Name: assessment of thermal damage from robot-drilled craniotomy for cranial window surgery in mice
Uploaded: 2022-11-11T13:20:00
Description: Watch this Scientific Journal Video about Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice at JoVE.com

This study was supported in part by Merit Review Awards GRANT12418820 (Capadona) and GRANTI01RX003420 (Shoffstall/Capadona), and Research Career Scientist Award # GRANT12635707 (Capadona) from the United States (US) Department of Veterans Affairs Rehabilitation Research and Development Service. Additionally, this work was also supported in part by the National Institute of Health, the National Institute of Neurological Disorders and Stroke GRANT12635723 (Capadona), and the National Institute for Biomedical Imaging and Bioengineering, T32EB004314, (Capadona/Kirsch). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. GRANT12635723. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.

All procedures and animal care practices were reviewed, approved by, and performed in accordance with the Louis Stokes Cleveland Department of Veterans Affairs Medical Center Institutional Animal Care and Use Committee.
1. Surgical robot hardware setup
<ol>
	<li>Before surgery, follow the surgical robot (see Table of Materials) manual and guide to set up the hardware and software. Perform frame calibration as detailed in the manual. If the drill or frame are...

<ol>
	<li>Kilic, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+cranial+windows+for+long+term+multimodal+neurovascular+imaging+in+mice.">Chronic cranial windows for long term multimodal neurovascular imaging in mice.</a> Frontiers in Physiology. 11, 612678 (2020).</li><li>Goldey, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removable+cranial+windows+for+long-term+imaging+in+awake+mice.">Removable cranial windows for long-term imaging in awake mice.</a> Nature Protocols. 9 (11), 2515-2538 (2014).</li><li>Augustinaite, S., Kuhn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+optical+signal+imaging+and+targeted+injections+through+a+chronic+cranial+window+of+a+head-fixed+mouse.">Intrinsic optical signal imaging and targeted injections through a chronic cranial window of a head-fixed mouse.</a> STAR Protocols. 2 (3), 100779 (2021).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+skull-removed+chronic+cranial+window+for+ultrasound+and+photoacoustic+imaging+of+the+rodent+brain.">A skull-removed chronic cranial window for ultrasound and photoacoustic imaging of the rodent brain.</a> Frontiers in Neuroscience. 15, 673740 (2021).</li><li>Wang, Y., Xi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+cranial+window+for+photoacoustic+imaging:+a+mini+review.">Chronic cranial window for photoacoustic imaging: a mini review.</a> Visual Computing for Industry, Biomedicine, and Art. 4 (1), 15 (2021).</li><li>Augustinaite, S., Kuhn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+cranial+window+for+imaging+cortical+activity+in+head-fixed+mice.">Chronic cranial window for imaging cortical activity in head-fixed mice.</a> STAR Protocols. 1 (3), 100194 (2020).</li><li>Kunori, N., Takashima, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+cranial+window+using+a+collagen+membrane+for+chronic+voltage-sensitive+dye+imaging.">An implantable cranial window using a collagen membrane for chronic voltage-sensitive dye imaging.</a> Micromachines. 10 (11), 789 (2019).</li><li>Beckmann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+deep-brain+imaging+in+mouse+using+visible-light+optical+coherence+tomography+through+chronic+microprism+cranial+window.">Longitudinal deep-brain imaging in mouse using visible-light optical coherence tomography through chronic microprism cranial window.</a> Biomedical Optics Express. 10 (10), 5235-5250 (2019).</li><li>Heo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+soft,+transparent,+freely+accessible+cranial+window+for+chronic+imaging+and+electrophysiology.">A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology.</a> Scientific Reports. 6, 27818 (2016).</li><li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neocortical+neurons+through+a+chronic+cranial+window.">Imaging neocortical neurons through a chronic cranial window.</a> Cold Spring Harbor Protocols. 2012 (6), 694-701 (2012).</li><li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li><li>Sundaram, G. 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The use of EB dye and imaging is straightforward, quick, and useful for evaluating vascular damage in the brain for new methods and techniques. Whether using a surgical robot or confirming methods currently done in the lab, it is important to validate surgical methods to isolate the effects of experimental treatments vs. surgical impact and improve animal welfare. A thermocouple setup is also useful in evaluating drilling methods to ensure no heating occurs. Increases in temperature due to bone drilling have been known t...

Cranial windows have become ubiquitously used throughout the fields of neuroscience, neural engineering, and biology to allow for direct visualization and imaging of the cortex in living animals1,2,3,4,5,6,7,8,9,10,11. The powerful combination of transgenic mice and multiphoton imaging has provided extremely valuable insights into circuit activity and other biological insights in the in vivo brain12,13,14,15,16,17,18. Miniature microscopes mounted on the skull have further extended these capabilities to enable recordings in awake, freely moving animals19. The process of creating a cranial window requires power-drilling to thin or completely remove the cranial bone to produce large enough craniotomies to secure a transparent piece of glass over the cortex20. Polydimethylsiloxane (PDMS) and other polymers have also been tested as cranial window materials9,21. Ultimately, the ideal cranial window is one that does not alter or interfere with normal endogenous activity underneath. However, it is commonly accepted that cranial window drilling aggravates underlying tissue, leading to damage to the brain, disruption of the environment, and effecting meninges to the point of occluding multiphoton imaging depth22. The resulting neuroinflammation has a wide array of effects ranging from permeability of the blood-brain barrier (BBB), to activation and recruitment of glial cells around the implant site23. Therefore, characterizing safer and more reproducible cranial window drilling methods is crucial for consistent imaging quality and reducing confounding factors.
While care is taken to minimize trauma to the underlying tissue, the act of drilling the bone has the potential to cause both thermal and mechanical perturbations to the brain24,25. Mechanical trauma from accidental drill penetration into the dura may further induce varying degrees of cortical injury24. In a study by Shoffstall et al.25, the heat from bone-drilling resulted in an increased BBB permeability, as indicated by the presence of Evans Blue (EB) dye in the brain parenchyma25. EB dye, injected intravenously, binds to circulating albumin in the bloodstream and therefore does not normally cross a healthy BBB in appreciable concentrations. As a result, EB dye is commonly used as a sensitive marker of BBB permeability26,27. While their study did not directly measure the impact of the BBB permeability on subsequent biological sequelae under study, prior studies have correlated BBB permeability to an increased neuroinflammatory response to chronically implanted microelectrodes and alterations in motor function28.
Depending on the goals of the study, the magnitude of thermal and mechanical damage may contribute a source of experimental error, negatively affecting the rigor and reproducibility of the study. There are dozens of cited methods for producing cranial windows, each using different drilling equipment, speeds, techniques, and users1,2,3,4,5,6,7,8,9,10,11. Shoffstall et al.25 reported that the observed variation in the heating outcomes was attributed to variability in the drill&#39;s applied force, feed rate, and angle of application, among other aspects that cannot be controlled for when drilling by hand25. There is a belief that automated drilling systems and other stereotaxic equipment can improve reproducibility and outcome consistency, but published method studies have not rigorously evaluated temperature or BBB permeability as one of the outcomes. Therefore, there is a need for more reproducible and consistently applied methods to produce cranial windows, as well as methods rigorously applied to assess the impact of cranial window drilling on underlying neural tissue.
The focus of this study is to determine and develop consistent and safe drilling methods for cranial windows. The size of the craniotomy for cranial window installation is significantly larger than standard craniotomies for brain implanted microelectrodes. Such craniotomies cannot be completed with a single burr hole when using standard equipment, thereby introducing more inter-surgeon technique variability when performed by hand20. Surgical drilling robots have been introduced to the field, but have not been widely adopted1,6,29. Automation of drilling offers control over variables contributing to observed trial-to-trial variation, suggesting that use of the equipment can reduce inter- and intra-surgeon effects. This is of particular interest given the added difficulty of the larger craniotomy needed for cranial window placement. While one could assume there to be clear benefits to the control provided by automating the drilling, there has been little assessment of the implementation of these equipment. Although visible lesions have not been observed5, the higher sensitivity test using EB is desired.
Here, BBB permeability is measured using a commercially available surgical drilling robot with corresponding software, which allows for programming of stereotaxic coordinates, craniotomy planning/mapping, and a selection of drilling styles (&#34;point-by-point&#34; vs &#34;horizontal&#34;), referring to the routed path of the drill bit. Initially, eight &#34;seed&#34; points are drilled (Figure 1A), outlining the cranial window. From here, the space in between the seeds is cut out using either the &#34;point-by-point&#34; or &#34;horizontal&#34; drill method. &#34;Point-by-point&#34; performs vertical pilot hole cuts (similar to a CNC drill press), while &#34;horizontal&#34; performs horizontal cuts along the circumference of the cranial window that outline the hole (similar to a CNC router). The result for both methods are a piece of skull that can be removed to reveal the cranial window. To isolate damage from drilling, the cranial window is not physically removed, so as to avoid any additional damage. A combination of EB dye coupled with fluorescent imaging is used to measure BBB permeability after performing craniotomies in mice, and an inserted thermocouple is used to directly measure temperature of the brain surface during drilling (Figure 1B,C). Previous observations indicated that pulsed drilling on/off with 2 s intervals was sufficient to mitigate drill heating25, and therefore is incorporated into the experimental approach for the surgical robot.
The intent of the presented work is to demonstrate methods of assessing thermal damage from craniotomy drilling. While the methods are presented in the context of automated drilling, such methods can be applied to manual drilling schemes as well. These methods can be used to validate the use of equipment and/or drilling schemes before adopting as a standard procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64188/64188fig01.jpg" /> 
Figure 1: Experimental pipeline schematic. Schematic demonstrating the process animals underwent for EB quantification post-cranial window procedure. (A) Schematic setup of the mouse with the stereotaxic frame and surgical robot drill. An example cranial window is shown over the motor cortex with seed points (green) and edge points (blue). (B) The perfusion setup includes injecting 1x Phosphate Buffered Saline (PBS) throughout the animal to remove any blood, followed by extraction of the brain. (C) The brain is then put into the EB fluorescent imaging system chamber to conduct fluorescent imaging on the Evans Blue dye. <a href="https://www.jove.com/files/ftp_upload/64188/64188fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Phosphate Buffered Saline 
			Type: Reagent</td>
			<td>VWR</td>
			<td>MRGF-6235</td>
			<td>For Evans Blue dilution</td>
		</tr>
		<tr>
			<td>Aura Software 
			Type: Tool</td>
			<td>Spectral Instruments Imaging</td>
			<td></td>
			<td>Open access imaging processing software for Lumina imaging sytems</td>
		</tr>
		<tr>
			<td>Buprenorphine 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbide Drill Bit, 0.6mm (Robot Drill) 
			Type: Tool</td>
			<td>Stoelting</td>
			<td>58640-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cefazolin 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Evans Blue Dye 
			Type: Reagent</td>
			<td>Millipore Sigma</td>
			<td>E2129</td>
			<td>Reconstituted in 1x phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>Isoflurane 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II 
			Type: Tool</td>
			<td>Perkin Elmer</td>
			<td>CLS136334</td>
			<td>IVIS Lumina III currently in place of Lumina II on the market</td>
		</tr>
		<tr>
			<td>Jenco Linearizing Thermometer 
			Type: Tool</td>
			<td>Jenco</td>
			<td>765JF</td>
			<td>For Thermocouple setup</td>
		</tr>
		<tr>
			<td>Ketamine 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LivingImage 
			Type: Tool</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Software for IVIS Lumina III</td>
		</tr>
		<tr>
			<td>Marcaine 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurostar Software 
			Type: Tool</td>
			<td>Stoelting</td>
			<td></td>
			<td>Comes with surgical robot purchase</td>
		</tr>
		<tr>
			<td>Physiosuite with MouseSTAT&#174; Pulse Oximeter &#38; Heart Rate Monitor 
			Type: Tool</td>
			<td>Kent Scientific</td>
			<td>PS-03</td>
			<td>Used to monitor vitals</td>
		</tr>
		<tr>
			<td>PrismPlus mice 
			Type: Animal</td>
			<td>Jackson Labortory</td>
			<td>031478, RRID:IMSR_JAX:031478, Male, ~8 months old</td>
			<td>Animals used for the study</td>
		</tr>
		<tr>
			<td>Stoelting Drill and Injection Robot for Motorized Stereotaxic Instruments 
			Type: Tool</td>
			<td>Stoelting</td>
			<td>58640</td>
			<td>Main robotic drill with stereotaxic frame</td>
		</tr>
		<tr>
			<td>Thermocouple 
			Type: Tool</td>
			<td>TC Direct</td>
			<td>206-557</td>
			<td>For Thermocouple setup</td>
		</tr>
		<tr>
			<td>USB-6008 Multifunction I/O DAQ 
			Type: Tool</td>
			<td>National Instruments</td>
			<td>USB-6008</td>
			<td>For Thermocouple setup</td>
		</tr>
		<tr>
			<td>Xylazine 
			Type: Drug</td>
			<td>Sourced from Animal Facility</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of thermal damage from robot-drilled craniotomy for cranial window surgery in mice

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a &#34;2 s on, 2 s off&#34; drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 &#176;C &#177; 1.35 &#176;C) group compared to the horizontal (16.66 &#176;C &#177; 2.08 &#176;C) and point-by-point (18.69 &#176;C &#177; 1.75 &#176;C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field.

Thermal evaluation 
Potential for thermal damage was evaluated by measuring the change in temperature from baseline due to drilling using horizontal (Figure 2A), point-by-point (Figure 2B), and pulsed point-by-point (Figure 2C) methods. Figure 2D displays the experimental setup for obtaining thermal data. A sample size of N = 4 cranial windows was used for thermal evaluation. Horiz...

Watch this Scientific Journal Video about Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice at JoVE.com

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice

Cranial windows have become a ubiquitously implemented surgical technique to allow for intravital imaging in transgenic mice. This protocol describes the use of a surgical robot that performs semi-automated bone drilling of cranial windows and can help reduce surgeon-to-surgeon variability and partially mitigate thermal blood-brain barrier damage.

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, ...

This protocol provides a method for evaluating vascular damage as a result of cranial window drilling. A surgical robot offers improved consistencies across procedures while reducing the amount of training to learn the technique. During this procedure, we utilized cranial injections which can be very challenging.We recommend extensive training for those new to the technique. To begin, set up the system as described in the manuscript and perform frame calibration following the surgical robot manual. Navigate to the surgical software and create a new project by selecting start with a clean project.Then set the subject as mouse at the top to designate the drilling coordinate atlas to be used. Next, select start new project, and click on planning in the bottom left corner to navigate to the drilling coordinate planning screen. Create the drilling scheme for the cranial window technique to be performed by clicking anywhere on the stereotaxic atlas.Use bregma as the reference and input the coordinates for the motor cortex as described in the manuscript. Press Enter on the keyboard to update the selected coordinates. Click store target to save these coordinates and input an appropriate name.Then click on the move button in the bottom left to navigate back to the main drilling screen. Click on tools, then project, and save as option to reuse this template project for later projects, as it will automatically retain the drilling coordinates. First, administer subcutaneous injections of antibiotics, cefazolin, analgesic, carprofen, and buprenorphine to the anesthetized mouse.Next, place the mouse on a heating pad and maintain anesthesia with 0.5%to 2%isoflurane via inhalation through a nose cone. Mount the animal on the surgical robot stereotaxic frame using supplied ear bars and routinely assess vitals and anesthetic depth via toe pinch. Then scrub the surgical area with chlorhexidine gluconate and 70%isopropanol for sterilization.And place a sterile plastic wrap over the mouse and stereotactic frame to maintain sterility during surgery. Using a scalpel blade, perform a one-inch incision on the midline of the skull, starting at the back of the eyes, dry out and clean the skull using 3%hydrogen peroxide with cotton tipped applicators. To prepare the tail for easy injection, wipe down with alcohol.Using the thumb and forefinger, bend the tail to expose the tail vein on top of the bend of the tail. Insert the syringe parallel with the vein, and slowly inject the volume of Evans blue. Once injected, wait for five minutes to allow Evans blue to circulate.A successful injection is immediately verified as the mouse's extremities and surgical window turn blue. Once the skull is prepared for drilling, navigate back to the surgical software and click on tools. Then select project, followed by new, and select a template project from the dropdown menu to choose a template project.Select same protocol elements. Click on planning, then select drill parameters to carry over to this new project and click start new project. Next, correct the drill and frame to account for the tilt and scaling of the mouse skull of the current animal by clicking tools, and select correct for tilt and scaling to open the corrections screen.At the top of the screen, ensure that the drill is active by clicking on the light red drill button After correction has been performed, come out of the correction window by clicking close in the bottom middle of the screen and navigate to the drilling screen by clicking tools and then selecting drill to begin the drilling procedure. Confirm the drill site by selecting the craniotomy shape window at the top of the screen, followed by viewing the drilling target details in the dropdown below. To carry out manual pulse drilling, turn off the auto-stop feature by unchecking the checkbox next to the auto-stop option in the drill menu, which allow for controlling when the drill is off for the pulsing.In the drill menu, select 100 micrometers as the drill depth advancement. Open up the drill site menu and navigate to the first drill site. Use the advance button or manual controls to lower the drill until it touches the skull and press set surface once the drill bit reaches the skull.Once ready, click advance to commence drilling. And after the drill has advanced 100 micrometers, press Escape twice to stop the drill. After two seconds, repeat this cycle for the depth of the skull.For each seed and edge point during drilling, ensure to set dura using the button in the drill menu once the dura has been reached. Measure the changes in temperature of the skull and brain using a thermocouple in combination with the three different drilling schemes connected to a data acquisition system, that allows for the measurement to be read into MATLAB. Mount a cadaver mouse onto the stereotaxic frame and robotic drill set up.Manually drill a small hole approximately two millimeter away from where the cranial window will be made into the side of the skull, which allows for thermocouple to be slid into position under where drilling of the cranial window occurs. Begin the drilling process for each of the three schemes as demonstrated previously, resulting in spikes due to temperature change, indicating heating occurring near the brain as the drill goes through the skull. Potential for thermal damage was evaluated by measuring the change in temperature from baseline due to drilling using horizontal, point by point, and pulsed point by point methods.Both the horizontal and point by point drilling schemes show non-significant differences in thermal changes. However, changing to a pulsed point by point method resulted in significantly less heating of the brain than both horizontal and point by point drilling. The horizontal edge drilling took 300 seconds, while point by point edge drilling took 200 seconds.The pulse method took the longest with seed and edge drilling, taking approximately 500 seconds each. To monitor the relationship between the drilling scheme and vascular damage, Evans blue fluorescent imaging was performed. The drilling via horizontal and point by point cutting was seen to be damaging to the vasculature in the brain.In comparison to the control groups, the pulsed point by point method had less localized damage at the seed and edge point, but still had visible Evans blue presence within the cranial window. When manually drilling it is crucial to check the craniotomy after advancing the drill to ensure the dura is not breached. In addition to cranial windows, a surgical robot can be used for standard craniotomies to improve surgical outcomes.

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Research

JoVE Journal

Neuroscience

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.

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, ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

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 ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs&#39; sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode.
Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs&#39; behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical ...

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...