Controlled Cortical Impact Model for Traumatic Brain Injury

Summary

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Abstract

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Introduction

Traumatic brain injury (TBI) is defined as an alteration in brain function, or other evidence of brain pathology, caused by an external force¹. TBIs remain a serious health problem throughout the world, particularly in the United States. According to the Centers for Disease Control and Prevention, at least 1.7 million TBIs occur annually in the United States resulting in 30.5% of all injury-related deaths. In 2000, the direct medical costs and indirect costs of TBIs totaled an estimated $76.5 billion in the United States alone. Although technological and therapeutic advancements in preceding decades have improved the quality and length of life for those suffering from TBIs, no effective pharmaceutical or preventative treatments currently exist. Due to the complexity and wide-reaching effects of TBIs, including tissue lesions, cell death, and axon degeneration, no two injuries are identical; thus, no current TBI model for animals accurately reproduces all aspects of TBI as seen in humans. However, animal models do provide the ability to produce nearly identical injuries necessary to investigate various effects of TBI with the hope of further understanding the clinical manifestations of TBIs.

The controlled cortical impact (CCI) model uses an impact system to deliver physical impact to the exposed dura of an animal. It induces TBIs ranging from mild to severe similar to those experienced by humans. This injury was first characterized in the ferret² and was later adapted for use in the rat^3,4, mouse^5-7, and sheep⁸. Since the first characterization, the site of injury has been placed both over the midline^2,9 and the lateral cortex¹⁰. CCI provides an easy and accurate method of investigating the effects and potential treatments for TBIs.

In addition to the CCI model, the fluid percussion and weight drop models are commonly used to produce TBIs. However, these models present limitations, including less control over injury parameters, producing histopathalogical changes not seen in human TBIs, and greater incidence of accidental death in mice^3,5,10. The blast wave model is also used to produce TBIs. Although the blast wave model does not reproduce the histopathalogical changes seen following a mechanical impact, this model does accurately produce TBIs experienced particularly by military personnel¹¹. The controlled cortical impact model is easy to control due to the precise control over deformation parameters such as time, velocity, and depth of impact⁵. Such accuracy makes replicating nearly identical injuries across an entire group of animals more feasible. Most importantly, CCI reproduces TBIs with features seen in human TBIs¹². However, there is no single animal model that is entirely successful in reproducing the complete spectrum of pathological changes observed after TBI. Further research is necessary to fully reveal the acute and chronic changes that occur after TBI.

Two types of injuries occur following a TBI: primary and secondary injuries. The primary injury occurs at the moment of impact and is not sensitive to therapeutic treatments; however, the secondary injuries that persist after the initial injury are subject to treatments¹³. The controlled cortical impact model produces the primary injury, thus allowing researchers to investigate the effects of TBI and potential therapeutic treatments for the potentially long-lasting effects of secondary injuries. Areas of potential research using the CCI model include neuronal death, cerebral edema, neurogenesis, vascular effects, histopathalogical changes, and memory deficits and more^3,13-16.

Protocol

Animal Care
Male C57 BL/6 mice were group-housed and kept in a 12/12 hr light/dark cycle with free to access to food and water ad libitum. The animals used in this protocol were 10-12 weeks old. All procedures were performed under protocols approved by Indiana University’s Animal Care and Use Committee.

1. Surgical Preparation

1. Anesthetize the mouse using a Ketamine/xylazine mixture (87.7 mg/ml Ketamine and 12.3 mg/ml Xylazine) and administer (1 ml/kg) via IP injection.
2. Shave the head of the mouse between the ears.
3. Apply a petroleum-based jelly to the eyes of the mouse to prevent drying out during surgery.
4. Clean the shaven area with 10% iodine. Then use 70% ethanol to clean off the iodine.
5. Fix the mouse head in the stereotactic frame using the ear bars and bite plate. Ensure the brain is stable.

2. Craniectomy

1. Make a longitudinal incision in the middle of the head with scissors. Use a hemostat to hold skin off to the left side.
2. Use a cotton-tipped applicator to remove the blood and tissue on the bone to expose the skull. Allow exposed skull to dry for 1 min.
3. Use forceps to apply pressure and ensure that the skull remains immobile. Identify anatomical landmarks Lambda (caudal aspect) and Bregma (frontal aspect). Draw a circle in the center of Lambda and Bregma with a 4 mm diameter and 0.5 mm away from the midline.
4. Use a drill to cut along the marked circle. Gently blow bone dust away. Do not drill completely through the bone to prevent damaging the dura mater.
5. Use forceps to remove bone and expose the dura mater.

3. Impaction

The impact system includes a control box to set impact parameters, an actuator to perform the impaction, and a stereotactic frame to secure the actuator and mouse head for impact.

1. Pre-set the velocity of the actuator to 3 m/sec before surgery.
2. Pre-set different deformation depth to induce different injury severities. Deformation depths of 0.0-0.2 mm, 0.5-1.0 mm, and 1.2-2.0 mm would result in mild, moderate, and severe TBIs, respectively. This protocol explained how to achieve a moderately severe brain injury with a deformation depth of 1 mm by using a velocity of 3 m/sec.
3. Attach the actuator to the holder in the stereotactic frame and use the micromanipulators moving it to secure the round, flat tip of the actuator (3 mm diameter) in the center of the open skull area. Then adjust the tip at an angle parallel to the surface of impact site.
4. Establish the zero point by moving down the actuator in the extending model until the tip touches the surface of the impact site. Then set the Z channel on the stereotactic control panel to zero.
5. Retract the impactor tip while simultaneously moving the actuator down 1 mm.
6. Hit the impact button to strike the injury site and achieve a deformation depth of 1 mm.

4. Injury Site Closure

1. Use cotton-tipped applicators to remove any blood following impact, but do not touch the injury area.
2. Place mouse on a warm pad to maintain body temperature.
3. Once bleeding has stopped, suture the wound closed. Put the animal back into the clean cage and allow it to recover from the surgery overnight on the warm pad.
4. Administer Buprenorphine 0.05-0.10 mg/kg SQ every 8-12 hr for 2 days after surgery.

Results

The controlled cortical impact model produces TBIs ranging in severity from mild to severe. Post-impact the amount of cranial swelling, bleeding, and cranial distortion at the impact site will reveal the injury severity resulting from the speed and deformation depth parameters. Mild TBIs result in cranial swelling at the impact site and slight bleeding due to the limited dura breach. A moderate TBI exhibits cranial swelling and increased bleeding due to dura breach upon impaction (Figure 1). The differen...

Discussion

The most critical steps for successfully generating consistent TBIs using an electronic magnet impact system to cause a CCI are: 1) stably fixing the mouse head in the stereotactic frame; 2) generating the same size of bone window between mice and removing the bone without damaging the dura under it during craniectomy; 3) correctly positioning the impact tip in the center of the open area and establishing the zero point before impacting.

A mouse head must be fixed in the stereotactic frame ver...

Materials

Name	Company	Catalog Number	Comments
Povidone-iodine 7.5%	Purdue product L.P.		Surgical scrub
Cotton tipped applicators	Henry Schein	100-6015	Remove blood and debris
Scissor	Fine Science Tools	14084-08	Surgery
Forcept	Fine Science Tools	11293-00	Surgery
Hemostat	Fine Science Tools	13021-12	Surgery
Rechargeable Cordless Micro Drill	Stoelting	58610	Combine with Burrs for generating the bone window
Burrs for Micro Drill	Fine Science Tools	19007-05
Suture monofilament	Ethicon	G697	Suture
tert-Amyl alcohol	Sigma	152463-250ML	Making 2.5% Avertin
2,2,2-Tribromoethanol	Sigma	T48402-25G	Making 2.5% Avertin