We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.
Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.
The most common spinal cord injury (SCI) occurring in humans is a contusive SCI1. To investigate the mechanisms of injury and the various therapeutic strategies following SCI, a precise, consistent, and reproducible contusive SCI model in rodents is needed.
Many spinal cord contusive injury models with various injury-producing mechanisms have been used in experimental SCI research2,3,4,5,6. Three contusive SCI models – specifically, the weight drop-based New York University (NYU)/ Multicenter Animal Spinal Cord Injury Studies (MASCIS) impactor3,6, the Ohio State University (OSU) impactor/electromagnetic SCI device (ESCID)5,7, and the Infinite Horizon (IH) impactor4,8 – are widely accepted in the SCI research field. The NYU/MASCIS impactor or an equivalent produces injury by dropping a fixed weight from different heights onto the target spinal cord to create multiple injury severities3,6. The OSU/ESCID causes injury by inducing tissue displacement5,7. The IH impactor produces injury by applying different forces to the spinal cord4,8. Each impactor uses a different velocity, which is an important parameter that influences the injury outcomes. The NYU/MASCIS apparatus generates velocities ranging from 0.33-0.9 m/s. The IH device has a maximum velocity of 0.13 m/s4. The OSU/ESCID impactor has a fixed velocity of 0.148 m/s5. Notably, the velocities of these models are lower than that observed in clinical velocities, which usually exceed 1.0 m/s9.
Here, we introduce a novel displacement-based contusive SCI device, called the Louisville Injury System Apparatus (LISA), to produce SCI in mice with a high impact velocity10. This system includes a vertebral stabilizer, which firmly stabilizes the vertebra at the site of injury, allowing the production of a constant, reproducible SCI. The laser sensor of the device ensures the precise determination of tissue displacement and the resultant severity of the SCI. The velocity of the plunger at the point of contact with the spinal cord can be adjusted from 0.5 to 2 m/s. These injury parameters closely replicate traumatic SCI seen clinically.
All surgical and animal handling procedures were performed as approved under the Guide for the Care and Use of Laboratory Animals (National Research Council) and the Guidelines of the Indiana University School of Medicine Institutional Animal Care and Use Committee.
1. Preparing the Animal and Performing the T10 Spinal Laminectomy
2. Performing the T10 Contusion Injury Using the LISA Impactor
This device consists of five main components: (1) a body with an impactor tip (Figure 1C), (2) a computer with software (Figure 1B), (3) an electrical control box (Figure 1B), (4) a vertebral stabilizer (Figure 2A), and (5) compressed air for the pneumatic control system (Figure 1A). To induce precise tissue displacement, the system relies on a laser sensor to measure the distance between the fully-extended plunger tip and the targeted spinal cord dorsal surface. The software takes the 4-mm thickness of the tip into account due to the fact that the laser beam only reaches the reflecting surface of the impactor (Figure 2B and Figure 3A-1). There are two positions at which the plunger tip may be placed: (1) in the path of the laser beam (Figure 3A-1) or (2) in a lateral position away from the laser beam (Figure 3B-1). When the plunger is in the laser beam path (Figure 3A-1), it measures the distance from the impactor tip and monitors the velocity of the impactor tip during motion between extension and retraction. When the plunger is in the lateral position away from the laser beam path (Figure 3B-1), the distance between the laser and the spinal cord is measured.
The stabilization of the T10 vertebra using our vertebral stabilizer is an integral component of the procedure (Figure 2A)10,11. Reliable distance measurements using the laser sensor depend on the stability of the target, which can be distorted if motion is present. To determine the accuracy and consistency of this system, 8 mice were subjected to 0.5-mm displacement injuries. These animals showed a displacement variability of ± 0.001 mm (± SD), indicating that the system is highly accurate and reproducible. Figure 4 demonstrates the immobilized target vertebrae in the stabilizer (Figure 4A) and the exposed T10 spinal cord prior to (Figure 4B) and after (Figure 4C) contusion under a surgical microscope.
The pressure of the compressed air controls the velocity of the impactor at the moment of injury. Our data demonstrate that the impact velocity is 0.81 ± 0.0345 m/s (mean ± SD) at a pressure of 138 kPa. The knob (Figure 1B) on the electrical box controls the duration of the tip-cord contact (dwell time) following the injury, and it can be adjusted between 0 and 5,000 ms. The tip-cord dwell time in most experiments is set at 0.32 ± 0.0147 s (mean ± SD) (Figure 5). Using this device, severity-dependent contusive injuries can be produced with tissue displacements of 0 mm (sham control), 0.2 mm (mild injury), 0.5 mm (moderate injury), and 0.8 mm (severe injury) in adult mice (Figure 6).
Figure 1: The Louisville Injury System Apparatus (LISA). (A) The system consists of an impactor, a control system, and a source of compressed air. (B) The control system includes a control box and a laptop computer. The software and control buttons of the control box allow the user to establish injury parameters. (C) The laser sensor is the key component of the device and measures the position of the injury target, the distance from the spinal cord to the sensor, and the injury velocity. The quick down-and-up movement of the impact tip is powered by compressed air. The location of the injury and the severity of the tissue displacement are adjusted by microdrivers, which control movement in three dimensions. Please click here to view a larger version of this figure.
Figure 2: The stabilizer and Mouse Holder. (A) The spine stabilizer consists of a U-shaped trough and two metal arms to hold the mouse vertebra. (B) The stabilizer is then mounted on the impactor device. The red line indicates the laser beam path. Please click here to view a larger version of this figure.
Figure 3: Method to Produce a Contusive SCI. (A–C) The Graphical User Interface (GUI) software with three injury parameters/zones are shown. (A, A-1) The green zone (SET ZERO LEVEL) calibrates the distance of the plunger tip. The red line indicates the laser beam path. (B, B-1) The blue zone is used to set the injury level (SET INJURY LEVEL). The impactor is lifted up and moved laterally to the right side to allow the laser beam to reach the dorsal surface of the spinal cord to set the zero level. The red line indicates the laser beam path. (C, C-1) Prior to impact, the tip is moved back onto the laser beam path to execute the injury (RUN). The injury parameters are under the red zone (RUN EXPERIMENT). The red line indicates the laser beam path. Please click here to view a larger version of this figure.
Figure 4: Injury Exposure and Assessment. (A) The metal arms of the spine stabilizer stabilize the T10 vertebra. (B) T10 laminectomy to expose the spinal cord, with the dorsal vessels clearly seen. (C) The impact-induced contusion (arrow) on the dorsal surface of the spinal cord confirms the injury. Scale bar = 2 mm. Please click here to view a larger version of this figure.
Figure 5: Injury Parameters. Consistent injury parameters include tissue displacement (mm), injury velocity (m/s), and tip dwell time (s). N = 8, Mean ± SD. Please click here to view a larger version of this figure.
Figure 6: Histologic Assessment. Representative cross sections of spinal cords, stained with Cresyl Violet and Eosin, show displacement-severity-dependent injuries following (A) sham (0 mm), (B) mild (0.2 mm), (C) moderate (0.5 mm), and (D) severe (0.8 mm) contusive SCIs at T10 using the LISA device. Images were taken at the injury epicenter. Scale bar = 500 µm. Please click here to view a larger version of this figure.
In 1911, Allen described the first weight drop model using a fixed weight to induce injuries on the exposed spinal cords of dogs12. Similar weight drop models have been developed based on the Allen model, including the NYU/MASCIS impactor3,6,13,14. In addition to the weight drop model, other SCI devices have been created. The OSU/ESCID5,7 model uses a tissue displacement mechanism to control injury severity, and the IH model4,8 uses force to create a gradable SCI. In these systems, vertebral stabilization is obtained by clamping the spinous processes rostral and caudal to the injury site. These devices utilize low injury velocities, specifically 0.33 – 0.9 m/s (NYU/MASCIS), 0.148 m/s (OSU/ESCID), and 0.13 m/s (IH). Stabilizing the rostral and caudal spinous processes may cause spine flexibility and spine movement during the impact, which may affect injury accuracy.
The LISA method attempts to overcome the shortcomings of existing models, particularly in regard to spine instability and low injury velocity. This method uses bilateral facet stabilization and avoids movement artifacts associated with the injury. This device utilizes a high-impact velocity that can be set between 0.5-2 m/s11,15. The laser sensor is more advanced than the Ling Vibrator used in the ESCID model and precisely measures the distance from the surface of the spinal cord without requiring any tissue contact. The model was originally developed to produce a rat SCI, and it has now been adapted to produce SCI on mice and on non-human primates16, with modifications.
Spine stabilization reduces variability in all experimental SCI methods, particularly in tissue displacement models. The laser distance sensor determines the magnitude of tissue displacement of the spinal cord during respiratory movements. It is important that the point of the spinal cord on which the laser is focused should be the identical point struck by the impactor. This step is accomplished during the calibration step (Figure 3), when the impactor tip and the laser beam are aligned. A potential weakness of this model is that the magnitude of tissue displacement is measured from the dural surface. Although the thickness of the dura constitutes a negligible difference between animals, significant variability may exist in the subarachnoid space filled with cerebrospinal fluid (CSF). Variability in injury outcomes may occur when producing a very mild contusion injury using a small tissue displacement. Overall, the consistency of the injury is mainly dependent upon the accuracy of tissue displacement and also upon the velocity and tissue contact time of the plunger.
The range of tissue displacement is wide (accuracy: 0-10 ± 0.005 mm). Based on previous pilot data and published information in rodents and non-human primates, a displacement of 20% of the anteroposterior diameter of the SC yields a mild SCI, a 30-40% displacement produces a moderate SCI, and a >50% displacement produces severe SCI at a 1 m/s velocity. There will be slight differences depending on the animal species. The dwell time is adjustable from 0 to 5 s using a time relay. In our study, the dwell time was set at 300 ms. This can be easily adjusted to replicate the dwell times of other SCI devices, including the NYU and IH models.
In summary, we have developed a displacement-based model of contusive SCI in adult mice. The model uses a U-shaped stabilizer to stabilize the bilateral spinal facets, avoiding the cord movement artifacts associated with the laser-guided measurement of the cord surface. This model can produce high-velocity cord injuries from 0.5-2 m/s. The laser sensor is more accurate than the conventional method to determine the velocity and the distance to the impact surface. The model can produce injuries of the spinal cord at all levels, from mild to severe. When modified, this device can also produce injuries in rats and large animals such as non-human primates.
The authors have nothing to disclose.
This work was supported in part by NIH NS059622, NS073636, DOD CDMRP W81XWH-12-1-0562; Merit Review Award I01 BX002356 from the U.S. Department of Veterans Affairs; Craig H Neilsen Foundation 296749; Indiana Spinal Cord and Brain Injury Research Foundation and Mari Hulman George Endowment Funds (XMX); Norton Healthcare, Louisville, KY (YPZ); the State of Indiana ISDH 13679 (XW); and the NeuroCures Foundation.
Ketamine (7.2mg/ml)/Xylazine (0.475mg/ml)/Acepromazine | Patterson Veterinary | 07-890-8598/07-869-7632/07-808-1947 | Anesthetic agent |
Buprenorphine(0.03mg/ml) | Patterson Veterinary | 07-891-9756 | Pain relief agent |
Carprofen | Patterson Veterinary | 07-844-7425 | antibiotic agent |
Purdue Products Betadine Surgerical Scrub | Fisher Scientific | 19-027132 | for sterilizing skin |
Dukal Gauze Sponges | Fisher Scientific | 22-415-490 | for sterilizing skin |
Decon Ethanol 200 Proof | Fisher Scientific | 04-355-450 | for sterilizing skin |
1ml NORM-JECT | HENKE SASS WOLF | D-78532 | for anethesia/pain relief/antibiotic agent injection |
10ml Syringe | TERUMO | REF SS-10L | for saline injection |
Artificial Tears Eye Ointment | Webster Veterinary | 07-870-5261 | provent eyes from dry |
Antiobiotic Ointment | Webster Veterinary | 07-877-0876 | provent surgery cut from infection |
Cotton Tipped Applicators | Fisher Scientific | 1006015 | stop bleeding |
Instrument Sterilizer | Fine Science Tools | 18000-50 | for sterilizing surgery tool |
Fine Forceps | Fine Science Tools | 11223-20 | grasp tissue |
Scalpel | Fine Science Tools | 10003-12 | skin cut |
Scalpel Blade #15 | Fisher Scientific | 10015-00 | skin cut |
Hemostat | Fine Science Tools | 13004-14 | stop bleeding |
Rongeur | Fine Science Tools | 16021-14 | laminectomy |
Agricola Retractor | Fine Science Tools | 17005-04 | keep the surgery view open |
Fine scissors | Fine Science Tools | 14040-10 | for muscle seperated from spine |
Sterile sutures | Fine Science Tools | 12051-10 | skin closure |
Mouse Vertebral stabilizer | Louisville Impactor System | N/A | Stabilize and expose the vertebra |
LISA | Louisville Impactor System | N/A | Produce an experimental contusion injury of the spinal cord in mice |