Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the pathophysiological mechanism and to develop effective therapeutic strategy for this devastating neurological disorder. Intraparenchymal hemorrhage and post-traumatic inflammatory response induced by initial physical impact can aggravate microglia/macrophage activation and neuroinflammation which subsequently worsen brain pathology. We provide here a controlled cortical impact (CCI) protocol that can reproduce experimental cortical contusion in mice by using a pneumatic impactor system to deliver mechanical force with controllable magnitude and velocity onto the dural surface. This preclinical model allows researchers to induce moderately severe focal cerebral contusion in mice and to investigate a wide range of post-traumatic pathological progressions including hemorrhage contusion, microglia/macrophage activation, iron toxicity, axonal injury, as well as short-term and long-term neurobehavioral deficits. The present protocol can be useful for exploring the long-term effects of and potential interventions for cerebral contusion.
Cerebral contusion is a form of traumatic brain injury that ranks high among the deadliest health issues in modern society1. It is primarily caused by accidental events such as traffic accident that results in external forces applying mechanical energy to the head. Traumatic brain injury affects an approximate of 3.5 million people and accounts for 30% of all acute injury-related deaths in the US each year2. Patients who survive cerebral contusion oftentimes suffer from long-term consequences including focal motor weakness, sensory dysfunction, and mental illness1.
The primary injury of cerebral contusion is induced by mechanical factors including stretching and tearing forces, leading to immediate parenchymal structure deformation and focal CNS cell death3. Hemorrhage contusion is a general term for brain hemorrhages due to vascular tear at the site of head trauma4. Specifically, intraparenchymal hemorrhage occurs immediately after a cerebral contusion leading to delayed hematoma formation. Within the hematoma, hemoglobin and free iron released from the lysed red blood cells can further trigger blood-related toxicity5,6 which cause herniation, brain edema, and intracranial pressure elevation5,6. The collaborative functions of neurons (axons), glia, blood vessels, and supportive tissue are also compromised by the mass effect of hematoma7. Additionally, persistent and diffuse neuroinflammation with progressive neurodegeneration continue for months and cause secondary damage in the brain8.
Microglia activation is one of many important pathological features of cerebral contusion9,10. After sensing the damage-associated molecular patterns (DAMPs) and leaked blood in the injured tissue, activated microglia trigger neuroinflammation which furthers secondary brain damage11. In addition, chemoattractant released from microglia promotes peripheral immune cell infiltration into the traumatic territory resulting in production of reactive oxygen species and pro-inflammatory cytokines. This creates a self-perpetuating pro-inflammatory environment which triggers progressive brain injury9,12. Meanwhile, microglia with an alternatively activated phenotype can contribute to tissue homeostatic restoration and brain repair through clearing debris from the injured tissue13. Prevention of secondary neuroinflammation by reducing detrimental microglial immune responses has been shown to be particularly useful for promoting brain recovery from cerebral contusion3,9,10,12.
Several preclinical models have been developed for studying traumatic brain injury including weight-drop model, lateral fluid percussion injury, and blast wave model14,15. However, these models each have their weakness including high mortality rate during the procedure, low reproducibility of histological results, and high variability of inflicted injury between laboratories16,17. In comparison, the controlled cortical impact (CCI) model is more adequate for studying focal cerebral contusion because of its precise control and high reproducibility14,15,18,19.
Furthermore, through manipulating the biomechanical deformation parameters such as velocity and depth of impact, the severity of the induced damage can be controlled to produce a wide range of injury magnitudes, allowing researchers to mimic different levels of impairment oftentimes seen in patients17. The preclinical model of CCI was first developed in 189620. Since then, CCI has been the broadest applicable model to be modified for the use in primates21, swine22, sheep23, rats24, and mice25. Together these features make CCI one of the most suitable experimental cerebral contusion models26.
Our laboratory uses a commercially available pneumatic CCI impact system and tested biomechanical deformation parameters to produce moderately severe focal cerebral contusion that territorializes the primary sensory and motor cortical areas without damaging the hippocampus27,28. We and others demonstrated that this CCI procedure can be used to study clinical features of human cerebral contusion including brain tissue loss, neuronal injury, intraparenchymal hemorrhage, neuroinflammation, and sensorimotor deficiency24,25,27,28,29,30. Here, we detail a standard protocol to perform mouse CCI which allows one to ask questions regarding CCI-induced myelin loss, iron deposition, CNS inflammation, hemorrhagic toxicity and the responses of microglia/macrophages in the aftermath of focal cerebral contusion.
All procedures described in this protocol were conducted under the approval of the Institutional Animal Care and Use Committee at Cheng Hsin General Hospital and National Taiwan University College of Medicine. Eight- to ten-week-old male C57BL/6 wild type mice were used in this protocol.
1. Anesthesia induction
- Anesthetize the mouse with ~4% isoflurane mixed with room air at ~0.2 L/min in an induction chamber connected to the isoflurane vaporizer.
- Ensure the respiratory pattern is smooth. Check the depth of anesthesia by confirming a lack of toe-pinch reflex in the animal.
2. Pre-surgical preparation
- Shave the mouse head with electrical clippers in a caudal to rostral direction. Do not trim the mouse whiskers.
NOTE: Loss of whiskers may influence the accuracy of subsequent behavioral test results.
- Place the mouse onto the stereotaxic frame. Carefully insert the ear bars into the ear canals. Ensure the mouse head is stabilized by both ear bars equally.
- Bring in the nose cone and maintain anesthesia at 1% - 2% isoflurane for the duration of the surgery.
- Apply petroleum jelly to both eyes to prevent drying out during the surgery. Keep the animal on a heating pad to maintain a body temperature of 37 ˚C.
- Disinfect the shaved head with betadine followed by 70% alcohol using sterile cotton swabs. Repeat three times.
3. CCI surgery
- Administer 100 µL of Bupivacaine (0.25%) subcutaneously using a 31 G insulin needle prior to the incision. Gently massage the injection site for better absorption.
NOTE: This local anesthetic provides pain relief directly at the site of surgery.
- Make a longitudinal incision (~1.5 cm) along the midline on the scalp with a scalpel or scissors. Use a hemostat to hold the skin off to the right side and allow the exposed skull to dry for 1 min. Use a sterile cotton swab to clean away any residual blood and tissues on the skull.
- Check that the mouse head is level in the horizontal plane.
- Identify anatomical landmarks Bregma and Lambda and mark both locations with a pencil.
- Ensure that the head of the animal is level in the rostral-caudal direction. Do this by measuring the Z coordinates of both Bregma and Lambda using a 31 G insulin needle attached to the stereotaxic frame.
NOTE: Adjust the ear bar vertically if necessary.
- Perform for the horizontal positioning of the animal head by following the same procedure of checking the Z coordinates at the midline along with two corresponding locations on the left and right side of the midline and adjust the ear bars if needed.
NOTE: A level and stable placement of the animal head is crucial for the reproducibility and reliability of the CCI model.
- Use the same 31 G insulin needle to identify the craniectomy site. Set the XY origin to Bregma and laterally move the needle 3 mm to the right. Mark this position as the site of craniectomy and draw a circle 4 mm in diameter on the skull with a pencil.
- Use a high-speed micro drill with a trephine (4 mm diameter) to cut along the pencil-outlined circle to create a 4 mm diameter open hole. Use a speed setting of 20,000 rpm. Avoid applying excess pressure.
NOTE: Perform this step quickly (usually within 30 s to 1 min) to prevent any thermal damage to the brain. Applying excess pressure while drilling may lead to accidental penetration that could compress and injure the brain surface.
- Carefully remove the bone flap with tweezer and temporally store it in ice cold normal saline. Gently rinse the hole with normal saline before applying pressure on the brain surface with the cotton swab tip to stop bleeding.
- Set the 2.5 mm diameter rounded impactor tip on the CCI device to an angle of 22.5˚. Zero the impact tip to the dural surface. Set the impact parameters on the control box to a velocity of 4 m/s and a deformation depth of 2 mm. Retract the metal tip.
NOTE: Zeroing the tip while it is statically and slightly pressed against the dural surface in the full stroke position improves the accuracy of the zero point and the reproducibility of the injury level.
- Discharge the piston to generate impaction on the brain. Place a sterile cotton swab onto the injured area to stop bleeding.
- Place the bone flap back to the mouse brain and secure with dental cement. Close the scalp with tissue adhesive (e.g., 3M Vetbond).
4. Postoperative recovery
- Place the mouse in a clean recovery cage with bedding under the heat lamp until full recovery.
- Provide moistened chow food and subcutaneously administer ketoprofen (5 mg/kg) for two consecutive days after surgery.
- Perform the above procedures except steps 3.7 and 3.8 for sham control animals.
5. Mouse euthanasia
- Euthanize mice on the day of study by isoflurane overdose and then decapitation.
NOTE: Several strategies can be used to euthanize the experimental animals prior to the sample collection.
- Collect brain samples for histological analysis.
Illustration of stereotactic placement and craniotomy procedure.
The CCI model is known for its stability and reproducibility in producing injury ranging from mild to severe18. Proper stereotactic technique and craniotomy procedure are major determinants in producing stable and reproducible CCI-induced brain injury (Figure 1A,B). An ideal craniotomy procedure would cause minimal histological injury in the sham-operated brain27,28 as shown (Figure 1C).
CCI induces long-term brain damage, blood-related toxicity, and neuroinflammation.
To evaluate brain damage, axonal loss, hemorrhagic pathology, and microglia/macrophages activation induced by CCI, we performed a battery of histological analysis including Nissl (neurons), luxol fast blue (myelin), Ly76 (erythrocytes), Iba1 (microglia/macrophages), and Perls (ferric iron) staining on injured brain slices from acute to chronic phases of CCI25,27,28,29,30,31,32. Changes in brain damage (Figure 2A) and corpus callosum (axon) loss (Figure 2B) were observed at days 1, 3, 7, and 28 after CCI. In addition, unilateral brain atrophy on the contusion side was seen at day 28 post-CCI (Figure 2). A successful CCI procedure produces focal injury between Bregma +1.42 mm and -1.34 mm at the coronal level27,30. Injury severity peaks around days 3 to 5 post-CCI the core of which centers approximately around Bregma +0.02 mm at the coronal level along with corpus callosum and striatal injuries25,28,31,32. Neuroinflammation was revealed by Iba1-positive activated microglia/macrophages accumulation around the border of the brain contusion area where the intraparenchymal hemorrhage was also detectable by the Ly76-positive staining from days 1 to 7 post-CCI (Figure 3B). A mixture of red blood cells (RBCs) with ruptured and intact cell morphology was observed at day 7 post-CCI (Figure 3B) suggesting the RBCs were lysed at this stage. This phenomenon is consistent with the observation that ferric iron deposition was detectable in the contusion region from days 7 to 28 post-CCI (Figure 3C). As CCI also induces corpus callosum and striatal injuries, activated microglia/macrophages are observed in the striatum far away from the contusion site (Figure 3D) from days 3 to 28 post-CCI. These observations indicate that the CCI method can reproduce the pathological features closely resembling human cerebral contusion including neuronal/axonal injury, intraparenchymal hemorrhage, and CNS inflammation.
Figure 1: Representative images of stereotactic and craniotomy procedures and histology of sham-operated brains.
(A) The mouse head was levelly and stably placed on the stereotactic frame. Notice the readings on the horizontal and vertical scales of the ear bars are the same. (B) The bone flap was removed after craniotomy (left) and representative images of brain surface after proper (middle) and faulty (right) craniotomy are shown. (C) Representative images of cresyl violet (left) and luxol fast blue (right)stained sham-operated brain sections at day 3. Please click here to view a larger version of this figure.
Figure 2: Brain tissue damage and myelin loss in CCI-induced cerebral contusion.
(A) Representative images of cresyl violet-stained brain sections after CCI at days 1, 3, 7, and 28. The dotted line indicates the contusion area. Scale bar = 1 mm. (B) Representative luxol fast blue-stained CCI brain sections at days 1,3,7, and 28. The residual myelin at corpus callosum are outlined by dotted line. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 3: CCI-induced intraparenchymal hemorrhage and neuroinflammation.
(A) Representative cresyl violet stained CCI brain section shows boxed regions selected for Iba1/Ly76 (red), Perls (yellow), and Iba1 (green) imaging. (B) Representative immunostaining of activated microglia/macrophage marker Iba1 (green), RBC marker Ly76 (red), and nuclear marker DAPI (blue). The dotted line indicates the border of contusion region. Scale bar = 100 µm in the full images and 50 µm in the inset images. (C) Representative Perls staining show iron deposition in the contusion region at days 1, 3, 7, and 28 post-CCI. Scale bar = 100 µm. (D) Representative images of activated microglia/macrophage marker Iba1 (green) expression at days 1, 3, 7, and 28 post-CCI. Scale bar = 100 µm in the full images and 50 µm in the inset images. Please click here to view a larger version of this figure.
The CCI protocol produces highly reproducible mechanical injury to the brain for cerebral contusion research. The following steps are crucial for generating consistent brain injury in animals using this CCI protocol.
First, the mouse head should be stably mounted on the stereotaxic frame and the anatomical landmarks Bregma and Lambda always in the same horizontal plane. Unsteady or unlevel head placement oftentimes result in varied injury levels between animals. To ensure the animal head is safely secured, use a pair of forceps to gently nudge the mouse head laterally. The head needs to be laterally immobile and any movement is indicative of an incorrect placement of the ear bars. Last, gently suspend the mouse body by lifting its tail to confirm that the head is firmly fixed on the stereotaxic frame.
Next, avoid damaging the dura mater when drilling into the skull by applying even pressure along the circular path as mechanical damage to the dura mater can cause unnecessary injury to the brain. Use great care to inspect the depth of drilling by gently tapping the bone window with a pair of fine forceps. In addition, heat generated during prolonged drilling process also induces cortical injury. In our protocol we use trephine rather than the round tip drill bit seen in most other CCI protocols33,34 to shorten the duration of craniotomy. Ice cold saline can be applied onto the skull during the drilling step for additional protection against heat. After drilling, the bone flap must be gently lifted without damaging the brain tissue underneath. Keeping a consistent size of the drilled bone window between animals is crucial for ensuring a consistent level of intracranial pressure and degree of brain deformation.
Furthermore, the impactor tip must be properly positioned at the center of the bone window and slightly pressed against the dural surface in order to generate consistent brain damage. Depth variance of tip zeroing on the dural surface can affect the extent of CCI injury.
Lastly, the bone flap should be repositioned and well secured after impaction. In fact, removing the bone flap prior to the mechanical impact is not relevant to human cerebral contusion. Therefore, repositioning bone flap immediately after the CCI helps maintain a consistent intracranial pressure in the closed head between animals during the subsequent disease progression.
High reproducibility, low mortality, and ease of controlling severity are among the many strengths of using a CCI model14,15,16,18,34. The requirement of anesthesia and craniotomy during the procedure are the major limitations of CCI model. Meanwhile, the neurologic deficits are usually resolved within days to weeks in rodents whereas human patients’ deficits can persist for months. However, no single animal model can mimic all clinical features. The CCI model is still highly reliable and oftentimes the preferred method to study short- and long-term pathophysiological events and to search for potential therapeutics for cerebral contusion14,15. Here we described the application of a CCI model to study hemorrhage contusion and secondary CNS inflammation after cerebral contusion. This protocol demonstrates a moderately severe CCI that produces focal brain injury in the primary sensory and motor cortical areas without damaging the hippocampus27,28. Depending on the research purpose, velocity and depth of impact, size of impactor tip, and position of the impaction can all be modified to create various severity of injury in different brain regions35.
Although we have applied this protocol in studying traumatic intraparenchymal hemorrhage, this CCI model can also be used to study other types of focal injury such as traumatic subarachnoid hemorrhage (tSAH), subdural hemorrhage (SDH), and epidural hemorrhage (EDH)1,7. While adult animal was used for the representative study, this CCI protocol with modification can also be applied to neonates to study the effect of cerebral contusion in the immature brain based on the goal of study36,37. A CCI model can not only facilitate study of single cerebral contusion but also repetitive mechanical cerebral impact that is relevant to people at high risk of multiple brain injuries such as military personnel19. Finally, we suggest that researchers perform pilot experiment to fine-tune the biomechanical parameters in the CCI model to better meet their research goal.
The authors have nothing to disclose.
We thank Danye Jiang for editing the manuscript and insightful input. We thank Jhih Syuan Lin for assisting in manuscript preparation. This work was supported by the Ministry of Science and Technology of Taiwan (MOST 107-2320-B-002-063-MY2) to C.F.C.
|4mm Short Trephine Drill||Salvin Dental Specialties, Inc.||TREPH-SHORT-4|
|anti-Iba1 antibody||Wako chemicals||#019-19741|
|cortical contusion injury impactor||Custom Design & Fabrication, Inc.||S/N 49-2004-C, eCCI Model 6.3||CCI device (S/N 49-2004-C, eCCI Model 6.3)|
|cresyl violet acetate||Sigma-Aldrich||C5042|
|DAB staining kit||Vector||SK-4105|
|goat anti-rabbit IgG secondary antibody, Alexa Fluor 488||Invitrogen||A11034|
|goat anti-rat IgG secondary antibody, Alexa Fluor 594||Invitrogen||A11007|
|tweezers||fine science tools||11252-20 NO. 5|
|isoflurane||Panion & BF Biotech Inc.|
|scissors||fine science tools||14068-12|
|solvent blue 38||Sigma-Aldrich||S3382|
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