Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Podsumowanie

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

Streszczenie

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Wprowadzenie

Spinal cord injury usually results in permanent spinal cord dysfunction below the injured segment. It is mostly caused by objects striking the spine and hyperextension of the spine, such as traffic accidents and falls¹. Due to the limited availability of effective treatment options for spinal cord injury, elucidation of the pathogenesis of spinal cord injuries using animal models will be informative for the development of appropriate treatment approaches. The contusion model of spinal cord injury caused by impact on the spinal cord results in the development of animal models with similar pathologies to most clinical spinal cord injury cases^2,3. Therefore, it is important to produce accurate, reproducible, and convenient animal models for spinal cord injury contusion.

Since Allen's invention of the first animal model of spinal cord injury in 1911, there have been major advances in the development of instruments for establishing spinal cord injury animal models^4,5. Based on injury mechanisms, spinal cord injury models are classified as contusion, compression, distraction, dislocation, transection, or chemical⁶. Among them, the contusion models, which use external forces to displace and injure the spinal cord, are closest to the clinical etiology of most spinal cord injury patients. Therefore, the contusion model has been used by many investigators in spinal cord injury studies^3,7. Various instruments are used to develop spinal cord injury contusion models. The New York University (NYU)-multicenter animal spinal cord injury studies (MASCIS) impactor produces spinal cord injury contusions by weight-drop device⁸. After several updated versions, the MASCIS impactor is widely used to develop spinal cord injury contusion animal models⁹. However, when the impact rod of MASCIS falls and hits the spinal cord, multiple injuries may occur, which affects the degree of injury in spinal cord injury models. Moreover, achieving mechanical precision to ensure the accuracy of the instrument and the repeatability of the manufacturing model is also challenging. The infinite horizon impactors cause contusions by controlling the force applied to the spinal cord rather than heavy drops¹⁰. It uses a computer connected to a sensor to directly measure the impact force between the impactor and the spinal cord. When the threshold is reached, the impactor is immediately retracted, thereby avoiding weight rebound and improving accuracy^10,11. However, the use of this fine motor modality to inflict damage can result in inconsistent damage and functional deficits⁶. The Ohio State University (OSU) device compresses the dorsal surface of the spinal cord at a transient rate by an electromagnetic driver^12,13. This device is similar to the infinite horizon impactors, as it uses short-distance compressions to cause spinal cord injuries. However, it has various limitations in that initial determination of the zero point will cause errors due to the presence of the cerebrospinal fluid^6,14. In summary, there are many instruments that can be used to develop spinal cord injury contusion animal models, but they all have some limitations that lead to insufficient accuracy and reproducibility of animal models. Therefore, in order to more accurately, conveniently and reproducibly create mouse contusion models of spinal cord injury, an automated and intelligent spinal cord injury impactor is needed.

We present a novel spinal cord injury impactor, Guangzhou Jinan University smart spinal cord injury system (G smart SCI system; Figure 1), for producing spinal cord injury contusion models. The device uses a laser rangefinder as a positioning device, combined with an automated mobile platform to automate strikes according to set strike parameters, including strike speed, strike depth and dwell time. Automated operation reduces human factors and improves the accuracy as well as reproducibility of animal models.

Protokół

The studies involving animals were reviewed and approved by the Ethics Committee of Jinan University.

1. Anesthetization of animals and T10 spinal laminectomy

1. Use 8 weeks-old female young adult C57/6J mice for this study. Anesthetize the mice by intraperitoneal injection of ketamine (100 mg/kg) and diazepam (5 mg/kg). Check for successful anesthetization indicated by loss of pain reflex. Apply vet ointment on eyes to prevent dryness under anesthesia.
2. Shave the hair on the back of the mice using a shaver to reveal the skin. Disinfect the skin with three alternating rounds of iodophor and alcohol.
3. Make 2.5 cm medial longitudinal incision in the dorsal skin using a scalpel and expose the spine at the T9-T11 level using tweezers.
4. Bilaterally fix T10 facets using a spinal fixator. Ensure the spine is stably fixed. Ensure that the paravertebral muscles are stripped and remove the spinous process as well as laminae using micro-grinding drill to expose the spinal cord of the T10 segment.

2. Contusion of the T10 spinal cord using the G smart SCI system

1. Turn on the switch and wait for the device to automatically return to its original state. Place the spinal fixator into the G smart SCI system and secure it using screws.
2. Using the operation touch screen (Figure 2A), set damage parameters, including impact speed (1 m/s), impact depth (0.5 mm, 0.8 mm, and 1.1 mm for three different sets of mice) and dwell time (500 ms)¹⁵.
3. Align the laser rangefinder at the center of the exposed spinal cord by moving the platform. (Figure 2B)
4. Click the Ready button on the touch screen (Figure 2C). The impact head will automatically adjust to a specific height based on setting parameters. The carrier table automatically moves the spinal cord impact site below the impact head.
5. Manually press the impact head to further determine the impact site. Click the Start button, the impact head will hit the spinal cord based on set parameters.
6. Remove the mice from the device and observe under a Stereomicroscope (20x) to determine spinal cord injury (Figure 3). To determine the success of model development, observe local congestion, collapse, and spinal membrane rupture.
7. Suture the muscle, fascia and skin layer by layer using 3-0 sutures. Place the mice in a warm box and wait for their recovery.

3. Post-operative care

1. Inject meloxicam (5 mg/kg) subcutaneously daily for 7 days after surgery. Manually empty the bladder every 8 h until bladder functions are restored.
2. At 14 days after operation, remove the suture threads.

4. Testing effects of spinal injury

1. Calculate the BMS scores for mice from the first postoperative day^16,17.
2. On the 30^th postoperative day, perform animal behavioral experiments, including catwalk, foot fault and rotarod^16,17. Catwalk: Record distance of 45 cm; Maximum run duration 8 s; Camera gain 28.02; Intensity threshold 0.01. Foot fault: Record 60 steps for each mouse. Rotarod: Speed 20 rpm. Record the time for the mouse to fall and record it as 120 s for more than 120 s.
3. On the 31st postoperative day, anesthetize the mice by intraperitoneal injection of ketamine (100 mg/kg) and diazepam (5 mg/kg) and then euthanize the mice by perfusion using 4% PFA. Remove the spinal cord carefully, and intercept 5 mm above and below the injury site for paraffin embedding. Make a 5 µm section of the center of the mouse spinal cord injury and perform Hematoxylin and eosin staining¹⁷.
4. For statistical analysis use commercial software. Express data as mean ± standard error of the mean (SEM) and compare using one-way ANOVA; p < 0.05 was considered significant.

Wyniki

Laminectomy was performed on 24 female mice (8 weeks old) as described above. Mice in the sham group (n=6) were not subjected to spinal cord injury, while the rest of the mice, including 0.5 mm group (n=6), 0.8 mm group (n=6), and 1.1 mm group (n=6) were subjected to different depths of spinal cord impingement. The BMS scores were regularly recorded until 1 month postoperatively (Figure 4). There were significant differences in postoperative BMS scores of mice in different groups. After 1 mo...

Dyskusje

Spinal cord injury can lead to sensory and motor deficits, which can result in severe physical and mental impairments. In China, incidences of spinal cord injuries in different provinces vary from 14.6 to 60.6 per million¹⁸. The increase in the prevalence of SCI will put more pressure on the healthcare system. Currently, there are limited effective treatment option for spinal cord injury, injuries because its pathomechanisms and repair processes are yet to be fully understood¹⁹

Materiały

Name	Company	Catalog Number	Comments
0.01M PBS (powder, pH7.2-7.4)	Solarbio Life Sciences	P1010
2,2,2-Tribromoethanol	Macklin	75-80-9
4% paraformaldehyde tissue fixative	Biosharp life science	BL539A
Biomicroscope	Leica	LCC50 HD
CatWalk	Noldus Information Technology	CatWalk XT 9.1
Cover glass	CITOTEST Scientific	10212432C
Embedding machine	Changzhou Zhongwei Electronic Instrument	BMJ-A
Ethanol absolute	DAMAO	64-17-5
FootFaultScan	Clever Sys Inc.	-
Glass slide	CITOTEST Scientific	80302-2104
Hematoxylin and Eosin Staining Kit	Beyotime Biotechnology	C0105S
micro-grinding drill	FEIYUBIO	19-7010
Mouse spinal fixator	RWD Life Science	68094
Paraffin microtome	Thermo	shandon finesse 325
RotaRod for Mice	Ugo Basile	47600
Stereomicroscope	KUY NICE	SZM-7045
Tert-Amyl alcohol	Macklin	75-85-4
Xylene	China National Pharmaceutical	#10023418