CN102038560A - Construction method of operation model for collecting cerebrospinal fluid repeatedly - Google Patents

Abstract

The invention discloses a construction method of an operation model for collecting cerebrospinal fluid repeatedly, comprising the following steps: positioning an operation drilling hole on the planar structure of an animal skull; puncturing into the drilling hole by an external sleeve puncturing needle; burying and fixing a flow guiding pipe; and collecting the cerebrospinal fluid for repeatedly by the flow guiding pipe. The method provides a new model for collecting the cerebrospinal fluid in a more effective and reasonable manner for a pharmacological experiment of toxic generation and pharmacological generation and the like. According to the method, the survival rate of operation of the animal can reach 80%, the success rate of the model can reach 60%, and thus the construction method of the model is reasonable in design, and is beneficial to collecting the cerebrospinal fluid repeatedly.

Description

Construction method of surgical model for multiple collection of cerebrospinal fluid

technical field

The invention relates to the field of cerebrospinal fluid collection methods, in particular to a method for constructing an operation model for multiple collection of cerebrospinal fluid.

Background technique

Cerebrospinal fluid (CSF) is a colorless and transparent liquid filled in the ventricles, subarachnoid space, and central canal of the spinal cord. It is similar in nature to plasma and lymph, slightly viscous, and is used to supply certain nutrients to brain cells. , Carry away the metabolites of brain tissue, regulate the acid-base balance of the central nervous system and buffer the pressure of the brain and spinal cord, and have a protective and supportive effect on the brain and spinal cord. Therefore, whether the drug can effectively pass through the blood-brain barrier can be explored by detecting the content of the drug in the cerebrospinal fluid.

The collection of cerebrospinal fluid is a common surgical problem in clinical trials and non-clinical research. The brain is the life center of mammals and the place where many important centers gather. Methods often choose spinal puncture method. At present, the collection of cerebrospinal fluid is mostly one-time collection, and the animal models are mainly rats and mice. In non-clinical studies, most experiments choose to use a puncture needle to pierce from the neck of experimental animals and reach the cisterna magna for collection.

When doing research on the metabolic kinetics of drugs in the body, the method of collecting different individual animals in batches is often used to meet the drug data at multiple time points. This alternative approach limits the scientific validity of pharmacokinetic/pharmacodynamic (PK/PD) studies. Therefore, using dogs or other animals to establish new non-rodent cerebrospinal fluid multiple collection surgical models is particularly important for biomedical research.

Contents of the invention

The invention provides a method for constructing an operation model for multiple collections of cerebrospinal fluid. The method is simple to operate and easy to control, and realizes multiple collections of cerebrospinal fluid.

A method for constructing a cerebrospinal fluid multiple collection operation model, comprising the following steps:

After the animal is anesthetized, a surgical drill hole is positioned on the plane structure of the animal's skull, and the puncture needle is punctured through the outer cannula in the drill hole, a drainage tube is embedded and fixed, and cerebrospinal fluid is collected through the drainage tube multiple times.

The diameter of the drilled holes should not be too large to avoid excessively large wounds, preferably 2 mm to 5 mm.

In order to ensure that the puncture needle passes through the skull smoothly and accurately reaches the cerebellar fossa, the drill hole is aimed at the physiological slit, and the direction of the drill hole should be consistent with the puncture direction.

The burr hole is preferably located in the center of the skull plane.

Before puncturing, the outer cannula puncture needle is filled with a blunt-tipped inner needle, and the needle is placed in the drilled hole. When puncturing, the puncture needle penetrates downward along the physiological slit at 30°-45° to reach the cerebellar fossa, and penetrates deep into the cerebellar fossa. About 3-5cm.

After the needle head of the puncture needle reaches the cerebellar fossa, pull out the inner needle, insert the drainage tube into the needle hole, fix the drainage tube and slowly pull out the puncture needle, fix the outer end of the skull of the drainage tube with medical biological glue, and expose several centimeters of drainage The tube was prepared for collecting cerebrospinal fluid, and the muscles and scalp were sutured to ensure that the drainage tube was firmly fixed and the interior was unobstructed. Dogs recover for more than 12 hours after surgery.

The drainage tube can be selected as drainage polyethylene plastic (PE) tube.

Said animals are non-rodent animals, preferably dogs.

The time for collecting cerebrospinal fluid is more than 12 hours after the animal recovers, and the device used for collecting cerebrospinal fluid can be a suction device such as a syringe.

Compared with prior art, the present invention has following advantage:

The method of the present invention realizes the collection of cerebrospinal fluid at the cerebellar fossa, because the cerebellar fossa is a relatively large cavity in the skull, and its ability to store cerebrospinal fluid is relatively high; Therefore, no matter whether the animal is awake or anesthetized, the cerebrospinal fluid will not be lost due to the surgical gap.

The method of the invention provides a more effective and reasonable new model for collecting cerebrospinal fluid for pharmacological tests such as toxicology and pharmacokinetics, and realizes multiple collections of cerebrospinal fluid. In this method, the surgical survival rate of animals can reach 80%, and the success rate of the model can reach 60%, which shows that the design of the model construction method is reasonable, and it is beneficial to the multiple collection of cerebrospinal fluid.

Description of drawings

Fig. 1 is the top view structure schematic diagram of canine head skeleton;

Figure 2 is a schematic diagram of the side structure of the dog's head skeleton;

Among them, 1 is the frontal bone; 2 is the temporal process of the zygomatic bone; 3 is the eye socket; 4 is the interparietal bone; 5 is the parietal bone; 6 is the drilling site; 7 is the zygomatic process of the temporal bone; Fossa; 10 is the remaining cerebellar fossa.

Detailed ways

Specific embodiments of the present invention are further described below.

Example 1

As shown in Figure 1 and Figure 2, the structure of the canine head skeleton includes: frontal bone 1; zygomatic temporal process 2; eye socket 3; interparietal bone 4; parietal bone 5; drilling site 6; temporal bone zygomatic process 7; masseter muscle Fossa 8; Cerebellar Fossa 9 and 10.

Beagle dogs were anesthetized by intravenous injection of 3% pentobarbital sodium solution (1ml/kg), and fixed on the operating table in prone position. The throat is padded with a soft pillow so that the occipital bone is facing up, and the head of the Beagle dog is fixed. With the Beagle dog occipital bone as the center, the hair on the left rear side of the dog occipital bone was shaved. Use a scalpel to make a transverse incision of about 2 cm in the shaved area 2 to 3 cm away from the occipital bone. The fascia above the plane of the dog's skull was bluntly dissected with a surgical dissection instrument to expose the muscle group above the skull. Separate the muscles vertically to the plane of the skull along the muscle lines until the skull is exposed. Determine the center of the skull plane, that is, the drilling site 6, drill a hole of 2-5 mm in the center with a skull drill, and surround the hole with a skull plane with a diameter of about 2 cm. In order to ensure that the puncture needle passes through the skull smoothly and accurately reaches the cerebellar fossa 9 or 10, the drilling direction should be consistent with the puncture direction, and the drilling should be aimed at the physiological slit, which is the link between the cerebellum and the brain in the canine brain tissue.

Use a puncture needle with an outer cannula diameter of 1.6mm, fill the needle with a blunt inner needle before puncture, put the needle into the drilled hole, and penetrate the cerebellum 30-45° downward along the physiological slit during puncture Nest, about 3 to 5 cm deep.

When the puncture needle reaches the cerebellar fossa, the inner needle can be pulled out, and the cerebrospinal fluid can be seen overflowing from the needle hole (the effect of intracranial pressure). Insert the drainage tube into the needle hole, fix the drainage tube and slowly pull out the puncture needle, fix the outer part of the skull of the drainage tube with medical bio-protein glue, and expose a few centimeters of the drainage tube for the collection of cerebrospinal fluid. Antibiotics are sprinkled on the surgical site, the muscles and scalp are sutured, and the head is placed in a low position under anesthesia. Seal the open end of the drainage tube to prevent cerebrospinal fluid from overflowing. Wound care the next day after surgery.

Twelve hours after the operation, the seal of the drainage tube was opened, a syringe was inserted into the drainage tube, and 50 μl of liquid in the tube was drawn and discarded. Wait for the cerebrospinal fluid to seep out from the drainage tube by itself; immediately take the cerebrospinal fluid and slowly draw it with a syringe. Cerebrospinal fluid was extracted daily after operation to check the success rate of cerebrospinal fluid operation, and observed for 7 days. The extraction volume is set as follows: 4.5ml on the first day (point-based collection under postoperative anesthesia); 0.5ml after waking up; and 0.5ml per day for the remaining 13 days.

The success rate of the operation is based on the postoperative healthy survival of Beagle dogs. The success rate of the model is that the continuous collection of cerebrospinal fluid without blood contamination by Beagle dogs after the operation is the success criterion. 10 Beagle dogs were operated on, and the success rate of the operation was 80%. The cerebrospinal fluid was collected every day after the operation, and the cerebrospinal fluid could be collected smoothly within 7 days, and the success rate of the model was 60%.

Example 2

Four model animals were constructed as described in Example 1. Take a drug ethylenediamine (EDA) injection that can penetrate the blood-brain barrier, configure the drug in NaCl injection with a concentration of 0.9% by mass, and infuse it intravenously for 3 hours at a rate of 1 mg/kg/h. 2ml of whole blood and 0.3ml of cerebrospinal fluid were regularly collected at 0, 30, 60, and 180 minutes after administration (end of administration) and 0.5, 1, 2, 3, and 4 hours after drug withdrawal. The established method was applied to detect the EDA concentration in biological samples. The results were analyzed using DAS 2.0 pharmacokinetic software. According to the results of pharmacokinetics, EDA was eliminated in a three-compartment model after intravenous infusion of 3 mg/kg to Beagle dogs.

Claims (9)

1. a cerebrospinal fluid is repeatedly gathered the construction method of surgery models, may further comprise the steps:

After Animal Anesthesia, positioning operation boring on animal skull planar structure, in described boring through the trocar sheath lancet puncture, imbed drainage tube and fixing drainage tube, by drainage tube collect cerebrospinal fluid repeatedly.

2. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, the diameter of described boring is 2mm～5mm.

3. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, the physiology slit is aimed in described boring, and the direction of boring is consistent with the puncture direction.

4. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, described boring is positioned at the skull planar central.

5. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, in pin, fill before the described trocar sheath lancet puncture blunt nosed in pin, syringe needle is put to the hole of having bored, puncture needle thrusts the arrival cerebellar fossa downwards for 30 °～45 ° along the physiology slit during puncture, gos deep into 3cm～5cm.

6. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, described trocar sheath puncture needle syringe needle is extracted interior pin after arriving cerebellar fossa, drainage tube is inserted in the pin hole, and fixedly drainage tube is slowly extracted puncture needle, uses fixedly drainage tube skull outer end portion of medical bio glue, expose several centimetres of drainage tubes in order to collect cerebrospinal fluid, suture muscles and scalp guarantee the drainage tube fixation, and be inner unobstructed.

7. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, described animal is non-rodent.

8. cerebrospinal fluid according to claim 7 is repeatedly gathered the construction method of surgery models, it is characterized in that, described animal is a dog.

9. cerebrospinal fluid according to claim 1 is repeatedly gathered the construction method of surgery models, it is characterized in that, the time of described collect cerebrospinal fluid is that animal recovers more than 12 hours.

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