Disparo reactiva Gliosis<em> En Vivo</em> Por un Prosencéfalo Lesiones Puñalada
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 neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects.
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.
Protocol
Representative Results
Discussion
Es fundamental que el cráneo o dura subyacente no se dañen durante la perforación. Utilice una ligera presión durante la perforación para asegurar el cráneo no está pinchado. Además, se debe tener cuidado al levantar la pieza del cráneo para asegurar la duramadre no se levanta con el hueso.
La lesión de arma blanca del cerebro anterior describe aquí los modelos de una lesión penetrante en el SNC. Aunque menos clínicamente traducible que los modelos de TBI como FPI o CCI, el mode…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03
Materials
| 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 12cmx7mm | Fisher | NC9841478 | |
| Antibodies and other reagents | Manufacturer | Catalog Number | Dilution (brightfield) Dilution (fluorescence) |
| Rb anti-GFAP | DAKO | Z033429-2 | 1:20k 1:1k |
| Shp anti-BrdU | Abcam | ab1893 | 1:2k 1:500 |
| Biotinylated goat anti-rabbit | Vector Laboratories | BA-1000 | 1:400 |
| Biotinylated rabbit anti-sheep | Vector Laboratories | BA-6000 | 1:400 |
| Alexafluor 488 goat anti-rabbit | Life Technologies | A-11008 | 1:400 |
| Alexafluor 568 donkey anti-sheep | Life Technologies | A-21099 | 1:1000 |
| DAPI Nucleic Acid Stain | Life Technologies | D3571 | 1:1000 |
| Cresyl Violet Acetate | Sigma Aldrich | C5042-10G | 1% |
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