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.

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The authors do not have any conflicts of interest to report. The contents do not represent the views of the U.S. Department of Veterans Affairs, the National Institutes of Health, or the United States Government.

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 to cause tissue damage, and even an increase of 5 &#176;C is enough to cause large vascular damage in the brain32,33,34,35,36. It is recommended to utilize the methods detailed here to improve laboratory and surgical techniques.
While useful for evaluation, thermocouple evaluation has a few limitations. Thermocouple data is acquired using cadaver mice due to the necessity for drilling a hole in the side of the skull to fit the thermocouple into the brain and possible damage to the brain as a result. As a result, temperature difference is measured across drilling instead of the physiological temperature of the animal. Additionally, there may be physiological temperature regulation functions that are not included in the analysis.
Several steps during the protocol are critical to ensure proper drilling. First, skull alignment, if done incorrectly, will lead to poor drilling accuracy along with damage to the brain (if auto-stop does not work). Ensure mounting of the animal is as straight as possible before tilt correction to avoid this issue. Correct any tilt offsets by following the tilt correction process slowly and surely. In a few cases during this study the tilt was off, leading to the drill system believing that it was drilling into the skull even though the drill bit had not even contacted the skull. Largely, this is an issue for accurately recording skull thickness, and if egregious enough, may cause inaccuracy in the drilling coordinates. Additionally, the auto-stop feature was inconsistent and must be used with care. Do not rely solely on the auto-stop feature to prevent damage to the brain. Always check the drilling hole to ensure over-drilling does not occur.
Regardless of auto-stop, there are a few optimizations that can be performed for the point-by-point and horizontal drilling methods. To ensure no incidental damage to the brain, point-by-point uses a drill offset during edge cutting, but the user must predetermine this setting beforehand through testing. A linear interpolation method could be incorporated with the shallowest seed point as the basis, so that at thicker seeds around the skull, damage will not occur in the brain. If needed, the user can always return to a thicker area of the skull and drill deeper. The horizontal cutting step uses a depth-cutting interval (default of 100 &#956;m) for each rotation around the edge points. This can also be determined based on skull thickness to avoid drilling too deep and damaging the brain.
Transgenic mice are a powerful experimental model for intravital multiphoton imaging. While the use of a surgical robot for cranial windows in transgenic mice is highlighted in this study, it is important to note the use of a surgical robot in other cranial surgeries. The ability to control and standardize drilling offers benefits to craniotomies in larger animal studies across the field. Even though some mechanical damage was visually observed, this is most likely due to the extremely small separation between the brain and skull in mice. Larger animals, such as rats, have more subarachnoid space and thicker dura, lending to less risk of mechanical damage because of robotic drilling25. In combination with the reduction in thermal damage shown using the pulsed method here, the surgical robot has potential to significantly reduce damage incurred from drilling across various animal models.
Overall, the pulsed point-by-point method showed the least amount of damage, whether it be as a result of less heating or less mechanical damage to the brain. Drilling by hand may offer a more controlled method for avoiding damage, but it is important to highlight the benefits of a surgical robot. A robot needs less training, can help reduce surgeon-to-surgeon variability, and once optimized fully, can lend to a more standardized procedure across labs. Additionally, the learning curve for a surgical robot is much lower than that of surgery by hand. This not only reduces the time needed to learn the technique, but also reduces the number of animals that are used for training purposes. The prevalence of cranial window drilling has increased with the innovation of multiphoton imaging through the brain as seen in published papers20,37. Employing characterizing methods such as thermocouples and EB dye imaging will help optimize the drilling technique, while the use of robots will make difficult surgeries more accessible and widespread.

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><tbody><tr><td>1x Phosphate Buffered Saline&lt;br/&gt; Type: Reagent</td><td>VWR</td><td>MRGF-6235</td><td>For Evans Blue dilution</td></tr><tr><td>Aura Software&lt;br/&gt; 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&lt;br/&gt; Type: Drug</td><td>Sourced from Animal Facility</td><td></td><td></td></tr><tr><td>Carbide Drill Bit, 0.6mm (Robot Drill)&lt;br/&gt; Type: Tool</td><td>Stoelting</td><td>58640-1</td><td></td></tr><tr><td>Carprofen&lt;br/&gt; Type: Drug</td><td>Sourced from Animal Facility</td><td></td><td></td></tr><tr><td>Cefazolin&lt;br/&gt; Type: Drug</td><td>Sourced from Animal Facility</td><td></td><td></td></tr><tr><td>Evans Blue Dye&lt;br/&gt; Type: Reagent</td><td>Millipore Sigma</td><td>E2129</td><td>Reconstituted in 1x phosphate-buffered saline</td></tr><tr><td>Isoflurane&lt;br/&gt; Type: Drug</td><td>Sourced from Animal Facility</td><td></td><td></td></tr><tr><td>IVIS Lumina II&lt;br/&gt; 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&lt;br/&gt; Type: Tool</td><td>Jenco</td><td>765JF</td><td>For Thermocouple setup</td></tr><tr><td>Ketamine&lt;br/&gt; Type: Drug</td><td>Sourced from Animal Facility</td><td></td><td></td></tr><tr><td>LivingImage&lt;br/&gt; Type: Tool</td><td>Perkin Elmer</td><td></td><td>Software for IVIS Lumina III</td></tr><tr><td>Marcaine&lt;br/&gt; Type: Drug</td><td>Sourced from Animal Facility</td><td></td><td></td></tr><tr><td>Neurostar Software&lt;br/&gt; Type: Tool</td><td>Stoelting</td><td></td><td>Comes with surgical robot purchase</td></tr><tr><td>Physiosuite with MouseSTAT&amp;reg; Pulse Oximeter &amp;amp; Heart Rate Monitor&lt;br/&gt; Type: Tool</td><td>Kent Scientific</td><td>PS-03</td><td>Used to monitor vitals</td></tr><tr><td>PrismPlus mice&lt;br/&gt; 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&lt;br/&gt; Type: Tool</td><td>Stoelting</td><td>58640</td><td>Main robotic drill with stereotaxic frame</td></tr><tr><td>Thermocouple&lt;br/&gt; 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&lt;br/&gt; Type: Tool</td><td>National Instruments</td><td>USB-6008</td><td>For Thermocouple setup</td></tr><tr><td>Xylazine&lt;br/&gt; 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. Horizontal and point-by-point use the same seed drilling schematic but vary on how the edge points are cut. Pulsed point-by-point employs a pulsed method for both seed and edge drilling portions. For the horizontal method, seed drilling showed a maximum temperature change of 16.66 &#176;C &#177; 2.08 &#176;C, while edge drilling showed 9.08 &#176;C &#177; 0.37 &#176;C. For the point-by-point method, seed drilling showed a maximum temperature change of 18.69 &#176;C &#177; 1.75 &#176;C, while edge drilling showed 8.53 &#176;C &#177; 0.36 &#176;C. For the pulsed point-by-point method, seed drilling showed a maximum temperature change of 6.90 &#176;C &#177; 1.35 &#176;C, while edge drilling showed 4.10 &#176;C &#177; 0.51 &#176;C. Both the horizontal and point-by-point drilling schemes show non-significant differences for thermal changes. However, changing to a pulsed point-by-point method resulted in significantly less heating (p &#60; 0.05) of the brain than both horizontal and point-by-point drilling (Figure 2E,F). The duration of surgery was also recorded, as that may have an impact on animal survivability for live surgeries. For both automated methods, the seed drilling took 360 s on average. Horizontal edge drilling took 300 s, while point-by-point edge drilling took 200 s. The pulsed method took the longest, with seed and edge drilling taking approximately 500 s each. Nevertheless, these differences are not large enough to warrant any consideration as surgeries can commonly last over 2-3 h.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64188/64188fig02.jpg" /> 
Figure 2: Thermal evaluation. Potential for thermal damage was evaluated based on maximum temperature changes in the brain as a result of drilling methods. (A) Horizontal drilling and (B) point-by-point drilling generated similar amounts of heat, whereas (C) a pulsed 2 s on, 2 s off point-by-point method showed minimal heating. (E) Seed drilling and (F) edge drilling resulted in significantly less thermal change in the pulsed point-by-point method of drilling (p &#60; 0.05, N = 4 per condition). (D) The thermocouple is placed underneath the skull of the mouse cadaver where the drilling is done. Data is acquired through a DAQ and fed into a computer for analysis. <a href="https://www.jove.com/files/ftp_upload/64188/64188fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Vascular damage 
Figure 3 indicates the relationship between drilling scheme and vascular damage. Table 1 indicates the p-value for each drilling scheme following statistical analysis as indicated in step 10. A sample size of N = 4 per group was used for EB dye evaluation. The presence of a higher amount of EB is a direct indicator of damage to the BBB, of which the point-by-point, horizontal, and pulsed drilling methods are significantly larger than that of the control (all with p = 0.043; Table 1). The point-by point method does not show any significant difference in terms of EB presence compared to the horizontal drilling (p = 0.411). Both these schemes employed the auto-stop function to prevent drilling into the brain; however, this auto-stop function often failed to prevent damage. This failure of auto-stop in the shared seed drilling portion could have caused unknown excess damage, complicating differentiation between the techniques. Therefore, a pairwise comparison to a pulsed point-by-point method without auto-stop was performed to evaluate the other two methods without incorporating auto-stop. There was no significant difference when pulsed point-by-point was compared to point-by-point (p = 0.486), whereas the pulsed point-by-point method had significantly less EB presence than the horizontal method (p = 0.043). Non-significance between pulsed point-by-point and point-by-point methods may be attributed to the large variation in point-by-point drilling (Figure 4).
Figure 3 shows representative images of both horizontal (Figure 3C) and point-by-point (Figure 3D) drilling with proper auto-stop features. Visually, and through EB fluorescent imaging, drilling via point-by-point and horizontal cutting was seen to be damaging to the vasculature in the brain in comparison to control groups (Figure 3A,B). The pulsed point-by-point method (Figure 3E) had less localized damage at the seed and edge point, but still had visible EB presence within the cranial window.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64188/64188fig03.jpg" /> 
Figure 3: Vascular damage. EB fluorescence images of explanted brains (1) and corresponding ROIs (2) utilized to determine the mean radiance of the area affected by cranial window craniotomy. (A) The mouse was injected with EB with no cranial window surgery to acquire baseline background EB presence in the brain vasculature. (B) The mouse was injected with saline only and a cranial window craniotomy was performed. This established that the mean radiance being measured was credited to the EB accumulation due to leaky blood vessels and vascular trauma near the site of the cranial window. (C) The mouse was injected with EB and the cranial window was created by the horizontal method of automatic drilling. (D) The mouse was injected with EB and the cranial window was created by the point-by-point method of automatic drilling. (E) Two representative images of cranial window produced with the point-by-point pulsed method of drilling after the mice (n = 2) were injected with EB. <a href="https://www.jove.com/files/ftp_upload/64188/64188fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Visual inspection of damage 
Visually inspecting the brains shows physical damage to the surface of the brain (Figure 4). The panels A-D demonstrate the EB presence of the horizontal drilling, panels E-H the point-by-point method, and panels I-L are the pulsed point-by-point method. &#34;Point-by-point&#34; performs vertical pilot hole cuts while &#34;horizontal&#34; performs horizontal cuts along the circumference of the cranial window that outline the hole. The &#34;pulsed point-by-point&#34; employs the same methods as the point-by-point without the use of the auto-stop feature, and depends on the user stopping the drilling at set increments of depth. Although a method has been found that will minimize the amount of thermal damage to the brain, there is still the issue of mechanical damage from the drill. Ideally, an auto-stop feature that detects CSF and stops drilling before damaging brain tissue would work here, but did not seem to work consistently. Even with extreme care taken in pulsed manual drilling, there was still visual damage on the brain. This could be the result of two factors: 1) the lack of control and feel that comes with hand drilling and 2) the separation depth between the skull and the brain for a small animal such as a mouse. Hand drilling may offer a more controlled method for getting through the skull without damaging the brain with enough practice and expertise. However, there is much higher skill and training needed compared to a plug-and-play robot, which would allow for several &#34;surgeons&#34; to contribute to the same study-not a common practice in the intracortical microelectrode field. With mice, the distance between the brain and skull is extremely thin, so even the slightest over-drill of 10 &#181;m can lead to mechanical damage to the brain.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64188/64188fig04.jpg" /> 
Figure 4: Visual inspection of damage. Digital images of all brains acquired for visual inspection and representation for each of the three drilling methods. (A-D) Horizontal consistently showed damage around the cranial window, whether from mechanical or thermal damage. (E-H) Point-by-point showed considerable variance in results, indicating a less reliable method for drilling. (I-L) Pulsed point-by-point was more consistent and showed less visual damage than the other methods, matching the differences in EB fluorescent analysis and thermocouple results. Scale bar = 2 mm. <a href="https://www.jove.com/files/ftp_upload/64188/64188fig04large.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></td>
			<td>Horizontal</td>
			<td>Point</td>
			<td>Pulsed</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Horizontal</td>
			<td>-</td>
			<td>0.411</td>
			<td>0.043*</td>
			<td>0.043*</td>
		</tr>
		<tr>
			<td>Point</td>
			<td>0.411</td>
			<td>-</td>
			<td>0.486</td>
			<td>0.043*</td>
		</tr>
		<tr>
			<td>Pulsed</td>
			<td>0.043*</td>
			<td>0.486</td>
			<td>-</td>
			<td>0.043*</td>
		</tr>
	</tbody>
</table>
Table 1: Statistical analysis of EB fluorescent imaging results. Results from the EB fluorescent imaging system for different drilling techniques were analyzed using&#160;a Kruskal-Wallis rank sum test with Benjamin-Hochberg correction followed by pairwise comparisons using the Wilcoxon rank sum exact test (N = 4 per group). Significant differences between groups are indicated with an asterisk *.

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, ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.0K Views.  Case Western Reserve University. 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.

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

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

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

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 ...

Medicine

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

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.

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 ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

Generating a Traumatic Brain Injury Mouse Model Using an Impactor Probe

Source: Furmanski, O., et al. <a href="https://app.jove.com/v/59561">Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells.</a> J. Vis. Exp. (2019).
This video demonstrates the procedure for inducing an open-skull traumatic brain injury in a mouse by making a midline skull incision, drilling through the skull and removing the bone flap to expose the brain cortex, and applying high pressure to induce trauma. The wound is then sutured to prepare the mouse model for subsequent experiments.

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