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.

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No conflicts of interest are declared.

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.

<table><tbody><tr><td>Adjustable Precision Applicator Brushes</td><td>Parkell</td><td>S379</td><td></td></tr><tr><td>BD insulin syringe</td><td>BD</td><td>NDC/HRI#08290-3284-38</td><td>5/16&quot; x 31G</td></tr><tr><td>Betadine</td><td>Purdue</td><td>NDC67618-151-17</td><td>including 7.5% povidone iodine</td></tr><tr><td>Buprenorphine</td><td>PAR Pharmaceutical</td><td>NDC 42023-179-05</td><td></td></tr><tr><td>Cefazolin</td><td>HIKMA Pharmaceutical</td><td>NDC 0143-9924-90</td><td></td></tr><tr><td>Ceramic Mixing Dish</td><td>Parkell</td><td>SKU: S387</td><td>For dental cement preparation</td></tr><tr><td>Cotton Tipped Applicators</td><td>ZORO</td><td>catlog #: G9531702</td><td></td></tr><tr><td>Catalyst</td><td>Parkell</td><td>S371</td><td>full name: &quot;C&quot; Universal TBB Catalyst</td></tr><tr><td>Dental cement powder</td><td>Parkell</td><td>S396</td><td>Radiopaque L-Powder for C&amp;amp;B Metabond</td></tr><tr><td>Dental drill</td><td>Foredom</td><td>H.MH-130</td><td></td></tr><tr><td>Dental drill controller</td><td>Foredom</td><td>HP4-310</td><td></td></tr><tr><td>Dexamethasone</td><td>Phoenix</td><td>NDC 57319-519-05</td><td></td></tr><tr><td>EF4 carbide bit</td><td>Microcopy</td><td>Lot# C150113</td><td>Head Dia/Lgth/mm 1.0/4.2</td></tr><tr><td>Ethonal</td><td>Fisher Scientific</td><td>04355223EA</td><td>75%</td></tr><tr><td>FG1/4 carbide bit</td><td>Microcopy</td><td>Lot# C150413</td><td>Head Dia/Lgth/mm 0.5/0.4</td></tr><tr><td>FG4 carbide bit</td><td>Microcopy</td><td>Lot# C150309</td><td>Head Dia/Lgth/mm 1.4/1.1</td></tr><tr><td>Headpost</td><td>N/A</td><td>N/A</td><td>Custom-manufactured</td></tr><tr><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></tr><tr><td>Heating blanket</td><td>CVS pharmacy</td><td>E12107</td><td>extra heating device and be used after surgery</td></tr><tr><td>Isoflurane</td><td>Pivetal</td><td>NDC 46066-755-03</td><td></td></tr><tr><td>Isoflurane induction chamber</td><td>Vetequip</td><td>89012-688</td><td>induction chamber for short</td></tr><tr><td>Isoflurane volatilizing machine</td><td>Vetequip</td><td>911103</td><td></td></tr><tr><td>Isoflurane volatilizing machine holder</td><td>Vetequip</td><td>901801</td><td></td></tr><tr><td>Leica surgical microscope</td><td>Leica</td><td>LEICA 10450243</td><td></td></tr><tr><td>Lubricant ophthalmic ointment</td><td>Picetal</td><td>NDC 46066-753-55</td><td></td></tr><tr><td>Marker pen</td><td>Delasco</td><td>SMP-BK</td><td></td></tr><tr><td>Meloxicam</td><td>Norbrook</td><td>NDC 55529-040-10</td><td></td></tr><tr><td>Microinjection pump and its controller</td><td>World Precision Instruments</td><td>micro4 and UMP3</td><td></td></tr><tr><td>Microliter syringe</td><td>Hamilton</td><td>Hamilton 80014</td><td>1701 RN, 10 &amp;mu;L gauge for syringe and 32 gauge for needle, 2 in, point style 3</td></tr><tr><td>Mosquito forceps</td><td>CAROLINA</td><td>Item #:625314</td><td>Stainless Steel, Curved, 5 in</td></tr><tr><td>Depilatory agent</td><td>McKesson Corporation</td><td>N/A</td><td>Nair Hair Aloe &amp;amp; Lanolin Hair Removal Lotion</td></tr><tr><td>Microscope 1</td><td>Nikon</td><td>SMZ745</td><td>Nikon microscope for cranial window preparation</td></tr><tr><td>Microscope 2</td><td>Zeiss</td><td>LSM 7 MP</td><td>two-photon microscope</td></tr><tr><td>Multiphoton laser</td><td>Coherent</td><td>Chameleon Ultra II, Model: MRU X1, VERDI 18W</td><td>laser for two-photon microscopy</td></tr><tr><td>Non-absorbable surgical suture</td><td>Harvard Apparatus</td><td>catlog# 59-6860</td><td>6-0, with round needle</td></tr><tr><td>Norland Optical Adhesive 81</td><td>Norland Products</td><td>NOA 81</td><td></td></tr><tr><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>&quot;B&quot; Quick Base For C&amp;amp;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 ...

intravital-imaging-fluorescent-protein-expression-mice-with-closed

Research

JoVE Journal

Neuroscience

1.1K Views.  University of Massachusetts Chan Medical School. Ons protocol biedt een manier om de neuronale reactie op mechanische stress in een levende muis te visualiseren, wat direct bewijs kan leveren van hoe traumatisch hersenletsel de hersenen beïnvloedt. Deze techniek kan het doeleiwit op dezelfde hersenlocatie in hetzelfde dier monitoren voor zowel de acute als de chronische fase na traumatisch hersenletsel. Het gen van belang en de viruspromotor kunnen dienovereenkomstig worden gewijzigd om andere eiwitten in de specifieke celtypen in de herse...

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Medicine

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.

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 ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

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

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

Delivery of a Traumatic Brain Injury to a Mouse Brain

Source: Zhong, J., et al. <a href="https://app.jove.com/v/64701">Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope.</a> J. Vis. Exp. (2023)
This video demonstrates the delivery of Traumatic Brain Injury (TBI) in mice. After exposing the skull, the TBI site is marked. The mouse is then positioned under the TBI device. The impact is delivered by lifting the metal rod and allowing it to drop freely onto the impactor, which is in contact with the skull.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

 Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature 

Source:<a style='text-decoration:none;' href='https://app.jove.com/v/53582/quantification-of-cerebral-vascular-architecture-using-two-photon-microscopy-in-a-mouse-model-of-hiv-induced-neuroinflammation'><span style='-webkit-text-decoration-skip:none;font-family:Roboto,sans-serif;font-size:10pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Nishimura, C.&#160;et al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2016).</a> &#160;This video demonstrates the in vivo visualization of cortical capillaries in the mouse brain using two-photon microscopy. It outlines the steps for injecting a fluorescent dye into an anesthetized mouse with a thin-skull cortical window, preparing the setup for imaging, and acquiring detailed images of brain microvascular structures for analysis.

Source:&#160;Nishimura, C.&#160;et al. Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced ...

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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