We are deeply thankful to Dr. Frank Kirchhoff for providing GFAP-EGFP and CX3CR1-EGFP mouse strains. The work was supported by grants from the Centre of International Mobility of Finland, Tekes, Finnish Graduate School of Neuroscience (FGSN) and the Academy of Finland.

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 (EL&#196;INKOELAUTAKUNTA-ELLA). Adult mice of 1-3 months age, weight 24-38 g, were kept in individual cages in the certified University&#8217;s animal facility and provided with food and water ad libitum.
1. Brain Injury Imaging Through a Cranial Window
<o...

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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&#8211;brain barrier. To study primary and secondary pathogenesis, as well as recovery after trauma, this injury model was combined with longitudinal...

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 towards a stage where drug efficacy can be predicted in patients with brain injuries. At present, several well established animal models of head trauma exist, including controlled cortical impact1, fluid percussion injury2, dynamic cortical deformation3, weight-drop4, and photo injury5. A number of experimental models have been used to study certain morphological, molecular and behavioral aspects of head trauma-associated pathology. However, no single animal model is entirely successful in validating new therapeutic strategies. Development of reliable, reproducible and controlled animal models of brain injury is necessary to assess the complex pathological processes.
The novel combination of the latest microscopic imaging technologies and genetically-encoded fluorescent reporters offers an unprecedented opportunity to investigate all phases of brain injury, which include primary injury, spreading of the primary injury, secondary injury, and regeneration. In particular, in vivo two-photon microscopy is a unique nonlinear optical technology that allows real-time visualization of cellular and even subcellular structures in deep cortical layers of rodent brain. Several types of cells and organelles can be imaged simultaneously by combining different fluorescent markers. Using this powerful tool, we can visualize dynamic morphological and functional changes in living brain under posttraumatic conditions. The advantages of in vivo two-photon microscopy in studying brain injury were recently demonstrated by Kirov and colleagues6. Using a mild focal cortical contusion model, these authors showed that acute dendritic injury in the pericontusional cortex is gated by the decline in the local blood flow. Moreover, they demonstrated that the metabolically compromised cortex around the contusion site is further damaged by the spreading depolarization. This secondary damage affects synaptic circuitry, making the consequences of traumatic brain injury more severe.
Here, we propose the method of stereotaxic prick with a syringe needle, which could be combined with simultaneous topical drug application, as an advanced model for local brain injury and as a tool to study pathophysiological consequences of acute trauma in mammalian brain in vivo.

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acute brain trauma in mice followed by longitudinal two-photon imaging

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.

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 &#956;m below the pial surface (Figure 1B), whereas skull thinning tends to impose a ...

Watch this Scientific Journal Video about Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging at JoVE.com

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

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.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

acute-brain-trauma-in-mice-followed-by-longitudinal-two-photon-imaging

Research

JoVE Journal

Medicine

12.3K visualizações.  University of Helsinki.  Trauma cerebral aguda é uma lesão grave que não tem tratamento adequado à data. Microscopia Multiphoton permite estudar longitudinalmente o processo de desenvolvimento agudo trauma cerebral e sondagem estratégias terapêuticas em roedores. Dois modelos de trauma cerebral aguda estudou com in vivo de dois fótons de imagem de cérebro são demonstrados neste protocolo. 