Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Summary

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Abstract

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Introduction

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies¹. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration^2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined....

Protocol

Adult (3-4 months old) male mice on a mixed C57BL/6 background were used in this protocol. Animals were kept on a 12 hr light/dark cycle, and allowed free access to food and water. All procedures performed in this protocol were conducted according to protocols approved by the Drexel University Institutional Animal Care and Use Committee.

1. Preparing Surgical Area

1. Disinfect surgical table with 70% ethanol, then cover the entire surgical bench with absorbent pads and arrange surgical instruments adjacent to stereotaxic.
2. Set up stereotaxic equipment without manipulator arm. Arrange the heating pad on the stereotaxic and ....

Results

Because animals undergoing this procedure do not require specialized post-operative care, short or long-term time survival periods are easily incorporated into the study, depending on the need to investigate acute or chronic pathology following injury. Principal features of reactive gliosis, such as upregulation of GFAP and hypertrophy of soma, can be observed as early as 2-3 days following injury. The peak phase of proliferation for reactive astrocytes is during days 3-5 following injury¹⁰. The representative.......

Discussion

It is critical that the skull or underlying dura are not damaged during the drilling. Use light pressure while drilling to ensure the skull is not punctured. In addition, care should be taken while lifting the skull piece to ensure the dura is not lifted off with the bone.

The forebrain stab injury described here models a penetrating injury to the CNS. Though less clinically translatable than TBI models such as FPI or CCI, the forebrain stab lesion model serves as a useful tool for a broad ran.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereotax	Harvard Apparatus	726049
High speed micro drill	Harvard Apparatus	724950
stainless steel scalpel blade, #11	MedVet	JOR581S
5/45 angled forceps	Fine Science Tools	11251-35
Gelfoam sponge 12 cm x 7 mm	Fisher	NC9841478
Rb anti-GFAP	DAKO	Z033429-2	Dilution - 1:20,000 (bright-field); 1:1,000 (fluorescence)
Shp anti-BrdU	Abcam	ab1893	Dilution - 1:20,000 (bright-field); 1:500 (fluorescence)
Biotinylated goat anti-rabbit	Vector Laboratories	BA-1000	Dilution - 1:400 (bright-field)
Biotinylated rabbit anti-sheep	Vector Laboratories	BA-6000	Dilution - 1:400 (bright-field)
Alexafluor 488 goat anti-rabbit	Life Technologies	A-11008	Dilution - 1:400 (bright-field)
Alexafluor 568 donkey anti-sheep	Life Technologies	A-21099	Dilution - 1:1,000 (fluorescence)
DAPI Nucleic Acid Stain	Life Technologies	D3571	Dilution - 1:1,000 (fluorescence)
Cresyl Violet Acetate	Sigma Aldrich	C5042-10G	Dilution - 1% (bright-field)