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 ....

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 p.......

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