Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Summary

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Abstract

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Introduction

Acute brain injury is a significant public health problem with high incidence of injury in motor vehicle crashes, falls or assaults, and high prevalence of subsequent chronic disability. Therapeutic approaches to the treatment of brain injury remain totally symptomatic, thus limiting the effectiveness of prehospital, surgical and critical care. This makes the social and economic impact of brain injury particularly severe. For a variety of reasons, most of the clinical trials failed to demonstrate improvement in recovery after brain injury using novel therapeutic approaches.

Animal models are crucial for developing new therapeutic strategies....

Protocol

All the procedures presented here were performed according to local guidance for animal care (The Finnish Act on Animal Experimentation 62/2006). Animal license (ESAVI/2857/04.10.03/2012) was obtained from local authority (ELÄINKOELAUTAKUNTA-ELLA). Adult mice of 1-3 months age, weight 24-38 g, were kept in individual cages in the certified University’s animal facility and provided with food and water ad libitum.

1. Brain Injury Imaging Through a Cranial Window

1. Anesthetizing animals and preparing the operation field
   1. Anesthetize mice by peritoneal injection of a mixture of ketamine (Ketalar....

Results

We have optimized two operation procedures: 1) chronic cranial window and 2) skull thinning, for posttraumatic brain imaging in transgenic mice. Schematic view of the experimental preparations is presented in Figure 1. Traumatic prick by steel needle of 0.3 mm OD (30 G) is applied to the drilled well (Figure 1A). A successful cranial window preparation allows imaging at depths up to 650 μm below the pial surface (Figure 1B), whereas skull thinning tends to impose a limit.......

Discussion

Brain trauma is an abrupt, unpredictable event. Here, we describe the animal model that reproduces a spectrum of pathological changes observed in human patients after brain injury such as neurodegeneration, elimination of dendrites, brain edema, glial scar, hemorrhages in the cerebral cortex coupled with focal subarachnoid hemorrhaging and increased permeability of the blood–brain barrier. To study primary and secondary pathogenesis, as well as recovery after trauma, this injury model was combined with longitudinal.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2A-sa dumb Tweezers, 115 mm	XYtronic	XY-2A-SA
30 G ½ in needle	BD	REF 304000
Animal trimmer, shaving machine	Aesculap	Isis GT420
Binocular Microscope	Zeiss	Stemi 2000
Biological Temperature Controller with stainless steel heating pad	Supertech	TMP-5b
Blunt microsurgical blade	BD	REF 374769
Borosilicate tube with filament	Sutter Instruments	BF120-69-10	For glass pipette production
Carprofene	Pfizer	Rimadyl vet
Chlorhexidine digluconate	Sigma	C9394
Dental cement	DrguDent, Dentsply	REF 640 200 271
Dexamethasone	FaunaPharma	Rapidexon vet
Disposable drills	Meisinger	HP 310 104 001 001 008
Dulbeco’s PBS 10x	Sigma	D1408
Dumont #5 forceps, 110 mm	FST	91150-20
Ealing microelectrode puller	Ealing	50-2013	Vertical puller for glass pipette production
Eye ointment	Novartis	Viscotears
Foredom drill control	Foredom	FM3545
Foredom micromotor handpiece	Foredom	MH-145
Gas anesthesia platform for mice	Stoelting	50264	Assembled on stereotaxic instrument
Hemostasis Collagen Sponge	Avitene, Ultrafoam BARD	Ref 1050050
Imaris	Bitplane
Ketamine	Intervet	Ketaminol vet
Mai Tai DeepSee laser	Spectra-Physics
Metal holder	Neurotar
Microdressing forceps, 105 mm	Aesculap	BD302R
Microfil	WPI	MF34G-5	Micro syringe filling capillaries
Mineral oil	Sigma	M8410
Multiphoton Laser Scanning Microscope	Olympus	FV1000MPE
NanoFil Syringe 10 μl	WPI	NANOFIL	Hamilton syringe
Nonwoven swabs 5 x 5	Molnlycke Health Care	Mesoft	Surgical tampons
Polyacrylic glue	Henkel	Loctite 401
Round glass coverslip	Electron Microscopy Sciences
1.5 thickness
Small animal stereotaxic instrument	David Kopf Instruments	900
Stoelting mouse and neonatal rat adaptor	Stoelting	51625	Assembled on stereotaxic instrument.
Student iris scissors, straight 11.5 cm	FST	91460-11
Sulforhodamine 101	Molecular Probes	S-359
UMP3 microsyringe pump and Micro 4 microsyringe pump controller	WPI	UMP3-1	Microinjector and controller
Xylazine	Bayer Health Care	Rompun vet