A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

Summary

Intraparenchymal hemorrhage and neuroinflammation accompanied by cerebral contusion can trigger severe secondary brain injury. This protocol details a mouse controlled cortical impact (CCI) model allowing researchers to study hemorrhage contusion and post-traumatic immune responses and explore potential therapeutics.

Abstract

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.

Introduction

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 toxicity^5,6 which cause herniation, brain edema, and intracranial pressure elevation^5,6. 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 contusion^9,10. 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 injury^9,12. 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 contusion^3,9,10,12.

Several preclinical models have been developed for studying traumatic brain injury including weight-drop model, lateral fluid percussion injury, and blast wave model^14,15. 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 laboratories^16,17. In comparison, the controlled cortical impact (CCI) model is more adequate for studying focal cerebral contusion because of its precise control and high reproducibility^14,15,18,19.

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 hippocampus^27,28. 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 deficiency^{24,25,27,28,29,30}. 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.

Protocol

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

1. Anesthetize the mouse with ~4% isoflurane mixed with room air at ~0.2 L/min in an induction chamber connected to the isoflurane vaporizer.
2. 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

1. 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.
2. 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.
3. Bring in the nose cone and maintain anesthesia at 1% - 2% isoflurane for the duration of the surgery.
4. 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.
5. Disinfect the shaved head with betadine followed by 70% alcohol using sterile cotton swabs. Repeat three times.

3. CCI surgery

1. 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.
2. 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.
3. Check that the mouse head is level in the horizontal plane.
   1. Identify anatomical landmarks Bregma and Lambda and mark both locations with a pencil.
   2. 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.
   3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Discharge the piston to generate impaction on the brain. Place a sterile cotton swab onto the injured area to stop bleeding.
9. 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

1. Place the mouse in a clean recovery cage with bedding under the heat lamp until full recovery.
2. Provide moistened chow food and subcutaneously administer ketoprofen (5 mg/kg) for two consecutive days after surgery.
3. Perform the above procedures except steps 3.7 and 3.8 for sham control animals.

5. Mouse euthanasia

1. 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.
2. Collect brain samples for histological analysis.

Results

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 s...

Discussion

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 sa...

Materials

Name	Company	Catalog Number	Comments
4mm Short Trephine Drill	Salvin Dental Specialties, Inc.	TREPH-SHORT-4
anti-Iba1 antibody	Wako chemicals	#019-19741
anti-Ly76 antibody	abcam	ab91113
carboxylate cement	3M	70201136010
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
Mayer's Hematoxylin	ScyTek	HMM500
tweezers	fine science tools	11252-20 NO. 5
isoflurane	Panion & BF Biotech Inc.
lithium carbonate	Sigma-Aldrich	62470
steriotexic frame	stoelting
scissors	fine science tools	14068-12
solvent blue 38	Sigma-Aldrich	S3382