阅读说明：本技术 一种经头颅定位穿刺建立小鼠远端视神经损伤模型的方法 (Method for establishing mouse distal optic nerve injury model through skull positioning puncture ) 是由 钟一声 余欢 沈柄桥 钟慧敏 章敏贵 陈珺珏 于 2021-09-13 设计创作，主要内容包括：本发明提供了一种经头颅定位穿刺建立小鼠远端视神经损伤模型的方法,属于临床动物模型技术领域。本发明的造模方法包括以下步骤：(1)麻醉：于小鼠腹腔注射麻醉剂；(2)备皮定位：剔除小鼠头顶毛发,剪开头顶皮肤,钝性分离筋膜,暴露前囟；采用头颅立体定位仪找到视神经对应的体表位置,以前囟为起点,向尾部和右侧分别移动0.5mm作为进针点；(3)进针：27G针头缓慢进针,进针深度为6mm时到达颅底,后将针缓慢拔出,拔针后按压止血并缝合颅顶筋膜及皮肤,术后预防感染。本发明的模型对损伤后小鼠的存活率可达85.56％,对视神经损伤的造模成功率达95％,特别是对远端视神经损伤的造模成功率可高达90％。(The invention provides a method for establishing a mouse distal optic nerve injury model through skull positioning puncture, belonging to the technical field of clinical animal models. The molding method of the invention comprises the following steps: (1) anesthesia: injecting anesthetic into the abdominal cavity of the mouse; (2) skin preparation and positioning: removing hair at the top of the mouse head, cutting the skin at the top of the mouse head, separating fascia bluntly, and exposing bregma; finding the body surface position corresponding to the optic nerve by adopting a skull stereotaxic apparatus, and respectively moving 0.5mm to the tail part and the right side by taking bregma as a starting point to be used as a needle inserting point; (3) inserting needles: and slowly inserting a 27G needle, slowly pulling out the needle when the insertion depth reaches the skull base when being 6mm, pressing for hemostasis after pulling out the needle, suturing skull top fascia and skin, and preventing infection after operation. The survival rate of the mouse after injury can reach 85.56%, the molding success rate of the optic nerve injury can reach 95%, and particularly the molding success rate of the optic nerve injury at the far end can reach 90%.)

1. A method for establishing a mouse distal optic nerve injury model through transcranial positioning puncture is characterized by comprising the following steps:

(1) anesthesia: selecting a healthy mouse, and injecting an anesthetic into the abdominal cavity of the mouse;

(2) skin preparation and positioning: removing hair at the top of the mouse head, cutting the skin at the top of the mouse head, separating fascia bluntly, and exposing bregma; finding the body surface position corresponding to the optic nerve by adopting a skull stereotaxic apparatus, and respectively moving 0.5mm to the tail part and the right side by taking bregma as a starting point to be used as a needle inserting point;

(3) inserting needles: slowly inserting a 27G needle to the skull base when the insertion depth is 6mm, slowly pulling out the needle, pressing to stop bleeding and suturing skull top fascia and skin after pulling out the needle, and using the gatifloxacin eye ointment to prevent infection after operation.

2. The method of claim 1, wherein in step (1) male BALA/C mice are selected for 4-6 weeks.

3. The method of claim 1, wherein the anesthetic selected in step (1) is xylazine at 10mg/kg and ketamine hydrochloride at 25 mg/kg.

4. The method of claim 1, wherein the mold is formed in an environment of room temperature 22-24 ℃ and is well ventilated.

5. The method of claim 1, further comprising nursing the mice after step (3), wherein the nursing is performed by incubating the mice at 37 ℃ for 2 hours and returning the mice to the animal room after the mice are recovered.

Technical Field

The invention belongs to the technical field of clinical animal models, and particularly relates to a method for establishing a mouse distal optic nerve injury model through skull positioning puncture.

Background

Optic nerve injury (TON) is a serious complication of trauma to the cranium, orbit and face, with severe impairment of vision after injury, often leaving permanent vision impairment. Clinically, the incidence of TON is reported to be 0.5% in closed craniocerebral injury and 2.5% in maxillofacial injury, with motor vehicle and bicycle accidents predominating, followed by falls and assaults. The damage to the optic nerve can be classified into two types, direct damage and indirect damage. Direct optic nerve injury refers to an orbital wound that penetrates the inside or outside of the optic nerve canal and results from direct destruction of the optic nerve by bone fragments or hematomas; indirect optic nerve injury is caused by transmission of forces and/or trophic factors to the optic nerve from closed trauma, without significant damage to adjacent tissue structures.

Optic nerve collateral damage is the most common cause of ONT and is the leading cause of blindness in young adults. Clinical statistics show that 79% of ONT patients are middle-aged men under 31 years old, and 21% of patients are men under 18 years old.

Methods of treatment for indirect optic nerve damage have long been controversial. The existing treatment methods mainly comprise large-dose hormone shock, decompression surgery and the like, but researches show that the effect is little, and a new treatment method or an intervention measure is urgently needed to break through the current treatment limitation. Therefore, it is important to establish a normative, reliable, convenient, easy and repeatable experimental animal model to deeply research the pathophysiology process of the indirect ONT and develop a novel therapy.

The existing methods for making the optic nerve injury animal model mainly comprise the following four methods: optic Nerve Crush (ONC), optic neuritotomy, explosive injury, and ultrasound induced TON (SI-TON). Now, the following is introduced:

the optic nerve in the ONC model is constructed by exposing the optic nerve through operation and clamping the optic nerve by forceps or hemostats for different times. However, the compression model is prone to damage to adjacent tissue (peri-nerve plexus, etc.); furthermore, the model does not quantify the force applied to effect injury, the extent of which varies from person to person performing the procedure, with both clamping force and time becoming potentially variable factors in inducing injury, thus limiting its widespread use.

Second, axonotomy of the optic nerve is a complete transection of the optic nerve. This approach fails to mimic a clinically significant proportion of the optic nerve damage; in addition, in the axonotomy model, there is no opportunity to rescue or attenuate the inflammatory response, and thus it is difficult to be a powerful model for exploring a method for preventing or treating indirect optic nerve damage.

(III) explosion injury model is the mice placed in a firm PVC tube, and then compressed air is sprayed to the mouse eyes. This method can quantify the force delivered to the ocular surface, but frontal explosions can cause severe anterior and posterior segment damage with high mortality rates (varying from 24% to 46% depending on the level of applied air pressure).

The (fourth) SI-TON model is to place a microtip ultrasound apparatus on the orbit just above the entrance of the optic nerve into the bone canal to deliver ultrasound pulses, but the difference in acoustic velocity between air and bone makes it difficult to focus the sound waves in such a narrow space, easily leading to molding failures.

The above methods can simulate the optic nerve injury to a certain extent, but all have defects, and none of them relates to the injury of the distal optic nerve, so that it is necessary to develop a stable, effective and simple mouse distal optic nerve injury model to bear relevant basic research in the field.

Disclosure of Invention

The invention aims to solve the technical problems and provides a method for establishing a mouse distal optic nerve injury model through transcranial positioning puncture.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for establishing a mouse distal optic nerve injury model through skull positioning puncture comprises the following steps:

(1) anesthesia: selecting a healthy mouse, and injecting an anesthetic into the abdominal cavity of the mouse;

(2) skin preparation and positioning: removing hair at the top of the mouse head, cutting the skin at the top of the mouse head, separating fascia bluntly, and exposing bregma; finding the body surface position corresponding to the optic nerve by adopting a skull stereotaxic apparatus, and respectively moving 0.5mm to the tail part and the right side by taking bregma as a starting point to be used as a needle inserting point;

(3) inserting needles: slowly inserting a 27G needle to the skull base when the insertion depth is 6mm, slowly pulling out the needle, pressing to stop bleeding and suturing skull top fascia and skin after pulling out the needle, and using the gatifloxacin eye ointment to prevent infection after operation.

The invention utilizes the skull stereotaxic apparatus to puncture after skull positioning so as to achieve the purpose of damaging the far-end optic nerve, thereby constructing the DONT model. Firstly, a skull stereotaxic apparatus is utilized to accurately find a body surface position corresponding to an optic nerve, so that a damaged area is relatively fixed; secondly, a 27G needle is adopted for puncture, and the damage to surrounding tissues is minimized on the premise of ensuring the optic nerve injury; in addition, the model only causes partial optic nerve damage instead of complete truncation, and is more close to clinic; finally, the molding method is simple and easy to implement, does not involve expensive material instruments and complicated molding steps, and provides a stable, reliable, simple and reproducible research model for the research of the far-end optic nerve injury. Through the examination of the molding effect, the survival rate of the mouse after the injury of the model can reach 85.56 percent, the molding success rate of the optic nerve injury reaches 95 percent, and particularly the molding success rate of the optic nerve injury at the far end can reach 90 percent.

Further, in step (1), BALA/C mice are selected for 4-6 weeks in males.

Further, the anesthetic selected in step (1) is xylazine at 10mg/kg and ketamine hydrochloride at 25 mg/kg.

Furthermore, the molding environment of the model is 22-24 ℃ at room temperature, and ventilation is good.

Further, nursing the mouse after the operation of the step (3), wherein the operation is to keep the temperature at 37 ℃ for 2h, and the mouse is returned to an animal room after reviving.

The invention has the following beneficial effects:

(1) the invention provides a method for establishing a mouse far-end optic nerve injury model by skull positioning puncture, which achieves the purpose of damaging the far-end optic nerve by puncture after skull positioning and can relatively fix an injury area;

(2) the method minimizes the damage to peripheral tissues on the premise of ensuring the optic nerve damage, only causes partial optic nerve damage but not complete truncation, and is closer to clinic;

(3) the molding method is simple and easy to implement, does not relate to expensive material instruments and complicated molding steps, and through the inspection of molding effect, the survival rate of the damaged mouse can reach 85.56%, the molding success rate of the optic nerve injury can reach 95%, the molding success rate of the optic nerve injury at the far end can reach 90%, and a stable, reliable, simple and reproducible research model is provided for the research of the optic nerve injury at the far end.

Drawings

FIG. 1 is a schematic diagram of the anatomy of optic nerve injury of a mouse ONT model, wherein an arrow points to the optic nerve injury site.

FIG. 2 is a micro-CT diagram of optic nerve injury of a mouse ONT model, and an arrow points to the optic nerve injury part.

FIG. 3 is a graph showing that the density of ocular RGCs gradually decreases with the time of optic nerve injury in a mouse ONT model; a: retinas of control and ONT 1-, 2-, 3-and 4-week groups were plated, corresponding to regions of retinal radii 1/6, 3/6, 5/6 in each quadrant, with Brn3a stained for labeled RGCs (magnification 200X, ruler length 50 μm); b: the density of RGCs in various sites of the retina of the model eye decreased significantly with increasing ONT time (n ═ 6; P <0.05 compared to control; P <0.01 compared to control; P <0.001 compared to control; P <0.0001 compared to control); ONT 1 weeks: 1week after optic nerve injury; ONT2 weeks: 2weeks after optic nerve injury; ONT 3 weeks: 3weeks after optic nerve injury; ONT4 weeks: 4weeks after optic nerve injury.

FIG. 4 shows the variation of F-VEP P1 wave latency and amplitude in mice with optic nerve injury; a: F-VEP detection waveforms of rats in groups of 1, 2, 3 and 4weeks after the control group mice and the optic nerve injury modeling, wherein each group of rats can record typical NPN waveforms of the F-VEP in the experimental process, the recorded waveforms are calibrated according to the rules of the International clinical Vision electrophysiology Commission, the first negative wave appearing at the beginning of the waveforms is N1 waves, the positive wave appearing immediately after the N1 waves is P1 waves, and the negative wave appearing after the P1 waves is N2 waves; b: the latency of the P1 wave gradually increased with increasing time to optic nerve injury, with statistical significance for the latency increase after 1week of modeling (n-12;. P <0.05 compared to control); c: the amplitude of the P1 wave gradually decreased with increasing time to optic nerve injury, and the difference was significant in each group after modeling compared to the control group (n 28;. P < 0.05;. P <0.01, compared to the control group,. P < 0.0001); ONT 1 weeks: 1week after optic nerve injury; ONT2 weeks: 2weeks after optic nerve injury; ONT 3 weeks: 3weeks after optic nerve injury; ONT4 weeks: 4weeks after optic nerve injury.

FIG. 5 is a graph showing changes in proliferation and activation of ocular microglia in a mouse ONT model; a: retinal plating of control and ONT groups at 1, 2, 3 and 4weeks, corresponding to areas of retinal radius 1/6, 3/6, 5/6 in each quadrant, with Iba-1 staining of labeled microglia (magnification 200X, ruler length 50 μm); b: with increasing ONT time, the microglial cell density in various regions of the retina of the model eye increased significantly, peaking at 3weeks after molding and then declining.

FIG. 6 is a graph showing the progressive increase and activation of ocular astrocytes with increasing optic nerve injury time in a mouse ONT model; a: retinas of control and ONT groups at 1, 2, 3 and 4weeks were plated and labeled astrocytes stained with GFAP (magnification 200 x, ruler length 50 μm) in each quadrant corresponding to the area of retinal radii 1/6, 3/6, 5/6; b: as ONT time increases, the density of astrocytes in various regions of the retina of the model eye increases, resulting in increased cell-cell contact and gradual reticulation.

FIG. 7 is a graph showing that murine ONT model eye Muller cells gradually increase and activate with increasing time to optic nerve injury; a: control mice and ONT mice in groups of 1week, 2weeks, 3weeks and 4weeks retina sections, GS + GFAP co-stained labeled Muller cells (magnification 200X, ruler length 50 μ M); b: as the ONT time increased, the GFAP expression levels of Muller cells gradually increased and GS gradually decreased in the retina of the model eye, indicating increased Muller cell activation.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Example 1

The method for constructing the mouse distal optic nerve injury model provided by the embodiment is as follows:

(1) molding environment: the room temperature is 22-24 ℃, and ventilation is good;

(2) anesthesia: male BALA/C mice were anesthetized with 10mg/kg xylazine and 25mg/kg ketamine hydrochloride intraperitoneally at 4-6 weeks;

(3) skin preparation and positioning: removing hair at the top of the head, cutting the skin at the top of the head, separating fascia at blunt nature, exposing bregma, finding out the body surface position corresponding to optic nerve by using a skull stereotaxic apparatus, and moving 0.5mm to the tail and the right respectively by taking the bregma as a starting point to serve as a needle feeding point;

(4) inserting needles: the 27G needle head is inserted slowly, and the needle insertion can feel the breakthrough feeling and observe the vibration of the right eyeball when the needle insertion reaches about 3mm, which is the reaction caused by the optic nerve injury caused by the needle insertion. The depth of the needle can reach the skull base when the depth is about 6mm, and then the needle is slowly pulled out. Pressing to stop bleeding and suture cranial fascia and skin after needle withdrawal, and using gatifloxacin eye ointment to prevent infection after operation; (construction method of control group: same position needle insertion is penetrated through skull and withdrawn, and other operations are consistent with the model group)

(5) And (3) postoperative care: the temperature is kept for 2h at 37 ℃, and the mice are returned to an animal room after reviving.

Experimental example 1

1. Modeling effect of constructing ONT model by skull puncture

1) Survival rate: of 180 mice molded by the ONT, 154 survived the observation period of 4weeks, with a survival rate of 85.56%.

2) Success rate: after 1week of molding, 20 mice were exposed to optic nerve isolation and observed under a microscope after fixation, in which 18 of the right distal optic nerve lesions were visible (fig. 1), and the molding success rate was estimated to be 90%. In addition, 20 mice observed optic nerve damage by micro CT, of which 19 were visible (FIG. 2), and the success rate of model creation was estimated to be 95%. By combining the two evaluation methods, the estimated molding success rate is about 92.5%.

2. Retinal ganglion cell survival observed by staining with Brn3a

At 1, 2, 3, 4weeks after ONT molding, staining of the retinal tile Brn3a showed that the density of RGCs in the retinal radius 1/6, 3/6, 5/6 region was significantly lower than the control group, and gradually decreased with increasing ONT molding time (fig. 3, table 1).

Table 1 density of RGCs in different groups of mice (mean ± standard deviation, n ═ 6)

3. Eye optic nerve function detection condition of mouse optic nerve injury model

F-VEP detection was performed on the model and control groups 1, 2, 3, 4weeks after the optic nerve injury model creation, and F-VEP was recorded with a LKC-UTAS-SBMF System (Multi-focal Visual Diagnostic Test System, USA) at a stimulus light intensity of 0dB (3.0cd · s · m · s%^-2) The stimulation frequency is 2.0Hz, the waveform time is 250ms, the passband is 1-100 Hz, and the superposition is carried out for 80 times. The peak value (ms) and amplitude (μ V) of the P1 wave were observed, measured 3 times per eye, and the average value was calculated. The results show that the latency of the P1 wave is gradually increased and the amplitude of the P1 wave is gradually reduced with the increase of the ocular hypertension time (figure 4), which indicates that the chronic ocular hypertension model can effectively cause the damage of the optic nerve function.

4. Proliferation and activation of microglia of mouse optic nerve injury model eye

At 1, 2, 3, 4weeks after ONT molding, retinal tile Iba-1 staining showed a significant increase in microglial cell density in the region of retinal radii 1/6, 3/6, 5/6 over the control group, and with increasing ONT molding time, the density reached a maximum at 3weeks after molding and then decreased (fig. 5).

5. Proliferation and activation of mouse optic nerve injury model eye astrocytes

GFAP staining of retinal footprints at 1, 2, 3, 4weeks post ONT modeling showed a marked increase in astrocyte density in the area of retinal radii 1/6, 3/6, 5/6 over the control, and increased cell-cell contact and interaction as modeling time increased, with gradual reticulation from individual cells (fig. 6).

6. Eye Muller cell activation of mouse optic nerve injury model

At 1, 2, 3, 4weeks after ONT molding, GS + GFAP staining of retinal sections showed a clear increase in activation of retinal muller cells compared to the control group, and activation gradually increased with increasing molding time (fig. 7).