CN113208764A

CN113208764A - Laser transmitter for cerebral apoplexy model and simulation method thereof

Info

Publication number: CN113208764A
Application number: CN202110262839.2A
Authority: CN
Inventors: 赵晟; 虞美娟
Original assignee: Nanjing Aikeyi Biotechnology Co ltd
Current assignee: Nanjing Aikeyi Biotechnology Co ltd
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2021-08-06

Abstract

The invention belongs to the field of neurobiology, and particularly relates to a stroke model laser transmitter and a simulation method thereof, wherein the stroke model laser transmitter is a novel, efficient, stable and low-cost stroke model laser, and is based on a 520nm laser light source, and is additionally provided with a direct-current power supply and a fixed support manufactured by using a 3D printing technology as an auxiliary. The common photochemical plug method is adopted, the operation process is simple, and the molding rate is high. The obtained novel laser has the characteristics of maintenance-free performance and long service life.

Description

Laser transmitter for cerebral apoplexy model and simulation method thereof

Technical Field

The invention relates to the technical field of neurobiology, in particular to a stroke model laser transmitter and a simulation method thereof.

Background

Stroke is a cerebrovascular disease in which cerebral nerves are damaged due to the damage of cerebral vessels. Less than 2% of patients arrive at hospital care within a treatment thrombolysis window of 4 hours, so most stroke patients suffer from permanent cerebral ischemia. Functional recovery and improvement of stroke-disabling survivors is urgently needed, but no satisfactory treatment is available at present, and preclinical and clinical studies are needed to determine safe and effective treatment methods. Improving the function of damaged nerves is a major focus of clinical and basic research on stroke recovery studies, so the selection and nature of functional testing is crucial for the correct execution and interpretation of these studies. Many animal models of stroke are developed for testing the assessment of neurological deficits by assessing motor, somatosensory, cognitive or other functions. It is noteworthy that different stroke models simulate different forms of stroke-leading to different neurological dysfunctions, so not all tests are applicable to each stroke model. Furthermore, although many functional test formats have been adapted from rats, mice have become the main experimental animal in stroke studies, but the methods and principles of testing differ between these two species and make the test results challenging.

The successful construction of a disease model is the premise of successful disease research, and at present, the mouse stroke modeling method mainly comprises a photochemical induction method, a Middle Cerebral Artery Occlusion (MCAO) method and an endothelin contraction method. The former model mainly results in permanent interruption of blood flow to the target area of the brain, while in the latter two models there is a reperfusion component following ischemia. These several modes are central in various studies, and the focus is on identifying and verifying key molecular targets related to post-stroke recovery, testing various treatment methods for post-stroke recovery, belonging to the basic biology of post-stroke brain recovery. Currently, many experimental stroke neuroprotection studies are focused on photochemically induced ischemic stroke. The method is suitable for some studies using antiplatelet and antithrombotic drugs.

Photochemical induction methods, commonly known as the photo-plug method, utilize the properties of photosensitizers which initiate chemical reactions when irradiated by a specific light source. The rose bengal is injected into the tail vein or the abdominal cavity, and is irradiated by exciting light or a cold light source with specific wavelength to induce the rose bengal at the irradiated blood vessel part to generate photochemical reaction, generate and release active substances such as free radicals and the like to cause vascular endothelial cell injury, platelet adhesion and release, further stimulate intravascular coagulation, generate a space-limited ischemic region and finally cause thrombosis. This non-invasive approach tends to limit the infarct size to a specific range with low mortality. The advantages of this model are as follows. (1) Non-invasive and easy to handle. During the induction of infarction, no craniotomy is needed, and intracranial infection is reduced as much as possible. (2) The method can be used to generate infarcts of the same size (reproducibility) in similar locations between animals, with good stability of the ischemic focus. (3) The technical change can lead the subcortical stroke, and the model is suitable for rats and mice. (4) The induction of embolism is similar to human cerebral thrombosis.

Therefore, the ideal light source method should have the characteristics of good stability, strong targeting property, easy operation, wide application range and the like. The illumination light sources on the market mainly comprise halogen lamps, 546nm optical filters, krypton lasers, xenon lamp irradiation systems and the like. The laser wavelength emitted by the handheld laser (laser pen) is 532nm, and the handheld laser can be used as an experimental light source.

Because the stroke laser transmitter in the market is expensive or inconvenient to operate, a novel stroke model laser transmitter which is convenient to operate and has a moderate price is urgently needed to be developed.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a stroke model laser transmitter and a simulation method thereof, which aim to solve the problems in the background art.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: the utility model provides a stroke model laser emitter, includes laser emitter, its characterized in that: the outer surface of the laser emitter is wrapped and connected with a sleeve head, the sleeve head is fixedly connected above the supporting column, a bottom plate is fixedly connected to the lower end face of the supporting column, and the laser emitter is electrically connected with an adjustable direct-current power supply.

Preferably, the bottom plate is a cuboid, and the length, the width and the height of the bottom plate are respectively 180mm, 160mm and 8 mm.

Preferably, a groove is embedded in the middle of the bottom plate, and the length and the width of the groove are respectively 120mm, 10mm and 4 mm.

Preferably, the wavelength of the laser emitter is 520 nm.

Preferably, the sleeve head is internally embedded with a fixing groove for fixing the laser emitter, and the sleeve head is used for fixing and adjusting the angle of the laser emitter.

Preferably, the sleeve head and the bottom plate are manufactured and molded by using a 3D printing technology.

The method for simulating the stroke model laser transmitter comprises the following steps: based on a laser emitter, a photosensitive dye rose bengal is injected through a tail vein or an abdominal cavity, and then excitation light with the wavelength of 520nm is used for inducing the rose bengal at the irradiated blood vessel part to generate photochemical reaction, and active substances such as free radicals and the like are generated and released to cause vascular endothelial cell injury, platelet adhesion and release, so that intravascular coagulation is excited, and finally thrombosis is caused.

(III) advantageous effects

The invention provides a stroke model laser transmitter and a simulation method thereof, which have the following beneficial effects: 1. easy operation, small wound area, easy angle adjustment, wound aiming and light source fixation; 2. the assembly element is convenient to produce, so that the production cost is greatly reduced; 3. the stability is good, and the controllability is high; 4. the method has wide applicability, is not only suitable for common C57 mice, but also can obtain higher molding efficiency for other mice (such as ICR mice). In conclusion, the invention is the stroke model laser emitter which is easy to operate, low in cost, high in stability and efficiency and wide in applicable mouse variety range.

Drawings

FIG. 1 is a schematic view of the structure of a base plate and a support column of the device of the present invention;

FIG. 2 is a schematic view of the structure of the cuff of the present invention;

FIG. 3 is a schematic diagram showing the position of the head incision of a mouse according to the present invention;

FIG. 4 shows the results of the laser irradiation test at different time intervals;

FIG. 5 shows the results of the laser irradiation test under different illumination intensities.

Detailed Description

For better understanding of the technical features of the present invention, the following examples are given by way of illustration and are not intended to limit the scope of the present invention.

The main instruments and reagent sources used in the examples:

main instrumentation equipment

Adjustable DC power supply

Optical power meter

Laser emitter (Shanghai laser and optical century Co., Ltd.)

3D printer

Stereotaxic apparatus

Suture (Shenzhen Rui Ward Life science and technology Limited)

Flat-bottomed needle (Shenzhen Rui Ward Life science and technology Co., Ltd.)

Operation box special for animal (Shenzhen Riwode Life science and technology Co., Ltd.)

Disposable aseptic syringe (Jiangsu Suyun medical equipment Co., Ltd.)

Pointed forceps (Baishun medical equipment factory in Jiangsu industrial park)

Nuclear magnetic resonance apparatus 7.0T (Bruker, Germany)

Digital display constant temperature water bath HH-S2 (Jiangsu Jintan medical instruments company)

0.5-10ul, 20-200ul and 100-1000ul adjustable micropipette (Eppendorf, USA)

Milli-Q ultrapure Water System (Millipore, USA)

FA2004 electronic balance (Shanghai Liangping Co., Ltd.)

PH meter (Shanghai-Hengchun scientific instruments Co., Ltd.)

SPF level laboratory rat grain (Jiangsu cooperative medical bioengineering, LLC)

Primary reagent

Rose color (American sigma company)

Chlorhexidine acetate

Absolute ethanol (Shanghai chemical reagent company, Chinese medicine group)

Penbarbital sodium (Shanghai chemical reagent company, Chinese medicine group)

Depilatory cream (Shenzhen Ruiwande Life science and technology Co., Ltd.)

Isoflurane (Shenzhen Ruiwende Life science and technology Co., Ltd.)

Technical scheme

Laser emitter for assembling cerebral apoplexy model

Designing and printing laser emitter base and laser sleeve head

The bottom plate is a cuboid, and the length, the width and the height of the bottom plate are respectively 180mm, 160mm and 8 mm. The length and the width of the groove in the middle of the bottom plate are respectively 120mm, 10mm and 4mm in depth. Three posts are provided on the base plate for supporting the laser. Wherein the support is mainly a combination of the center coincidence of a cone and a cuboid. The long diameter of the bottom circle of the cone is 27mm, the short diameter is 21 mm, and the height is 124 mm. The length, width and height of the cuboid are respectively 18mm, 14mm and 62 mm. The pillar was hollowed out in the direction of the center of the cylinder having a diameter of 10mm and a height of 65mm coinciding with the pillar. Then, triangular columns (equilateral triangle with 10mm bottom waist) with the height of 65mm are used for hollowing and decorating in the direction parallel to the support columns. The front surface of the support is cut flat by a cuboid A with the length, width and height of 30mm, 10mm and 65mm respectively, and the part of the support higher than A is cut off by the cuboid. Finally, the pillars were hollowed out in a direction perpendicular to the pillars by 7 cylinders 3mm in diameter and 30mm in length, as shown in FIG. 1.

The laser sleeve head is mainly used for fixing the laser and adjusting the angle of the laser. The laser is a connected combination of two cuboids (B, C). The length, width and height of the cuboid B are respectively 36mm, 36mm and 38mm, and the middle of the cuboid B is dug out by a cuboid of 34mm, 34mm and 36mm, namely the cuboid B is 2mm thick. The bottom surface of the cuboid B is dug out by a cylinder with the diameter of 18 mm. Forming a square groove with a hole on the bottom surface. The length, width and height of the cuboid C are respectively 20mm, 12mm and 38 mm. Then, a cylinder with a diameter of 10mm and a height of 12mm is hollowed out 5mm from the bottom surface to prepare for supporting later, as shown in FIG. 2.

Assembled laser transmitter

The laser is placed in the cuff and secured to the holder. The laser wires are connected to a dc power supply. The intensity of the light was measured with a light intensity meter before starting the experiment to keep the light intensity stable within the moldable range.

Establishment of photochemical method induced cerebral ischemia model

Operation preparation: 100mg/kg rose bengal reagent is prepared, all surgical instruments are soaked in 0.5% chlorhexidine acetate for disinfection, and the surgical area is disinfected by 70% alcohol before starting surgery. The body weight of the mice was recorded and the volume of rose bengal injected was calculated (i.p. injected at 10. mu.l/g mouse weight, total volume injected each not exceeding 300u 1).

Anesthetizing the mice: 1% of sodium pentobarbital and 0.5% of chlorhexidine acetate are prepared to soak all surgical instruments for disinfection, and the surface of a surgical area is disinfected by 70% alcohol before starting surgery. The body weight of the mice was recorded and the dose of sodium pentobarbital injected was adjusted. The mice were injected at a weight of 10. mu.l/g (i.p., total injection volume of no more than 300u1 per mouse) and kept breathing 40-60 times per minute.

Target area irradiation: depilating the head with depilatory cream, fastening teeth with fine thread to fix the head firmly, and fixing limbs of mouse with air permeable medical adhesive tape. A cotton swab was used to wipe the skin over a 0.5% chlorhexidine acetate disinfected surface. An incision was made using a scalpel, along the horizontal line of the eye to the midline of the neck, exposing the skull, and drying the skull surface using a sterile cotton swab. At 2mm of the anterior cranial side (fig. 3), the cover is about 30mm²The optical fiber is brought into close contact with the surface of the skull to avoid light scattering, but care is taken not to apply pressure thereto.

Rose bengal injection: mice were injected rose-red neck-back according to a dose of 10. mu.l/g body weight. After 5min, the light source was turned on to avoid any other light source illuminating the animal. The laser was applied to the brain surface with a laser intensity of 40mW for 10 minutes.

And (3) wound suturing: the wound surface was cleaned and disinfected with a cotton swab stained with 0.5% chlorhexidine acetate to avoid dehydration. The scalp muscle layer is sutured with a reverse cutting needle and absorbable suture, the skin is sutured with nylon suture, and the wound surface is smeared with a little antibiotic ointment. The anesthetic gas was discontinued, the anesthetic drug was discontinued, the mouse was carefully removed from the stereotaxic apparatus and placed on a preheated heating pad until it was fully awake and returned to the cage.

3. Nuclear magnetic resonance imaging detection of molding efficiency

And 4h after the molding is finished, the nuclear magnetic resonance imaging can be carried out on the mouse brain. MRI acquisition was done on a 7.0T small animal magnetic resonance imager. Inhalation anesthesia is adopted during mouse MRI scanning period, the induction dose is 5% after isoflurane is mixed with oxygen, and the maintenance dose is 1%. The model mouse lies on the face of the mouse head in the coil, the head is positioned in the center of the coil, and the respiratory rhythm and amplitude are monitored by connecting with respiratory gating, the respiratory frequency is kept at 25-35 times/min by adjusting the dose of anesthetic, and the temperature of the water heating system is kept.

T1 weighted imaging (T1 weighted imaging, T1WI) liposuction imaging

T1WI employs a multi-slice multi-echo imaging sequence (MSME) with the following scanning parameters: TR 500.0ms, TE 15.0ms, FOV 2cm × 2cm, MTX 256 × 256, FA 180 °, NEX 4, layer thickness 1mm, 12 layers in total, and no-gap scanning. Fat inhibition was added, and the imaging range was mouse head, cross-sectional scanning. Images were analyzed using Image J software.

T2 weighted imaging (T2 weighted imaging, T2WI) liposuction imaging

Conventional T2WI employs Relaxation enhanced fast Acquisition (RARE), with the following scan parameters: TR is 3000.0ms, TE 36.0ms, FOV is 2cm × 2cm, MTX is 256 × 256, FA is 180 °, NEX is 2, 12 layers in total, layer thickness 1mm, and no-gap scanning. Images were analyzed using Image J software to measure high-signal cerebral infarct areas.

1: laser irradiation test at different time intervals

That is, the laser is set to irradiate the cerebral cortex of the mouse for different time lengths (5min, 10min, 15min) without changing other conditions, and the cerebral cortex of the C57 mouse is irradiated, compared with a large number of laser emitters on the market, when the irradiation time reaches 10min, the irradiation efficiency is obviously higher than the molding efficiency of the laser emitter on the market, and the result is shown in fig. 5.

2: laser irradiation test under different light intensities

That is, the laser is set to irradiate the cerebral cortex of the mouse with different light intensities (40mW, 80mW) without changing other conditions, and the cerebral cortex of the C57 mouse is irradiated, and compared with a large number of laser emitters on the market, when the irradiation light intensity reaches 40mW, the irradiation efficiency is obviously enough to achieve the molding efficiency of the laser emitter on the market, as shown in fig. 5.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The utility model provides a cerebral apoplexy model laser emitter, includes laser emitter, its characterized in that: the outer surface of the laser emitter is wrapped and connected with a sleeve head, the sleeve head is fixedly connected above the supporting column, a bottom plate is fixedly connected to the lower end face of the supporting column, and the laser emitter is electrically connected with an adjustable direct-current power supply.

2. The laser transmitter of claim 1, wherein: the bottom plate is a cuboid, and the length, the width and the height of the bottom plate are respectively 180mm, 160mm and 8 mm.

3. The laser transmitter of claim 2, wherein: the middle of the bottom plate is embedded with a groove, and the length and the width of the groove are respectively 120mm, 10mm and 4 mm.

4. The laser transmitter of claim 1, wherein: the wavelength of the laser emitter is 520 nm.

5. The laser transmitter of claim 1, wherein: the inside fixed slot that is equipped with fixed laser emitter that inlays of pullover, pullover is used for fixed and adjustment laser emitter's angle.

6. The laser transmitter of claim 1, wherein: the sleeve head and the bottom plate are manufactured and molded by using a 3D printing technology.

7. The method for simulating a stroke model laser transmitter according to any one of claims 1 to 6, wherein: based on a laser emitter, a photosensitive dye rose bengal is injected through a tail vein or an abdominal cavity, and then excitation light with the wavelength of 520nm is used for inducing the rose bengal at the irradiated blood vessel part to generate photochemical reaction, and active substances such as free radicals and the like are generated and released to cause vascular endothelial cell injury, platelet adhesion and release, so that intravascular coagulation is excited, and finally thrombosis is caused.