Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury

Summary

Simplified traumatic brain injury (TBI) models have facilitated the development of therapeutic approaches. This protocol outlines the creation of a stab-wound mouse cortex using needles, enabling the analysis of hemorrhage and inflammation. The stab-wound TBI mouse model offers the advantage of being performed without requiring specialized equipment.

Abstract

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.

Introduction

Traumatic brain injury (TBI) is caused by physical damage, often resulting from accidents, including traffic accidents and fall accidents. TBI is classified into two types: penetrating brain injury, which occurs when a sharp object perforates the skull as well as the brain, and closed brain injury, which is caused by violent shaking of the brain inside without a break in the skull¹.

The causes of TBI are very diverse, including concussions, brain contusions, hematomas, and skull fractures; therefore, TBI mouse models have been developed using various protocols to replicate these different causes. For example, a repet....

Protocol

All animal care protocols were approved by the Institutional Animal Care and Use Committee of Ochanomizu University, Japan, and were performed in accordance with the guidelines established by the Ministry of Education, Science, and Culture in Japan. Six-week-old adult C57BL/6J female mice (20-25 g) were used; however, this protocol can be applied to other mouse strains, including ICR. All mice were provided ad libitum access to food and water in a clean environment. Details of the reagents and equipment used are listed in the Table of Materials.

1. Stab-wound surgery to the cerebral cortex

1. Prepa....

Discussion

Here, a protocol for creating a TBI mouse model using needles was introduced. This protocol allows for a quantitative assessment of recovery from the breakdown of the BBB and inflammation after brain injury using histological and molecular biological approaches. Alternative protocols, such as the repetitive concussive TBI model and weight-drop TBI model, can also be used to analyze BBB breakdown and inflammation. These models replicate TBI pathology under controlled conditions, including specific parameters for impact st.......

Materials

Name	Company	Catalog Number	Comments
19 G x 1•1/2" needle	TERUMO	NN-1938R
27 G x 3/4" needle	TERUMO	NN-2719S
anti-GFAP antibody	Sigma-Aldrich	G9269
anti-Iba1 antibody	Wako	019-19741
Atipamezole Hydrochloride	Nippon Zenyaku Kogyo		Product name: Antisedan
Biotin-conjugated mouse IgG antibody	Vector Laboratories	BA-9200
Biotin-conjugated rabbit IgG antibody	Vector Laboratories	BA-1000
Bovine albumin	Nacalai tesque	01860-07
Brain Slicer	Visikol	BSLM-2
Butorphanol Tartrate	Meiji Animal Health		Product name: Vetorphale 5 mg
Confocal microscope	Zeiss	LSM700
Cryostat	Leica	CM1520
DAB	Sigma-Aldrich	D5637-1G
DAPI	Roche	10236276001
Evans blue	Wako	056-04061
Fluorescent-conjugated rabbit IgG antibody	Invitrogen	A-21206
Fluoromount-G	Invitrogen	4958-02	Water-based mounting medium
Isoflurane Inhalation Solution	Viatris	v002139
KOD SYBR qPCR Mix	TOYOBO	QKD-201	qPCR master mix kit
Medetomidine	Nippon Zenyaku Kogyo		Product name: Domitor
Microscope	Olympus	FSX100
Microvolume spectrophotometer	ThermoFisher Scientific	NanoDrop One
Midazolam 10 mg/2 mL	Sandoz	1124401A1060
MOUNT QUICK	Daido Sangyo	DM01	Water insoluble mounting medium
Newborn calf serum	Gibco	16010159
O.C.T. compound	Sakura Finetek Japan	45833	Embedding medium
Peel-A-Way, Truncated 22 mm Square Top	Ted Pella	27118	Tissue embedding mold
Peristaltic perfusion pump	ATTO	SJ-1211
Plate reader	Fisher Scientific	Cytation 3
Real-time qPCR machine	ThermoFisher Scientific	StepOne Plus
ReverTra Ace qPCR RT Kit	TOYOBO	FSQ-101	cDNA synthesis kit
Superfrost Plus Slide Glass	Fisher Scientific	12-550-15	Positive-charged slide glass
Suture with needle	Alfresa	HT2003NA75-KF2
TRIzol Reagent	Invitrogen	15596026
VECTASTAIN ABC Standard Kit	Vector Laboratories	PK-4000	Avidin/biotin-based peroxidase system kit