This work was supported by grants R01-HL87290, R01-HL87290-S1 and R01-AI082610 from the National Institutes of Health to LJMC.

1. Craniotomy
A craniotomy in 8-to-10-week old mice needs to be performed in advance as previously described1, except that a titanium bar is not placed in the head of the animal. The chronic cranial window is a stable preparation allowing examination of the pial microcirculation even months after being implanted. Usually, we perform our studies 2-3 weeks after the cranial window implantation.
2. Intravital microscopy
<ol>
 <li>Two-three weeks after craniotomy, body temperature is checked and then the mouse is lightly anesthetized with isoflurane (4% for induction, 1-2% for maintenance). Light anesthesia is used to prevent animal stress and discomfort during the experimental procedure.</li>
 <li>Animals are then placed in prone position on a stereotaxic frame and the head carefully secured using ear bars. The level is adjusted using the right and left levers. Core body temperature is maintained using a heating pad. Because mice with cerebral malaria develop hypothermia, the temperature settings need to be adjusted for each animal.</li>
 <li>The cover slip on the cranial window is gently cleaned with a cotton swab moistened with mineral oil and a panoramic picture of the vessels under the window is taken with the help of a stereomicroscope and a digital camera (Nikon Coolpix 995, Japan). The picture is printed, identified and dated and will be used as a map for the measurements of vessel diameter and red blood cell (RBC) velocities.</li>
 <li>The mouse is then transferred to an intravital microscope stage (customized Leica-McBain, San Diego, CA). A drop of water is placed on the cranial window taking advantage of the well formed by the dental acrylic, focus is adjusted. Measurements are carried out using a 20X (LUMPFL-WIR, numerical aperture 0.6, Olympus) water immersion objective. The same vessels are followed so that direct comparisons to their baseline levels could be performed allowing for more robust statistics for small sample populations. Images are captured using a digital low light high speed camera (Hamamatsu C9300-221, Japan), or low light analogue camera (COHU 4815, San Diego, CA) connected to a VCR tape (JVC, Japan), time stamped (MicroImage Video Systems, Boyertown, PA) and a color monitor (PELCO, Clovis, CA). The tape is properly identified before recording starts, and the time stamp allows identification of the events by documenting the timeframe during which such events were recoded.</li>
 <li>A first check of the vessels is made to evaluate the quality of the preparation and if the blood is flowing in all vessels. Then, selection of the vessels to be measured is made and it should include venules and arterioles of different diameters (in our mouse pial preparations, most vessels range between 20 and 80&mu;m) and cover different locations within the area exposed by the window. Arterioles and venules can be easily differentiated by checking the direction of the blood flow in the ramifications of the vessel (whether it distributes or collects blood). In our studies, measurements are made in 12 vessels. Precise location of each spot to be measured is annotated in the picture of the pial vasculature.</li>
 <li>For each spot, the vessel diameter is measured using an image shear device (Image Shear, Vista Electronics, San Diego, CA)2. Once the spot is selected, the image of the vessel is aligned in vertical position and the image is sheared until the opposed extremes are aligned and the reading is documented. This value can be translated into micrometers by previous calibration for each specific magnification using a Reticle Calibration Stage Micrometer (Edmund Optics Inc., Barrington, NJ).</li>
 <li>Each spot is recorded during at least 30 seconds for erythrocyte tracking. For tracking, a sample of blood is collected, and erythrocytes are fluorescently labeled with the carbocyanine dye Dil (Molecular Probes, Carlsbad, CA) and infused through the tail vein to obtain an in vivo concentration of ~0.4-0.5%3. In animals infected with Plasmodium berghei ANKA (PbA) expressing the green fluorescent protein (PbA-GFP, a donation from the Malaria Research and Reference Reagent Resource Center MR4, Manassas, VA; deposited by CJ Janse and AP Waters), no infusion of labeled cell is necessary. The video images are recorded at 150 frames per second. This rate is set to obtain one to six images of a cell on one video frame for determination of velocities up to 8 mm/s. The video images are digitalized with a personal computer using Adobe Premier 4.0 software, and x-y coordinate data for each cell image are obtained using SigmaScan Pro 4.0 software (SPSS Chicago, IL)4. Cell positions are determined manually rather than by image analysis, given that the eye of a trained observer was shown to give a good estimation of the location of the center of a cell, which in general corresponds to the location of maximal fluorescence observed for most cell orientations. Multiple determinations of position and velocity are made for each cell and averaged to obtain mean RBC velocity.</li>
 <li>When RBC velocity measurements are performed offline, the total observation time for each mouse does not exceed 10-15 minutes minimizing the cardiodepressive effects of anesthesia.</li>
 <li>Once vessel diameter and RBC velocity measurements are available, calculation of the blood flow in each vessel can be made by using the formula: Q = V x &pi;(D/2)2, where Q = blood flow, V = RBC velocity and D = vessel diameter.</li>
 <li>These procedures can be repeated over time. For instance, in the mouse model of cerebral malaria, we perform daily measurements starting on day 4 of infection. The values obtained every day for each mouse are normalized in relation to day 0 (pre-infection), which is considered to be 100%.</li>
 <li>In addition to vessel diameter and blood flow measurements, additional evaluations can be performed, for instance improved assessment of perfusion and the analysis of leukocyte and/or platelet rolling and adherence in pial vessels. These measurements are performed with the help of fluorescent-labeled markers. In the case of malaria, we can also detect circulating or adherent parasites by means of using the PbA strain that expresses GFP.</li>
 <li>For perfusion assessment we use a solution of FITC-labeled albumin (Sigma, St Louis, MO 50&mu;g/mouse), and to quantify leukocyte adherence to pial vessels we use antibodies against the pan-leukocyte marker CD45 labeled with Texas Red (TxR) (Invitrogen, Carlsbad, CA 4&mu;g/mouse). For this, 25&mu;L of the Albumin-FITC (2mg/mL) and 20&mu;L of the anti-CD45-PE antibodies (200&mu;g/mL) are mixed and injected in the pre-warmed tail vein. The mouse can then be imaged immediately, however if leakage measurements will take albumin-FITC is allowed to circulate for 15 minutes. Green fluorescence (518nm) emitted by albumin-FITC and GFP (PbA-GFP pRBC) is captured using and ALPHA Vivid: XF100-2 (Omega Optical, Brattleboro, VT), and anti-CD45-TxR fluorescence (615nm) is exited and captured with a Vivid Standard: XF42.</li>
 <li>The fluorescent-labeled albumin allows improved visualization of the vascular network, including penetrating vessels, and it is particularly useful in disease states such as cerebral malaria to check for non-perfused or under-perfused vessels. It can also be used to measure vascular leakage5.</li>
 <li>The fluorescent-labeled anti-CD45 antibodies make it easy to identify and quantify leukocyte rolling and adhesion to pial vessels. To avoid bias in quantification, we quantify rolling and adhesion in the same spots pre-defined to measure blood flow. Quantification of leukocyte adhesion is made by counting the number of leukocytes in a 100&mu;m-vessel length. Rolling is quantified by counting the number of leukocytes travelling at a velocity significantly slower than blood velocity in the same 100&mu;m length, during 30 seconds.</li>
</ol>
3. Representative results
<img src="/files/ftp_upload/2184/2184fig1.jpg" alt="Figure 1" /> 
Figure 1. (Step 2.6) Pictures of the mouse pial vascular network accessible through the cranial window.
<img src="/files/ftp_upload/2184/2184fig2.jpg" alt="Figure 2" /> 
Figure 2. (Steps 3.1-3.5) Schematic display of the set up for intravital microscopy of the mouse pial microcirculation. 1: intravital microscope; 2: 20X water immersion objective; 3: light source; 4a: digital low-light high-speed camera; 4b: analog camera; 5: mouse in the stereotaxic frame; 6: computer monitor; 7: analog shearer monitor showing how the image shearing (arrow) is done to measure vessel diameter.
<img src="/files/ftp_upload/2184/2184fig3.jpg" alt="Figure 3" /> 
Figure 3. (Step 3.6-3.8) Microvascular red blood cell velocity measurements by cell tracking from high speed fluorescence video recordings. Pictures A to F are sequence images of one pial vessel, showing a single moving RBC crossing the microscopic field delimited by the camera. Manual determination of frame by frame positions of 15 or more cells crossing the field, with its pre-calibrated distance, allows calculation of mean RBC velocity in each vessel with the help of an Excel spreadsheet.
<img src="/files/ftp_upload/2184/2184fig4.jpg" alt="Figure 4" /> 
Figure 4. (Step 3.9) Data of a representative experiment showing the changes in pial blood flow over time in Plasmodium berghei ANKA (PbA) infected mice (n = 5) and in uninfected control mice (n = 5). Whereas in control mice the pial blood flow is relatively stable over time, PbA-infected mice show a marked decrease in blood flow at the time of cerebral malaria development (day 6). Data are the mean &plusmn; SEM.
<img src="/files/ftp_upload/2184/2184fig5.jpg" alt="Figure 5" /> 
Figure 5. (Step 4.3) A large number of leukocytes adherent to pial vessels of a mouse infected with Plasmodium berghei ANKA, as revealed by staining with anti-CD45-Texas Red fluorescent antibodies.

<ol>
	<li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J. Vis. Exp. (15), (2008).</li><li>Intaglietta, M., Tompkins, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+measurements+by+video+image+shearing+and+splitting.">Microvascular measurements by video image shearing and splitting.</a> Microvasc. Res. 5, 309-312 (1973).</li><li>Briceno, J. C., Cabrales, P., Tsai, A. G., Intaglietta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+displacement+of+red+blood+cells+during+hemodilution+and+the+effect+on+arteriolar+oxygen+profile.">Radial displacement of red blood cells during hemodilution and the effect on arteriolar oxygen profile.</a> Am. J. Physiol. Heart Circ. Physiol. 286, 1223-1228 (2004).</li><li>Bishop, J. J., Nance, P. R., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+erythrocyte+aggregate+size+and+flow+rate+in+skeletal+muscle+venules.">Relationship between erythrocyte aggregate size and flow rate in skeletal muscle venules.</a> Am. J. Physiol. Heart Circ. Physiol. 286, H113-120 (2004).</li><li>Baldwin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+hemoglobins+produce+venular+interendothelial+gaps+and+albumin+leakage+in+the+rat+mesentery.">Modified hemoglobins produce venular interendothelial gaps and albumin leakage in the rat mesentery.</a> Am. J. Physiol. Heart Circ. Physiol. 277, H650-H659 (1999).</li><li>Cabrales, P., Zanini, G. M., Meays, D., Frangos, J. A., Carvalho, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cerebral+malaria+is+associated+with+a+vasospasm-like+microcirculatory+dysfunction,+and+survival+upon+rescue+treatment+is+markedly+increased+by+nimodipine.">Murine cerebral malaria is associated with a vasospasm-like microcirculatory dysfunction, and survival upon rescue treatment is markedly increased by nimodipine.</a> Am. J. Pathol. 176 (3), 1306-1315 (2010).</li></ol>

No conflicts of interest declared.

The intravital microscopy method described here provides a unique and powerful tool for detailed observation of the pial microcirculation in the mouse. It allows singling out individual arterioles and venules and measuring changes of a number of parameters such as vessel diameters, RBC velocities, blood flow, adherence and rolling of leukocytes, platelets and other blood elements, vascular leakage, tissue pH and pO2 and potentially many other applications. The in vivo vascular response can be promptly evaluated ...

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<td>Isoflurane</td>
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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, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Watch this Scientific Journal Video about Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window at JoVE.com

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

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

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

intravital-microscopy-mouse-brain-microcirculation-using-closed

Research

JoVE Journal

Neuroscience

17.3K ビュー.  University of California, San Diego. 軟膜微小循環の時間的空間的血行動態および炎症性イベントに追従するように生体顕微鏡。

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

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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 using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

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

Piezo High Accuracy Surgical Osteal Removal (PHASOR): A Technique for Improved Cranial Window Surgery in Mice

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated thinning of the skull. Cranial window surgery is typically performed with a high speed rotary drill, and many investigators find it challenging to prevent the drill from damaging the delicate dura and blood vessels. Extensive training and practice is required to remove the bone without damage to underlying tissue and thus cranial window surgery can be difficult, time consuming, and produce tissue damage. Piezoelectric surgery, which is extensively used for maxillofacial and dental surgery, utilizes ultrasonic vibrations to remove bone without damaging soft tissues. We have developed a method applying piezoelectric surgery to improve cranial window surgery in mice in preparation for multiphoton imaging. Comparisons within our lab find that the method requires less surgery time and has a lower average rate of complications due to dural bleeding than cranial window surgery with a rotary drill.

Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

Piezoelectric surgery has led to improvements in human maxillofacial and dental surgery. We have developed a protocol to optimize piezoelectric surgery for cranial window surgery in mice.

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...