Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Jianjun Zhong; Georgia Gunner; Nils Henninger; Dorothy P. Schafer; Daryl A. Bosco

doi:10.3791/64701

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain ...

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Neuroscience

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JoVE Journal Neuroscience

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

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08:25 min

April 21, 2023

DOI: 10.3791/64701-v

Jianjun Zhong^1,2, Georgia Gunner³, Nils Henninger¹, Dorothy P. Schafer³, Daryl A. Bosco¹

¹Department of Neurology,University of Massachusetts Chan Medical School, ²Department of Neurosurgery,The First Affiliated Hospital of Chongqing Medical University, ³Department of Neurobiology,University of Massachusetts Chan Medical School

Overview

This study demonstrates a method for visualizing the neuronal response to mechanical stress following traumatic brain injury (TBI) in a live mouse model. It details the implantation of a cranial window to facilitate intravital imaging of EGFP-expressing neurons using two-photon microscopy, enabling tracking of the neuronal response over time.

Key Study Components

Area of Science

Neuroscience
Traumatic Brain Injury
Imaging Techniques

Background

Traumatic brain injury (TBI) leads to significant neuronal changes.
Monitoring neuronal responses during acute and chronic phases is crucial for understanding TBI effects.
Two-photon microscopy allows for deep imaging in live tissues.
EGFP expression helps in tracking specific neuronal populations.

Purpose of Study

To visualize and quantify neuronal responses to mechanical stress in a live mouse model following TBI.
To provide insights into the effects of TBI on neuronal function over time.
To establish a protocol for changing gene targets to study various proteins in different cell types.

Methods Used

Two-photon microscopy for imaging neuronal responses.
Use of mice for in vivo experimentation with induced traumatic brain injury.
Detailed surgical protocol for cranial window implantation to provide accessibility for imaging.
Stable positioning and preparation techniques were emphasized to minimize damage during procedures.

Main Results

The technique allows for real-time monitoring of neuronal behavior following TBI.
Responses observed in EGFP-expressing neurons illustrate alterations in excitability following mechanical injury.
Data showing continuity in responses both acutely and chronically demonstrates the lasting effects of TBI on neuronal integrity.

Conclusions

This study establishes a valuable tool for exploring the dynamic responses of neurons to traumatic injury in vivo.
It enables investigation into specific molecular changes related to TBI over time, potentially leading to better understanding and treatment strategies.
The findings have implications for neuronal plasticity and recovery mechanisms following injury.

Frequently Asked Questions

What advantages does the cranial window provide?

The cranial window allows for non-invasive imaging of neuronal activity in real-time, enabling continuous observation of changes after traumatic brain injury.

How is traumatic brain injury induced in the model?

TBI is induced by a specific impact at marked coordinates on the mouse's skull, utilizing a device that delivers a controlled mechanical force.

What imaging outcomes can be expected?

The study allows monitoring of the neuronal response, specifically focusing on changes in excitability and EGFP expression levels after injury.

Can this method be adapted for other proteins?

Yes, the protocol can be adjusted by changing the gene of interest and the viral promoter to study different proteins in various neuronal types.

What are some key limitations of this approach?

Limitations may include the need for precise surgical techniques and potential variability in injury response based on individual animal factors.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

Our protocol provides a way to visualize the neuronal response to mechanical stress in a live mouse, which can provide direct evidence of how traumatic brain injury affects the brain. This technique can monitor the target protein at the same brain location in the same animal for both the acute and chronic phases after traumatic brain injury. The gene of interest and the virus promoter can be changed accordingly to study other proteins in the specific cell types in the brain.

To begin, apply ophthalmic ointment to the anesthetized mouse's eyes. Remove the hair from the top of the head by trimming with regular scissors. After establishing a 12 to 15 millimeters long midline incision, excise the skin over the left and right hemispheres of the skull using curved tip spring scissors.

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