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, 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 "2 s on, 2 s off" 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 °C ± 1.35 °C) group compared to the horizontal (16.66 °C ± 2.08 °C) and point-by-point (18.69 °C ± 1.75 °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.
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'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 ("point-by-point" vs "horizontal"), referring to the routed path of the drill bit. Initially, eight "seed" points are drilled (Figure 1A), outlining the cranial window. From here, the space in between the seeds is cut out using either the "point-by-point" or "horizontal" drill method. "Point-by-point" performs vertical pilot hole cuts (similar to a CNC drill press), while "horizontal" 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.
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. Please click here to view a larger version of this figure.
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
2. Software preparation
3. Preparation for surgery
4. Skull preparation
5. Evans Blue tail vein injection
CAUTION: EB is a possible carcinogen. Use gloves when handling.
6. Surgical robot drilling procedure
7. Perfusion and brain extraction
8. Evans Blue imaging and analysis
9. Thermocouple evaluation
10. Statistics
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 °C ± 2.08 °C, while edge drilling showed 9.08 °C ± 0.37 °C. For the point-by-point method, seed drilling showed a maximum temperature change of 18.69 °C ± 1.75 °C, while edge drilling showed 8.53 °C ± 0.36 °C. For the pulsed point-by-point method, seed drilling showed a maximum temperature change of 6.90 °C ± 1.35 °C, while edge drilling showed 4.10 °C ± 0.51 °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 < 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.
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 < 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. Please click here to view a larger version of this figure.
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.
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. Please click here to view a larger version of this figure.
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. "Point-by-point" performs vertical pilot hole cuts while "horizontal" performs horizontal cuts along the circumference of the cranial window that outline the hole. The "pulsed point-by-point" 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 "surgeons" 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 µm can lead to mechanical damage to the brain.
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. Please click here to view a larger version of this figure.
Horizontal | Point | Pulsed | Control | |
Horizontal | – | 0.411 | 0.043* | 0.043* |
Point | 0.411 | – | 0.486 | 0.043* |
Pulsed | 0.043* | 0.486 | – | 0.043* |
Table 1: Statistical analysis of EB fluorescent imaging results. Results from the EB fluorescent imaging system for different drilling techniques were analyzed using 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 *.
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 °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 μm) 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.
The authors have nothing to disclose.
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.
1x Phosphate Buffered Saline Type: Reagent |
VWR | MRGF-6235 | For Evans Blue dilution |
Aura Software Type: Tool |
Spectral Instruments Imaging | Open access imaging processing software for Lumina imaging sytems | |
Buprenorphine Type: Drug |
Sourced from Animal Facility | ||
Carbide Drill Bit, 0.6mm (Robot Drill) Type: Tool |
Stoelting | 58640-1 | |
Carprofen Type: Drug |
Sourced from Animal Facility | ||
Cefazolin Type: Drug |
Sourced from Animal Facility | ||
Evans Blue Dye Type: Reagent |
Millipore Sigma | E2129 | Reconstituted in 1x phosphate-buffered saline |
Isoflurane Type: Drug |
Sourced from Animal Facility | ||
IVIS Lumina II Type: Tool |
Perkin Elmer | CLS136334 | IVIS Lumina III currently in place of Lumina II on the market |
Jenco Linearizing Thermometer Type: Tool |
Jenco | 765JF | For Thermocouple setup |
Ketamine Type: Drug |
Sourced from Animal Facility | ||
LivingImage Type: Tool |
Perkin Elmer | Software for IVIS Lumina III | |
Marcaine Type: Drug |
Sourced from Animal Facility | ||
Neurostar Software Type: Tool |
Stoelting | Comes with surgical robot purchase | |
Physiosuite with MouseSTAT® Pulse Oximeter & Heart Rate Monitor Type: Tool |
Kent Scientific | PS-03 | Used to monitor vitals |
PrismPlus mice Type: Animal |
Jackson Labortory | 031478, RRID:IMSR_JAX:031478, Male, ~8 months old | Animals used for the study |
Stoelting Drill and Injection Robot for Motorized Stereotaxic Instruments Type: Tool |
Stoelting | 58640 | Main robotic drill with stereotaxic frame |
Thermocouple Type: Tool |
TC Direct | 206-557 | For Thermocouple setup |
USB-6008 Multifunction I/O DAQ Type: Tool |
National Instruments | USB-6008 | For Thermocouple setup |
Xylazine Type: Drug |
Sourced from Animal Facility |