Controlled Cortical Impact for Traumatic Brain Injury

Introduction

Traumatic brain injury (TBI) is referred to as alteration or dysfunctionality in brain function, or brain pathology resulting from external trauma or force. TBI is a serious health condition because of its complexity and wide-reaching effects, including lesions, necrosis, and axon degeneration. Research on traumatic brain injury has been challenging because no two injuries are identical, and it has been difficult to reproduce all the aspects of TBI in a single animal model. The controlled cortical impact (CCI) is a neurotrauma model that uses an impact system to produce graded, reproducible injury to the exposed dura of the test subject to mimic physiological, histological, and behavioral aspects of closed-head traumatic brain injury. It induces mild to severe TBIs similar to those experienced by humans. CCI employs an electronic pneumatic piston to deliver a precise contusion injury to the exposed dura. It provides an easy and accurate model to investigate the effects and potential treatments for TBIs.

Two types of injuries occur as a result of traumatic brain injury (TBI): primary and secondary injuries. The primary injury is developed at the moment of impact and is not subject to therapeutics. Whereas, the secondary injuries persist after the initial injury and are sensitive to therapeutics. The CCI model produces the primary injury and allows the researchers to investigate the effects of TBI and therapeutic agents for the potentially long-lasting effects of secondary injuries. CCI model is useful for research in several areas, including neuronal death, neurogenesis, cerebral edema, vascular effects, tissue changes, memory deficits, and more (Dixon et al., 1991).

History

The controlled cortical impact model for traumatic brain injury is relatively new. It was initially developed by J.W. Lighthall and colleagues in the late 1980s and early 1990s to deliver traumatic brain injury in ferrets. The control over important injury parameters and the ability to induce reproducible injuries have made the model valuable for use in rats during the early 1990s. Since then, CCI has been further developed for use in several other species, including pigs, mice, and non-human primates (Osier. & Dixon., 2016).

The scalability and reproducibility of the results have made the CCI one of the most popular and widely used preclinical TBI models as it provides quantitative control over essential physiological and biomechanical parameters of TBI including the impact extent, velocity, depth, and force of the tip. Taken together, the customization of the CCI model allows the researchers to address a multitude of questions as well as to scale the injury for the investigation of histopathological and functional brain deficits.

Apparatus

CCI model employs an electronically controlled pneumatic impactor to deliver a precise, focal contusion injury to the exposed brain surface of the test animal. The CCI injury device uses a small-bore, double-acting steel pneumatic cylinder for impact delivery. It is vertically mounted on a crossbar perpendicular to the brain surface (however the animal could be adjusted according to the experimental angle in the stereotactic device). A removable impactor tip (3-5 mm) is attached to the lower end of the rod, and the upper end is attached to a sensor system that adjusts and detects the impactor velocity. The impactor compresses the brain tissue at a user-selected speed, dwell time (i.e., the duration for which the cortical tissue remains depressed), and the depth of the injury (Osier. & Dixon., 2017).

Protocols

1. Anesthetize the animal and carefully place it on the stereotaxic frame.
2. Trim the fur from the head of the animal if needed.
3. Inject local anesthesia under the scalp and make a midline incision using a scalpel or scissors. Pull out the skin and expose the skull bone while keeping the scalp retracted with the help of hemostatic forceps.
4. Using Dumont forceps, clear the skull bone from the periosteum, and retract the muscle lateral to the lateral ridges to create sufficient space for the craniotomy.
5. Position the craniotomy using the midline and bregma sutures on the skull bone.
6. Perform the craniotomy using a drill or trephine without causing a rift to the dura mater.
7. Remove the bone piece and place it in a sterile, isotonic saline solution if it is to be used later.
8. Place the stereotaxic frame on the CCI device.
9. Retract the piston after identifying the null position. Lower the piston to the required distance.
10. Perform the controlled cortical impact.
11. Move the stereotaxic frame to the operating table.
12. Replace the bone piece and apply tissue adhesive to fix it in its former place.
13. Suture the scalp and return the animal to the recovery cage.

Alternate protocol

Surgical Preparation (J. Romine., X. Gao., & Chen., 2014)

1. Anesthetize the test animal with 1mg/kg intraperitoneal injection of Ketamine/xylazine mixture (87.7 mg/ml Ketamine and 12.3 mg/ml Xylazine).
2. Shave the mouse head between the ears.
3. Apply a petroleum-based jelly on the eyes of the mouse to prevent drying.
4. Wipe the shaven area with 10% iodine. Then clean it with 70% ethanol.
5. Fix the mouse head in the stereotaxic frame using the ear bars and bite plate.

Craniectomy

1. Create a longitudinal incision in the middle of the head using scissors. Hold the skin to the left side with the help of a hemostat.
2. Expose the skull by removing the blood and tissue on the bone using a cotton-tipped applicator. Allow the exposed skull to dry for 1 minute.
3. Stabilize the skull using the forceps.
4. Identify the anatomical landmarks Lambda (caudal) and Bregma (frontal).
5. Draw a circle in the center of the anatomical landmarks with a 4 mm diameter and 0.5 mm away from the midline.
6. Cut along the marked circle with the help of a drill.

Note: Do not drill completely through the bone to preserve the dura mater.

7. Remove the bone and expose the dura matter using forceps.

Impaction

1. Set the velocity of the impactor to 3 m/sec before surgery.
2. Adjust the deformation depth to induce the required injury severity. Deformation depths of 0.0-0.2 mm, 0.5-1.0 mm, and 1.2-2.0 mm result in mild, moderate, and severe traumatic brain injuries, respectively.
3. Attach the impactor to the holder in the stereotactic frame and position the impactor tip on the skull parallel to the impact site.
4. Move the actuator to establish the zero point until the tip touches the impact site.
5. Set the Z channel on the control panel to zero.
6. Retract the tip by moving the actuator down to 1 mm.
7. Hit the impact button to deliver the injury and achieve a deformation depth of 1 mm.

Injury Site Closure

1. Remove the blood using a cotton-tipped applicator, but do not touch the impact area.
2. Place the test subject on a warm pad to maintain the body temperature.
3. Once the bleeding has stopped, suture the wound.
4. Put the animal into the clean cage and allow it to recover overnight on the warm pad.
5. Administer 0.05-0.10 mg/kg SQ Buprenorphine every 8-12 hours for 2 days following surgery.

Applications

A well-suited model for concussion research (Osier. & Dixon., 2017)

Mild traumatic brain injury (mTBI) is a severe health problem that demands additional research. Controlled cortical impact (CCI) is the most commonly used and well-characterized model of traumatic brain injury (TBI) that has been utilized in research for three decades. CCI could efficiently be used on several common laboratory animals, including mice, rats, ferrets, and pigs. Also, the CCI model could be used to produce graded injuries ranging from mild to severe. CCI has been applied to study open and closed head mTBI, repeated injuries, and the long-term deficits following mTBI and concussion.

Assessment of excitotoxicity induced by traumatic brain injury (Palmer et al., 1993)

A controlled cortical impact model has been used to measure the interstitial concentrations of aspartate and glutamate (together with serine and glutamine) in the rat frontal cortex following traumatic brain injury. Histological analysis indicated that the severe TBI delivery results in almost twice the injury depth as compared to that following mild traumatic brain injury. In the experimental groups, a maximal increase in excitatory amino acid (EAA) concentration proportional to the severity of the injury was observed. Although these increases were normalized within 20-30 minutes following the moderate TBI, concentrations of aspartate and glutamate took >60 minutes to normalize in case of severe TBI. Changes in levels of non-transmitter amino acids were minimal.

In conclusion, the study presented that the elevations in [EAA] following TBI, are sufficient to kill neurons. This, along with the cytoprotective usefulness of EAA receptor antagonists and the similarity between the TBI-induced injury and the injury caused by EAA receptor agonists, implies that it is the TBI-induced increases in [EAA], which result in neuronal death. The controlled cortical impact (CCI) model has provided a powerful study model for the investigation of excitotoxicity induced by traumatic brain injury.

Mimicking TBI pathophysiology (Osier. & Dixon., 2016)

The CCI model is clinically relevant because of its ability to reproduce many of the histopathological changes following traumatic brain injury. These histological changes include cortical contusion, hippocampal cell loss, disruption of the blood-brain-barrier, and overall brain volume loss. Also, clinical injury is characterized by several secondary injury cascades, including apoptosis, inflammation, and oxidative stress that could be efficiently modeled using the CCI. The controlled cortical impact model is known to mimic chronic ventricular enlargement, necrosis, apoptosis, axonal injury, and inflammation. The pathophysiological conditions presented by the CCI have made the research and investigation of traumatic brain injury easier.

Testing therapies (Osier. & Dixon., 2016)

CCI represents an essential and useful model to identify promising therapies in preclinical studies and translate them to clinical trials and ultimately, clinical practice to cure traumatic brain injury. CCI studies, in combination with other models, have made it possible to characterize several therapies for clinical trials, though the success of the tests has been limited. For example, both preclinical studies and clinical trials of amantadine have shown promising neurobehavioral recovery after TBI. Also, several novel therapies (e.g., hypothermia; progesterone; cyclosporine) have been promoted to successful phase II trials using the CCI model.

Strengths and Limitations

• Controlled cortical impact provides a high degree of control over the impact depth, velocity, dwell time, and the volumetric characteristics associated with the impact tip sizes.
• CCI model is popular because of its scalability and uses in multiple species of test animals: ferrets, rats, mice, swine, and non-human primates.
• The controlled cortical impact model is considered a clinically relevant preclinical TBI model as it can reproduce almost all the aspects of clinical TBI, including the pathophysiological and functional consequences of head injury.
• Animals undergoing CCI do present important aspects of TBI pathophysiology and functional deficits, but they cannot completely replicate the symptom profiles experienced by humans.
• The controlled cortical impact model has some limitations, such as the production of focal contusion-type injury.

References

1. E. C. Dixon., L. G. Clifton., W. J. Lighthall., A. A. Yaghmai., & Hayes., L. R. (1991). A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Methods, 39(3), 253-262.
2. J. Romine., X. Gao., & Chen., J. (2014). Controlled cortical impact model for traumatic brain injury. J Vis Exp, 90.
3. M. A. Palmer., W. D. Marion., L. M Botscheller., E. P. Swedlow., D. S. Styren., & DeKosky., T. S. (1993). Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model. J Neurochem, 61(6), 2015-24.
4. N. D. Osier., & Dixon., C. E. (2016). The Controlled Cortical Impact Model: Applications, Considerations for Researchers, and Future Directions. Front Neurol, 7(134).
5. N. Osier., & Dixon., C. E. (2016). The Controlled Cortical Impact Model of Experimental Brain Trauma: Overview, Research Applications, and Protocol. Methods Mol Biol, 1462, 177-192.
6. N. Osier., & Dixon., E. C. (2017). Mini Review of Controlled Cortical Impact: A Well-Suited Device for Concussion Research. Brain Sci, 7(7).