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 ...

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.Once the skull is exposed, remove the periosteum by gently rubbing it with the sterile cotton tipped applicator, and flushing it with sterile saline. Once the skull area is dried, mark the Traumatic Brain Injury, or TBI, impact site at the coordinates 2.5 millimeters posterior to the bregma and two millimeters lateral from the sagittal suture to the right. Quickly remove the mouse from the stereotaxic frame and place the head on the buffer cushion under the TBI device.Align the impactor tip with the marked impact site. Lift the metal column by pulling the tethered nylon string 15 centimeters above the mouse head, and then release it, allowing the weight to fall freely onto the transducer rod, which is in contact with the skull top at the TBI site. Place the anesthetized mouse's head onto the stereotaxic frame kept under the surgical microscope.Gently and slowly drill the surface of the skull using an FG4 carbide bit at a low rotor speed to remove the remaining periosteum, and create a rough skull surface such that the dental cement binds securely with the skull. Use saline to flush and clear bone dust from the skull surface. To separate the lateral muscles from the skull, at approximately five millimeters posterior to the eye, where the suture connecting the parietal and temporal skull is located, gently insert the fine closed tips and gently move the closed tips in the posterior direction until the lambdoid suture.Separate the lateral muscles on the side where cranial window implantation will occur. Wash away the debris from the surgical site using saline and dry the area with gauze. To implant the head post, mark the center point 2.5 millimeters posterior to the bregma and 1.5 millimeters off the sagittal suture on the right hemisphere using a marker pen and a surgical caliper.Then trace the circumference of the craniotomy on the clean and dry skull. Loosen the ear bar and rotate the head, so that the craniotomy plane is perfectly horizontal, and then tighten the ear bar again. Use the wood stick of a cotton tipped applicator to add two small drops of superglue to the front and back edge of the head post.Position the titanium head post over the center of the craniotomy, and quickly adjust it to rest within the same plane where the cranial window will be implanted. Apply light pressure until the superglue is dried, which usually takes approximately 30 seconds. Prepare dental cement in a pre-cooled ceramic dish by thoroughly mixing 300 milligrams of cement powder, six drops of QuickBase liquid, and one drop of catalyst.Quickly apply a generous amount of dental cement mixture to the outside perimeter of the traced circumference and cover any exposed bone surface. However, do not cover the site of the craniotomy. Release the ear bar and secure the head post to the metal frame to ensure that the head is stable for precise drilling along the marked craniotomy circumference.To perform the craniotomy, use a surgical caliper to verify the diameter of the marked circle as demonstrated before, and adjust as necessary, so that the cranial window will fit snugly inside the craniotomy. Using an electric dental drill, etch and thin the skull along the outside of the marked circle using an FG4 carbide bit first, which creates a track within which to thin the skull. Irrigate the area with saline to wash away the bone dust.Continue to thin the skull using an FG 1/4 carbide bit until the skull is paper thin and transparent. Periodically, stop drilling, and irrigate the whole area with sterile saline again to reduce heating from the drill and to wash away the bone dust. Complete the skull thinning using an EF4 carbide bit, and continue thinning and drilling through the rest of the skull along the track.Insert a 0.5 millimeter fine-tipped forceps through the cracked place, and lift the bone flap gently upward without indenting the underlying brain. After removing the bone flap, irrigate the craniotomy area with saline. Using the curved tipped surgical forceps, gently remove the visible arachnoid matter.Use the straight tipped surgical forceps to pick up the sterile glass window with the three millimeter glass cover slip facing down. Place and adjust the glass window above the craniotomy site to make sure that the window can fit snugly to the craniotomy edge. The five millimeter glass cover slip is on top.Prepare the dental cement as demonstrated earlier, and wait approximately six minutes until the cement becomes pasty and thick. While waiting for the cement to become pasty and thick, apply an adequate amount of pressure to the window through a stereotaxic manipulator to check that the skull can securely and tightly contact the glass window. Use an adjustable precision applicator brush to add a small amount of cement alongside the window edge to seal the glass window with the skull.Wait approximately 10 minutes to let the cement completely dry, then gently release, and remove the manipulator above the window. Complete by trimming the dental cement using the dental drill with an FG4 carbide bit if excess cement covers the window. At approximately four hours after surgery, two-photon imaging was carried out, where at the superficial level vasculature appeared black, and at the deep level of approximately 400 micrometers, EGFP protein expression was diffusely distributed throughout the cell body.At one week and four months post TBI, the vasculature pattern observed at the day zero time point was used as a reference to locate the same imaging region. The vasculature pattern is similar to that at day zero. Similarly, the EGFP expression was detected throughout the cell body.The fluorescence intensity at day zero and four months was similar at both the superficial and deeper levels. However, the fluorescence intensity at one week was lower than that of day zero and four months, possibly due to the translational repression reported for other TBI models. It is crucial to take utmost care when operating the craniotomy step to avoid damaging the brain tissue.Do not get the drill tip inserted into the brain. Using this technique, researchers can visualize different proteins of interest in the same cell longitudinally across different phases post-TBI, providing information on the localization, expression, and solubility of that protein in the mammalian brain after trauma.

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 ...

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 ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...