Name: intravital imaging of fluorescent protein expression in mice with a closed-skull traumatic brain injury and cranial window using a two-photon microscope
Uploaded: 2023-04-21T14:05:00
Description: Watch this Scientific Journal Video about Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope at Jo

We thank Dr. Miguel Sena-Esteves at the University of Massachusetts Chan Medical School for gifting the AAV(PHP.eB)-Syn1-EGFP virus, and Debra Cameron at the University of Massachusetts Chan Medical School for drawing the mice skull sketch. We also thank current and past members of the Bosco, Schafer and Henninger labs for their suggestions and support. This work was funded by the Department of Defense (W81XWH202071/PRARP) to DAB, DS, and NH.

All the animal related protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (US) Committee. The protocols were approved by the Institutional Animal Care and Use Committee of University of Massachusetts Chan Medical School (UMMS) (Permit Number 202100057). In brief, as shown in the schematic of study (Figure 1), the animal receives a virus injection, a TBI, a window implantation, and then intravital imaging...

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In this study, AAV injection, TBI administration, and a headpost with cranial window implantation were combined for longitudinal imaging analysis of EGFP-labeled neurons within the mouse brain cortex (layers IV and V) to observe the effects of TBI on cortical neurons. This study notes that the TBI site chosen here, above the hippocampus, provides a relatively flat and broad surface for implantation of the cranial window. Conversely, the skull is relatively narrow anterior to this site, and therefore it is difficult to en...

Traumatic brain injury (TBI), which can result from sports injuries, vehicle collisions, and military combat, is a worldwide health concern. TBI can lead to physiological, cognitive, and behavioral deficits, and&#160;lifelong disability or mortality1,2. TBI severity can be classified as mild, moderate, and severe, the vast majority being mild TBI (75%-90%)3. It is increasingly recognized that TBI, particularly repetitive occurrences of TBI, can promote neuronal degeneration and serve as risk factors for several neurodegenerative diseases, including Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and chronic traumatic encephalopathy (CTE)4,5,6. However, the molecular mechanisms underlying TBI-induced neurodegeneration remain unclear, and thus represent an active area of study. To gain insight into how neurons respond to and recover from TBI, a method for monitoring fluorescently tagged proteins of interest, specifically within neurons, by longitudinal intravital imaging in mice after TBI is described herein.
To this end, this study shows how to combine a surgical procedure for the administration of closed-skull TBI that is similar to what that has been reported previously7,8, together with a surgical procedure for implantation of a cranial window for downstream intravital imaging, as described by Goldey et al9. Notably, it is not feasible to implant a cranial window first and subsequently perform a TBI in the same region, as the impact of the weight drop that induces the TBI is likely to damage the window and cause irreparable harm to the mouse. Therefore, this protocol was designed to administer the TBI and then implant the cranial window directly over the impact site, all within the same surgical session. An advantage of combining both the TBI and cranial window implantation in a single surgical session is a reduction in the number of times a mouse is subjected to surgery. Further, it allows one to monitor the immediate response (i.e., on the timescale of hours) to TBI, as opposed to implanting the window at a later surgical session (i.e., initial imaging starting on a timescale of days post-TBI). The cranial window and intravital imaging platform also offer advantages over monitoring neuronal proteins by conventional methods such as immunostaining of fixed tissues. For example, fewer mice are required for intravital imaging, as the same mouse can be studied at multiple time points, as opposed to separate cohorts of mice needed for discrete time points. Further, the same neurons can be monitored over time, allowing one to track specific biological or pathological events within the same cell.
As a proof of concept, the neuron-specific expression of enhanced green fluorescent protein (EGFP) under the synapsin promoter is demonstrated here10. This approach can be extended to 1) different brain cell-types by utilizing other cell-type specific promoters, such as myelin basic protein (MBP) promoter for oligodendrocytes and glial fibrillary acidic protein (GFAP) promoter for astrocytes11 , 2) different target proteins of interest by fusing their genes with the EGFP gene, and 3) co-expressing multiple proteins fused to different fluorophores. Here, EGFP is packaged and expressed via adeno-associated virus (AAV) delivery through an intracranial injection. A closed-skull TBI is administered using a weight-drop device, followed by implantation of a cranial window. Visualization of neuronal EGFP is achieved through the cranial window, using two-photon microscopy to detect EGFP fluorescence in vivo. With the two-photon laser, it is possible to penetrate deeper into the cortical tissue with minimal photodamage, allowing for repeated longitudinal imaging of the same cortical regions within an individual mouse for days and up to months12,13,14,15. In sum, this approach of combining a TBI surgery with intravital imaging aims to advance the understanding of the molecular events that contribute to TBI-induced disease pathology16,17.

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	<tbody>
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			<td>Parkell</td>
			<td>S379</td>
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			<td>BD</td>
			<td>NDC/HRI#08290-3284-38</td>
			<td>5/16&#34; x 31G</td>
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			<td>Purdue</td>
			<td>NDC67618-151-17</td>
			<td>including 7.5% povidone iodine</td>
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			<td>Buprenorphine</td>
			<td>PAR Pharmaceutical</td>
			<td>NDC 42023-179-05</td>
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			<td>HIKMA Pharmaceutical</td>
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			<td>Parkell</td>
			<td>S371</td>
			<td>full name: &#34;C&#34; Universal TBB Catalyst</td>
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			<td>S396</td>
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			<td>HP4-310</td>
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			<td>Phoenix</td>
			<td>NDC 57319-519-05</td>
			<td></td>
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			<td>EF4 carbide bit</td>
			<td>Microcopy</td>
			<td>Lot# C150113</td>
			<td>Head Dia/Lgth/mm 1.0/4.2</td>
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			<td>Ethonal</td>
			<td>Fisher Scientific</td>
			<td>04355223EA</td>
			<td>75%</td>
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			<td>FG1/4 carbide bit</td>
			<td>Microcopy</td>
			<td>Lot# C150413</td>
			<td>Head Dia/Lgth/mm 0.5/0.4</td>
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			<td>FG4 carbide bit</td>
			<td>Microcopy</td>
			<td>Lot# C150309</td>
			<td>Head Dia/Lgth/mm 1.4/1.1</td>
		</tr>
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			<td>Headpost</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-manufactured</td>
		</tr>
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			<td>Heating apparatus</td>
			<td>CWE</td>
			<td>TC-1000 Mouse</td>
			<td>equiped with the stereotaxic instrument and be used while operating surgery</td>
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			<td>Heating blanket</td>
			<td>CVS pharmacy</td>
			<td>E12107</td>
			<td>extra heating device and be used after surgery</td>
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			<td>Vetequip</td>
			<td>911103</td>
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			<td>Vetequip</td>
			<td>901801</td>
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			<td>Leica surgical microscope</td>
			<td>Leica</td>
			<td>LEICA 10450243</td>
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			<td>Lubricant ophthalmic ointment</td>
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			<td>Marker pen</td>
			<td>Delasco</td>
			<td>SMP-BK</td>
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			<td>Meloxicam</td>
			<td>Norbrook</td>
			<td>NDC 55529-040-10</td>
			<td></td>
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			<td>World Precision Instruments</td>
			<td>micro4 and UMP3</td>
			<td></td>
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			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>Hamilton 80014</td>
			<td>1701 RN, 10 &#956;L gauge for syringe and 32 gauge for needle, 2 in, point style 3</td>
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			<td>Mosquito forceps</td>
			<td>CAROLINA</td>
			<td>Item #:625314</td>
			<td>Stainless Steel, Curved, 5 in</td>
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			<td>Depilatory agent</td>
			<td>McKesson Corporation</td>
			<td>N/A</td>
			<td>Nair Hair Aloe &#38; Lanolin Hair Removal Lotion</td>
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			<td>Microscope 1</td>
			<td>Nikon</td>
			<td>SMZ745</td>
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			<td>Microscope 2</td>
			<td>Zeiss</td>
			<td>LSM 7 MP</td>
			<td>two-photon microscope</td>
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			<td>Multiphoton laser</td>
			<td>Coherent</td>
			<td>Chameleon Ultra II, Model: MRU X1, VERDI 18W</td>
			<td>laser for two-photon microscopy</td>
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			<td>Non-absorbable surgical suture</td>
			<td>Harvard Apparatus</td>
			<td>catlog# 59-6860</td>
			<td>6-0, with round needle</td>
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			<td>Norland Optical Adhesive 81</td>
			<td>Norland Products</td>
			<td>NOA 81</td>
			<td></td>
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			<td>No-Snag Needle Holder</td>
			<td>CAROLINA</td>
			<td>Item #: 567912</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick base liquid</td>
			<td>Parkell</td>
			<td>S398</td>
			<td>&#34;B&#34; Quick Base For C&#38;B Metabond</td>
		</tr>
		<tr>
			<td>Regular scissor 1</td>
			<td>Eurostat</td>
			<td>eurostat es5-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Regular scissor 2</td>
			<td>World Precision Instruments</td>
			<td>No. 501759-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Round cover glass 1</td>
			<td>Warner instruments</td>
			<td>CS-5R Cat# 64-0700</td>
			<td>for 5 mm of diameter</td>
		</tr>
		<tr>
			<td>Round cover glass 2</td>
			<td>Warner instruments</td>
			<td>CS-3R Cat# 64-0720</td>
			<td>for 3 mm of diameter</td>
		</tr>
		<tr>
			<td>Rubber rings</td>
			<td>Orings-Online</td>
			<td>Item # OO-014-70-50</td>
			<td>O-Rings</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Bioworld</td>
			<td>L19102411PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissor 1</td>
			<td>World Precision Instruments</td>
			<td>No. 91500-09</td>
			<td>tip straight</td>
		</tr>
		<tr>
			<td>Spring scissor 2</td>
			<td>World Precision Instruments</td>
			<td>No. 91501-09</td>
			<td>tip curved</td>
		</tr>
		<tr>
			<td>Stereotaxic platform</td>
			<td>KOPF</td>
			<td>Model 900LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel</td>
			<td>Item #: 1647358</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical Caliper</td>
			<td>World Precision Instruments</td>
			<td>No. 501200</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps 1</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>Catlog# 0508-5/45-PO</td>
			<td>style 5/45, curved</td>
		</tr>
		<tr>
			<td>Surgical forceps 2</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>catlog# 0103-5-PO</td>
			<td>style 5, straight</td>
		</tr>
		<tr>
			<td>Surgical forceps 3</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>catlog# 72912</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps 4</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>Catlog# 0508-5/45-PO</td>
			<td>style 5/45, curved</td>
		</tr>
		<tr>
			<td>Surgical gauze</td>
			<td>ZORO</td>
			<td>catlog #: G0593801</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Leica</td>
			<td>Leica KL300 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>UV box</td>
			<td>Spectrolinker</td>
			<td>XL-1000</td>
			<td>also called UV crosslinker</td>
		</tr>
		<tr>
			<td>Vaporguard</td>
			<td>Vetequip</td>
			<td>931401</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M Animal Care</td>
			<td>Part Number:014006</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital imaging of fluorescent protein expression in mice with a closed-skull traumatic brain injury and cranial window using a two-photon microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

As proof of concept for this protocol, viral particles expressing AAV-Syn1-EGFP were injected into the brain cortex of male TDP-43Q331K/Q331K mice (C57BL/6J background)19 at the age of 3 months. It is noted that wild-type C57BL/6J animals can also be used, however this study was carried out in TDP-43Q331K/Q331K mice because the laboratory is focused on neurodegenerative disease research. A TBI surgery was performed 4 weeks after AAV injection. Within the same surgical setting...

Watch this Scientific Journal Video about Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope at Jo

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

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.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Research

JoVE Journal

Neuroscience

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...