A method for constructing an animal model of long-term exposure to -gx and +gz compound acceleration

Abstract

The application provides a method for constructing an animal model of long-term exposure to -Gx and +Gz compound acceleration, which repeatedly exposes animals to an acceleration environment of -Gx and +Gz in a centrifugal manner. The method of the application truly reproduces the multi-axial compound load characteristics borne by special occupational groups, better simulates the biomechanical characteristics of the compound mechanical environment, and induces intervertebral disc degeneration that is closer to clinical practice in terms of imaging and histopathology, thereby providing a reliable biological experimental carrier for the study of injury mechanisms, prevention and treatment intervention means of exposed groups.

Description

Technical Field

[0001] This invention relates to the fields of aerospace medicine, sports medicine, and biomedicine, and specifically to a method for constructing an animal model of long-term exposure to combined -Gx and +Gz acceleration. Background Technology

[0002] Among special occupational groups such as astronauts, pilots, and race car drivers, there is a high incidence of acute and chronic spinal injuries caused by repeated exposure to compound acceleration. Constructing an animal model of intervertebral disc degeneration that can accurately simulate the characteristics of this compound acceleration load is of great significance for revealing the relevant injury mechanisms and developing effective protective measures.

[0003] Currently, animal models used to study acceleration-induced intervertebral disc degeneration are primarily constructed through repeated exposure to acceleration in a single direction. These models can only reproduce acceleration in a single vector direction within a combined acceleration environment and cannot realistically simulate the mechanical state coupled with the dorsothoracic impact (-Gx) and the head-foot overload (+Gz). Therefore, animal models based on either -Gx or +Gz alone cannot accurately simulate the actual stress state of the intervertebral disc under this combined acceleration environment, leading to potentially significant discrepancies between research conclusions and actual conditions, and thus failing to better guide injury prevention and treatment. Summary of the Invention

[0004] This invention aims to overcome the aforementioned shortcomings. Under actual compound acceleration conditions, the intervertebral disc is simultaneously subjected to axial compression and anterior-posterior shear and bending stresses. The coupling effect of this multiaxial load significantly exacerbates the accumulation of damage and degeneration of the intervertebral disc, and its pathological characteristics, degree of degeneration, and evolution law are fundamentally different from those under arbitrary single-vector acceleration. This invention proposes a method to construct a rabbit intervertebral disc degeneration model by repeated exposure to compound acceleration (-Gx, +Gz).

[0005] The present invention provides a method for constructing an animal model of long-term exposure to combined -Gx and +Gz acceleration, characterized in that: the animal is repeatedly exposed to the acceleration environment of -Gx and +Gz by centrifugation.

[0006] The exposed parameters of this construction method are as follows:

[0007] -Gx value: -4Gx to -7Gx

[0008] +Gz value: +2Gz to +6Gz;

[0009] Duration per session:

[0010] -Gx direction: 10 s~60 s;

[0011] +Gz direction: 10 s~20 s.

[0012] Daily frequency: 3 to 10 times, with an interval of ≥5 minutes between two exposures;

[0013] Total cycle: 3 to 12 weeks, 5 to 7 days per week.

[0014] The present invention also provides the application of the animal model obtained by the above construction method in studying the pathogenesis of acute and chronic spinal injuries in special occupational groups repeatedly exposed to compound acceleration.

[0015] This invention also provides the application of the animal model obtained by the above construction method in the study of intervertebral disc degeneration induced by biomechanical characteristics in a complex mechanical environment.

[0016] The present invention also provides the application of the animal model obtained by the above construction method in the study of prevention and treatment strategies for acute and chronic spinal injuries in special occupational groups repeatedly exposed to compound acceleration.

[0017] This invention also provides the application of the animal model obtained by the above construction method in the study of prevention and treatment strategies for intervertebral disc degeneration induced by biomechanical characteristics in a complex mechanical environment.

[0018] The screening method for the above-mentioned prevention and treatment strategies is characterized by including the step of applying the candidate strategies to the animal model obtained by the construction method described above.

[0019] The function and effects of this invention:

[0020] The method of this invention involves repeated exposure of animals to a centrifuge platform capable of simultaneously generating -Gx and +Gz, realistically replicating the multiaxial composite load characteristics experienced by the aforementioned special occupational groups. Compared to single-direction acceleration models, the composite acceleration model constructed in this invention can better simulate the biomechanical characteristics of this composite mechanical environment. The induced intervertebral disc degeneration is closer to clinical reality in terms of imaging and histopathology, providing a reliable biological experimental platform for studying the injury mechanisms, prevention, and treatment interventions in exposed populations.

[0021] This invention pioneered long-term combined exposure to -Gx and +Gz, which successfully induced degenerative lesions in animals that more closely resembled the actual lesion characteristics of carrier-based aircraft pilots, achieving a more realistic pathological simulation.

[0022] This invention is based on the construction of a model using New Zealand white rabbits, whose anatomical structure, namely the tissue structure and composition of the intervertebral disc, is highly related to that of humans. It found that the biomechanical response under combined acceleration load is closer to that of the human body, and the degeneration results are more clinically valuable.

[0023] The experiments of this invention are highly reproducible. That is, by specifying the exposure parameters (amplitude, interval, period), the model is standardized, and different laboratories can stably produce homogeneous degradation models using this method. Attached Figure Description

[0024] Figure 1 Animal centrifuge;

[0025] Figure 2 Histological staining of rabbit intervertebral discs;

[0026] Figure 3 MicroCT images of experimental rabbits. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1. Model Construction

[0029] 1.1. Animal selection and grouping

[0030] Healthy adult male New Zealand white rabbits, weighing 2.5 kg to 3.5 kg, were selected.

[0031] The group was divided into a control group (n=8) and an experimental group (n=8).

[0032] All animals were housed in separate cages under the same conditions (they were placed in the animal house a week in advance to adapt to the housing environment and to the handling by the experimenters).

[0033] 1.2. Experimental Method (Centrifuge Exposure Method)

[0034] Centrifugation exposure environment via animal centrifuge (e.g.) Figure 1 (As shown) Centrifugation produces.

[0035] Each animal is fixed to the centrifugal rotating platform of the animal centrifuge using a 3D-printed rabbit box. In the initial position, the back faces the center and the chest faces outward. When the equipment rotates, it generates a stable centrifugal force. This posture allows the acceleration generated by the equipment to act precisely on the rabbit's body at -Gx (from back to chest).

[0036] Subsequently, when it is necessary to switch to the +Gz direction, the animal centrifuge tray is rotated 90° clockwise under remote control so that the acceleration generated by the equipment can be precisely applied to the rabbit's body at +Gz (from head to feet). That is, when the tray is rotated 90° clockwise, the rabbit's head faces the center of the centrifuge and its feet face outwards. At this time, the centrifugal acceleration generated by the centrifuge is from head to feet at +Gz.

[0037] Through the above operations, the state of intervertebral disc degeneration caused by the human spine being subjected to both back-thoracic impact (-Gx) and head-foot overload (+Gz) in an aerospace environment was realistically reproduced.

[0038] Exposure parameter range:

[0039] -Gx direction: -4Gx to -7Gx;

[0040] +Gz direction: +2Gz~+6Gz;

[0041] Duration per session:

[0042] -Gx direction: 10 s~60 s;

[0043] +Gz direction: 10 s~20 s.

[0044] Daily frequency: 3 to 10 times (interval between two exposures ≥ 5 minutes)

[0045] Total duration: 3 to 12 weeks (5 to 7 days per week)

[0046] In a preferred case: the rabbits in the experimental group were exposed to the target G value of -6Gx for 45 s, +4Gz for 15 s, with a G value growth rate of 1 G / s, at 10 min intervals, 6 times a day, for 28 days.

[0047] In our comparative studies, we found that spinal injury is determined by the G-force amplitude, duration of a single application, daily loading frequency, interval, and total exposure period. Parameter deviations alter the cumulative mechanical load, causing gradient changes in the degree and type of injury (acute / chronic, reversible / organic).

[0048] For example: when -G X Below -4G X At this time, due to reduced shear stress, acute injury is rare; long-term exposure leads to slow progression of intervertebral disc degeneration, prolonged modeling time, and modeling failure, among other adverse outcomes. However, when -G X Above -7G X At this time, the transverse shear load increases significantly, making it easier for intervertebral disc annulus fibrosus traction injury, vertebral endplate micro-injury, and facet joint stress abnormalities to occur, increasing the risk of acute injury such as paralysis and death. For example, in one experimental case, -9GX A single exposure can cause acute damage to the intervertebral disc.

[0049] When +Gz is below +2Gz, the axial compressive load is small, the spinal mechanical stress is mild, and the probability of organic damage is low. However, above +6Gz, the vertebral body and intervertebral discs are subjected to high-intensity compression, and -G... X When shear loads are superimposed, the combined stress of compression and shear can significantly amplify the risk of acute injury. In one experimental case, at +7Gz, rabbits showed obvious intolerance and the mortality rate increased significantly.

[0050] If a single exposure time is less than 10 seconds, the shearing action time is short, and it is mostly a transient stress, rarely resulting in organic spinal cord injury. However, prolonged modeling time carries the risk of modeling failure. When it exceeds 60 seconds, the prolonged shearing action time carries the risk of aggravating acute injury.

[0051] Example 2. Experimental Results

[0052] 2.1 Data Collection and Sampling (using parameters from the preferred case)

[0053] After the acceleration exposure, the animals were anesthetized with 5% isoflurane and euthanized, and cervical intervertebral disc tissue was removed. Seven experimental rabbits were sent for histopathological and imaging examinations.

[0054] 2.2. Indicator Testing

[0055] 2.2.1 Histological assessment: HE staining was used to observe changes in the overall structure of the intervertebral disc, including the degree of porosity of the nucleus pulposus and the integrity of the annulus fibrosus laminae.

[0056] like Figure 2 As shown in the figure, HE staining revealed that the intervertebral disc in the control group (Figure A) had an intact structure, abundant and well-formed cells in the nucleus pulposus, and a dense, lamellar, regularly arranged annulus fibrosus with a clear boundary from the nucleus pulposus. The intervertebral disc in the experimental group (Figure B) showed mild degenerative changes, characterized by a reduced number of nucleus pulposus cells and lighter staining of the extracellular matrix; micro-cracks appeared in the inner layer of the annulus fibrosus, with a slightly disordered arrangement.

[0057] 2.2.2 Imaging assessment: CT scans were used to observe morphological changes in isolated cervical intervertebral discs, focusing on analyzing indicators such as disc height changes and T2 signal intensity changes to reflect water content and degree of degeneration.

[0058] 2.2.2.1. Experimental Methods:

[0059] Specimen type: Rabbit cervical vertebrae

[0060] Sample size: 7 samples

[0061] Machine Model: Venus® Micro CT (VNC 200)

[0062] Testing Unit: Shanghai Nuozhen Biotechnology Co., Ltd.

[0063] Objective: To conduct radiographic observation of microCT reconstructed images of cervical spine specimens from 7 rabbits, focusing on assessing bony indirect signs related to intervertebral disc degeneration, including changes in intervertebral disc height, endplate sclerosis and irregularity, osteophyte-like hyperplasia at the vertebral body margins, vertebral body alignment, and integrity of bone structure.

[0064] 2.2.2.2. Experimental Results

[0065] like Figure 3 MicroCT images of rabbit A showed uneven intervertebral disc height across multiple cervical segments, with mild to moderate narrowing of some discs. Increased density and mild marginal irregularities were observed near the endplates, and mild osteophyte-like hyperplasia was present at the vertebral body margins. Overall vertebral alignment was acceptable, with no obvious fractures or extensive bone destruction. Imaging assessment: Moderate degeneration tendency, approximately grade II-III.

[0066] like Figure 3 MicroCT images of rabbit B showed multi-segmental intervertebral space narrowing, with significant decrease in intervertebral space height in some segments. Localized sclerosis and irregular margins of the vertebral endplates suggested chronic degenerative changes. Three-dimensional reconstruction showed mild to moderate osteophyte formation at the vertebral body margins. No clear signs of acute fracture were observed. Imaging assessment: Moderate degenerative tendency, approximately grade II.

[0067] like Figure 3 MicroCT images of rabbit C showed significant narrowing of the intervertebral space and prominent unevenness in intervertebral space height. Adjacent endplate sclerosis and irregular changes were evident, and some vertebral body margins showed significant osteophyte-like hyperplasia or degenerative bone remodeling. Overall, this sample indicated a high degenerative load. Imaging assessment: Moderate to severe degenerative tendency, approximately grade III.

[0068] like Figure 3 MicroCT images of rabbit D showed mild to moderate intervertebral space narrowing in multiple segments, mild irregularity of the endplate margins, and relatively mild endplate sclerosis. Three-dimensional reconstruction showed no obvious osteophyte formation, and the vertebral bodies were generally continuous, with no obvious collapse or bone destruction. Imaging assessment: Mild to moderate degeneration, approximately grade II.

[0069] like Figure 3 In rabbit E, microCT images showed significant intervertebral space narrowing in multiple segments of the cervical spine, with endplate sclerosis and roughened margins in some segments. Three-dimensional reconstruction revealed osteophyte formation and degenerative remodeling at the vertebral body margins, with overall degenerative changes more pronounced than in rabbit 4. Imaging assessment: Moderate to severe degenerative tendency, approximately grade II-III.

[0070] like Figure 3 MicroCT images of rabbit F showed decreased or uneven intervertebral disc height across multiple segments, extensive endplate sclerosis, and osteophyte-like hyperplasia at local vertebral body margins. Overall bone structure continuity was acceptable, with no definite destructive bone defects or acute fractures observed. Imaging assessment: Moderate degenerative tendency, approximately grade II-III.

[0071] like Figure 3 MicroCT images of rabbit G showed uneven intervertebral space height across multiple segments, with significant narrowing in some spaces. Endplate sclerosis, irregularity, and vertebral body margin hyperplasia were prominent, and local measurements indicated significant differences between the vertebral bodies and intervertebral spaces. Overall, degeneration was evident. Imaging assessment: Moderate to severe degeneration, approximately grade III.

[0072] Grade I: Mild intervertebral space narrowing, with no obvious changes in the endplate or only slight sclerosis. Grade II: Clear intervertebral space narrowing, accompanied by endplate sclerosis, irregular margins, or mild to moderate osteophyte formation.

[0073] Grade III: Significant intervertebral space narrowing in multiple segments or focal areas, accompanied by significant endplate degeneration and remodeling, osteophyte-like hyperplasia, or bony changes at the vertebral body margins.

[0074] In summary, microCT images of the cervical spine in seven rabbits revealed varying degrees of intervertebral disc-related bony changes, primarily manifested as decreased or uneven intervertebral disc height, endplate sclerosis and irregularity, and osteophyte-like hyperplasia at the vertebral body margins. The overall degree of degeneration was moderate, with some samples showing moderate to severe changes.

Claims

1. A method for constructing an animal model of long-term exposure to combined -Gx and +Gz acceleration, characterized in that: The animals were repeatedly exposed to accelerations of -Gx and +Gz by centrifugation.

2. The construction method as described in claim 1, characterized in that: The exposure parameters are as follows: -Gx direction: -4Gx to -7Gx; +Gz direction: +2Gz~+6Gz; Duration per session: -Gx direction: 10s~60s; +Gz direction: 10s~20s; Daily frequency: 3 to 10 times, with an interval of ≥5 minutes between two exposures; Total cycle: 3 to 12 weeks, 5 to 7 days per week.

3. The application of the method described in claim 1 in the field of animal model construction.

4. The application of the animal model obtained by the method described in claim 1 in studying the pathogenesis of acute and chronic spinal injuries in special occupational groups repeatedly exposed to compound acceleration.

5. The application of the animal model obtained by the method described in claim 1 in the study of intervertebral disc degeneration induced by biomechanical characteristics in a complex mechanical environment.

6. The application of the animal model obtained by the method described in claim 1 in the study of prevention and treatment strategies for acute and chronic spinal injuries in special occupational groups repeatedly exposed to compound acceleration.

7. The animal model obtained by the method described in claim 1 can be used in the study of prevention and treatment strategies for intervertebral disc degeneration induced by biomechanical characteristics in a complex mechanical environment.

8. The method for screening prevention and treatment strategies as described in claim 6 or 7, characterized in that, This includes the step of applying a test candidate strategy to an animal model obtained by the establishment method as described in claim 1.

Images