability of model animals, it has a good prospect for popularization and use.

The present invention may be embodied in other specific forms without departing from the spirit or main characteristics of the invention. Therefore, no matter from which point of view, the above-mentioned embodiments of the present invention can only be considered as explanations of the present invention and can not limit the invention. The claims indicate the scope of the present invention, but the above description does not indicate the scope of the present invention. Therefore, any changes within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims.

Claims (9)

1. Model of Rat Spinal Cord Injury method for building up, is characterized in that in accordance with the following steps:

(1) rat is weighed, by every 100g body weight, with 0.4ml pentobarbital sodium inj, carry out lumbar injection;

(2) after rat anesthesia, back preserved skin, sterilization skin;

(3) determine T ₉-T ₁₂position, at T ₉-T ₁₂directly over otch, do a 2-3cm skin incision, separating muscle and fascia, expose T ₉-T ₁₂spinous process and vertebral plate;

(4) by T ₉t ₁₀muscle and the ligament on spinous process left side or right side are peeled off thorough exposure T ₉-T ₁₀spinous process and transverse process, determine T ₉-T ₁₀gap;

(5) avoid spinal cord center, the side of slightly taking back or right side, by staight needle from T ₉-T ₁₀gap is vertically inserted pulp cavity and is entered correspondent section spinal cord, extracts syringe needle, forms entry point; The syringe needle of broken broach is vertically inserted from entry point, and entry point is when left side, and during inserting needle, crooked needle point is towards left side; Entry point is when right side, and during inserting needle, crooked needle point is towards right side; The syringe needle that slightly swings after syringe needle enters pulp cavity, enters another Hemimyelia but definitely avoid crooked syringe needle to surpass spinous process center line, only destroys thus left side or right side spinal cord; Also still with vertical angle, extract brokenly the syringe needle of broach then light and slowly;

(6) suture muscles and skin, after rat is clear-headed, under dry, peace and quiet, natural light condition, normal diet is fed, and cleaned at regular intervals sterilization.

2. Model of Rat Spinal Cord Injury method for building up according to claim 1, is characterized in that: the concentration of described pentobarbital sodium inj is 1%.

3. Model of Rat Spinal Cord Injury method for building up according to claim 1, is characterized in that: the syringe needle of described staight needle is for putting down without sharp syringe needle.

4. Model of Rat Spinal Cord Injury method for building up according to claim 3, is characterized in that: described staight needle is for determining the entry point of broken broach.

5. Model of Rat Spinal Cord Injury method for building up according to claim 3, is characterized in that: described staight needle is for to polish the syringe needle of syringe to make.

6. Model of Rat Spinal Cord Injury method for building up according to claim 5, is characterized in that: described staight needle, also can be for injectable drug in spinal cavity for determining the entry point of broken broach.

7. Model of Rat Spinal Cord Injury method for building up according to claim 1, is characterized in that: the syringe needle of described broken broach is right angle syringe needle, described broken broach when vertical lower pin, the free end level of described right angle syringe needle.

8. Model of Rat Spinal Cord Injury method for building up according to claim 7, is characterized in that: described broken broach for by the needle slope of syringe backward 90-degree bent make.

9. according to the Model of Rat Spinal Cord Injury method for building up described in claim 5 or 8, it is characterized in that: described syringe is common 1ml disposable syringe.