Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Summary

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Abstract

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Introduction

Mild TBI, including concussion and sub-concussion, account for the majority of all TBI cases (>80% of all TBI)¹. Mild TBI commonly results from falls, traffic accidents, acts of violence, contact sports (e.g., football, boxing, hockey), and military combat^2,3. Mild TBI can lead to neurobiological events that affect neurobehavioral functions throughout the patient's lifetime and increase the risk of neurodegenerative diseases^4,5,6. Animal models provide an efficient and controlled means ....

Protocol

All procedures involved in this protocol were performed under the Institutional Animal Care and Use Committee approval (Zhejiang Normal University, Permit Number, dw2019005) and in compliance with the ARRIVE and the NIH Guide for the Care and Use of Laboratory Animals. Technical specifications can be found in the Table of Materials.

1. Animal handling procedure

1. House mice in a controlled environment with a temperature of 22-24 °C, humidity ran.......

Discussion

TBI refers to two primary types, closed and penetrating, with the latter characterized by a disruption of the skull and dura mater. Clinical data suggest that CHIs are more prevalent than penetrating injuries^1,2. After a single mild TBI, most patients experience PCS symptoms that typically resolve in a short period of time, and there is controversy regarding the proportion of patients whose PCS develop into long-term sequelae^23,.......

Materials

Name	Company	Catalog Number	Comments
75% ethanol	Shandong XieKang Medical Technology Co., Ltd.	220502
Buprenorphine hydrochloride	Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd	H12020272	Solution, Analgesic
Carprofen	Shanghai Guchen Biotechnology Co., Ltd	53716-49-7	Powder, Analgesic
Chlorhexidine digluconate	Shanghai Macklin Biochemical Co.,Ltd.	18472-51-0	19%-21% aqueous solution, Antimicrobial
Dental cement and solvent kit	Shanghai New Century Dental Materials Co., Ltd.	20220405, 3#	Powder reconsituted in matching solvent
Dissecting microscope	Shenzhen RWD Life Science Inc.	77019
Erythromycin ointment	Wuhan Mayinglong Pharmaceutical Group Co.,Ltd.	220412	Antibiotic
Fiber Optic Cold Light Source	Shenzhen RWD Life Science Inc.	F-150C
Flat-tipped micro-drill bit	Shenzhen RWD Life Science Inc.	HM31008	2 mm, steel
FPI device software	Jiaxing Bocom Biotech Inc.	Biocom Animal Brain Impactor V1.0
ICR mice	Jinhua Laboratory Animal Center	Stock#2023091	25 Male mice, 25-30g, 8 weeks old
Isoflurane	Shandong Ante Animal Husbandry Technology Co., Ltd.	2023090501
Isothermal heating pad	Wenzhou Repshop Pet Products Co., Ltd.
Luer Loc hup			Custom made using a 19G needle hub
Micro hand-held skull drill	Shenzhen RWD Life Science Inc.	78001	Max: 38,000rpm
Modified FPI device	Jiaxing Bocom Biotech Inc.
Morris water maze	Shenzhen RWD Life Science Inc.	63031	Evaluate mouse spatial learning and memory abilities
Open field	Shenzhen RWD Life Science Inc.	63008	Evaluate mouse locomoation and anxiety
Ophthalmic lubricant	Suzhou Tianlong Pharmaceutical Co., Ltd.	SC230724B
Sodium diclofenac ointment	Wuhan Mayinglong Pharmaceutical Group Co.,Ltd.	221207	nonsteroidal anti-inflammatory drug
Small animal anesthesia system-Enhanced	Shenzhen RWD Life Science Inc.	R530IP
Smart video-tracking system	Panlab Harvard Apparatus Inc., MA, USA	V3.0	Animal tracking and analysis
Stereotactic frame	Shenzhen RWD Life Science Inc.	68043
Vetbond Tissue Adhesive	3M, St Paul, MN, USA	202402AX	Suture the animal wound
Y maze	Shenzhen RWD Life Science Inc.	63005	Evaluate mouse spatial working memory