Tamara Zeric for helping to obtain the multiphoton image in this paper. Supported by the Brain &#38; Behavior, Parkinson&#39;s and JPB Foundations, R01 MH108186 and R01 DA07418. F31 fellowship 1F31MH109293-01A1 to S C.

All procedures involving animals were performed according to the standards set by the Columbia University Medical Center Institutional Animal Care and Use Committee (IACUC). Euthanasia was performed via cervical dislocation under anesthesia with ketamine 100 mg/kg and xylazine 10 mg/kg injected intraperitoneally. All surgical procedures were performed in a sterile manner (surgeon was wearing head cap, face mask, sterile gloves, and clean disposable lab coat) and surgical tools were autoclaved between each use.
1. Presurgical Steps
<ol>
	<li>Autoclave all surgical instruments to ensure sterility.</li>
	<li>Anesthetize C57bl6 mice with isoflurane (4% for induction and 1.5-2% for surgery). Pinch the hind toe every 10 min to ensure proper depth of anesthesia. Increase anesthesia if vocalization or hind limb retraction is seen.</li>
	<li>Apply veterinary eye lubricant to the eyes to prevent drying.</li>
	<li>Remove the hair over the scalp with a 3 min application of hair removal gel or by shaving with an electric razor.</li>
	<li>Place the mouse in the stereotaxic frame on a heating pad or calibrated water recirculating blanket set to 38&#176;C.</li>
	<li>Administer buprenorphine (0.1 mg/kg) subcutaneously to provide presurgical analgesia.</li>
	<li>Disinfect the scalp with three alternating applications of chlorhexidine or betadine and 70% ethanol using a cotton tipped applicator.</li>
	<li>Inject 100 mL of bupivacaine diluted 50% in saline under the scalp to provide local anesthesia.</li>
</ol>
2. Piezoelectric Cranial Window Surgery
<ol>
	<li>Using autoclaved sterile surgical instruments, remove a 1 cm circle of scalp over the skull by cutting with surgical scissors in a circular pattern. Lift the scalp off the skull exposing the periosteum tissue over the bone.</li>
	<li>Use the tip of forceps to scrape the exposed bone clear of any remaining periosteum tissue. This is important to ensure that the dental cement will not fall off later.</li>
	<li>Set the piezoelectric surgery unit to vibrate on the lowest setting. Note that higher settings may break blood vessels due to intense vibrations.</li>
	<li>Fix the sterile circular tip to the handpiece. 
	NOTE: The 4 mm circular tip is well suited for surgeries. The large tip size contributes to the speed of the surgery as the entire window can be thinned simultaneously.</li>
	<li>Fill a 10 mL syringe with ice cold artificial cerebrospinal fluid (ACSF) and hold it in the hand not holding the piezoelectric handpiece.</li>
	<li>Irrigate the skull by dripping the ACSF from the syringe onto the skull at a rate of 1 mL/min. Alternatively, use a peristaltic pump positioned over the skull to free the use of the other hand. 
	NOTE: Irrigation with cold ACSF is important to prevent overheating due to the friction from the high frequency vibrations.</li>
	<li>Gently apply the vibrating surgical tip to the skull with a light circular motion (3 circles per 1 s) and thin the bone to the desired depth to perform a thin skull preparation (5 to 10 min). 
	NOTE: Here, 4 mm windows were made that were thinned to 20 &#181;m. However, the size of the window will vary with the size of the surgical tip. The thickness of the bone can be adjusted to the desired depth based on the length of time spent applying the tip to the skull.</li>
	<li>Gently adjust the angle of the handpiece and change the angle of the tip while applying pressure to the skull to thin the skull uniformly.</li>
	<li>To perform a complete bone removal without leaving any thin bone behind, thin the bone until cracks are visible around the window area. Remove the remaining flakes of thin bone with forceps without damaging the underlying dura.</li>
	<li>Soak a 1 mm piece of hemostatic collagen foam in cold ACSF. Hold the wet foam with forceps and place it on the thin skull. Allow it to sit on the thin skull&#160;for about 30 s to stop any micro bleeds from developing.</li>
	<li>Use forceps to place a 4 mm glass coverslip laterally over the window that has been thinned into the bone.</li>
	<li>Use the back of a small plastic or wooden cotton tipped applicator to apply approximately 100 &#181;L of dental acrylic to the skull to hold the window in place.</li>
</ol>
3. Post Surgical Care
<ol>
	<li>Remove the mouse from the surgical apparatus and place it in a heated cage for recovery. Monitor it continuously until it has regained full consciousness (assessed by normal mobility, gait, and behavior).</li>
	<li>Assess the animals twice a day for three days for manifestations of post-surgical pain including decreased activity, grooming, food, and water consumption, guarding behavior (e.g. limping or a hunched posture) or increased aggression. Do not return the animal to the company of other animals until it has fully recovered.</li>
	<li>Administer Buprenorphine (0.05&#8211;0.1 mg/kg) subcutaneously as an analgesic 8&#8211;12 h after the surgery for 3 days. Monitor the area of the window closely following surgery for dehiscence, inflammation, and signs of infection. If the animal does not return to baseline feeding and grooming behaviors, consult a veterinarian regarding possible interventions or euthanasia.</li>
</ol>

<ol>
	<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=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4, 1128-1144 (2009).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long+term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long term imaging of the cortex in live mice.</a> Nat protoc. 12, 213-220 (2010).</li><li>Dorand, D. R., Barkauskas, D. S., Evans, T. A., Petrosiute, A., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+intravital+thinned+skull+and+cranial+window+approaches+to+study+CNS+immunobiology+in+the+mouse+cortex.">Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex.</a> Intravital. 3, e29728-1-e29728-8 (2014).</li><li>Drew, P. 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=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nat Methods. 7, 981-984 (2010).</li><li>Wang, J., Zhang, Y., Xu, T. H., Luo, Q. M., Zhu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+innovative+transparent+cranial+window+based+on+skull+optical+clearing.">An innovative transparent cranial window based on skull optical clearing.</a> Laser Physics Letters. 9, (2012).</li><li>Yang, 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=Skull+Optical+Clearing+Solution+for+Enhancing+Ultrasonic+and+Photoacoustic+Imaging.">Skull Optical Clearing Solution for Enhancing Ultrasonic and Photoacoustic Imaging.</a> IEEE Trans Med Imaging. 35, (2016).</li><li>Vercellotti, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezoelectric+surgery+in+implantology:+a+case+report--a+new+piezoelectric+ridge+expansion+technique.">Piezoelectric surgery in implantology: a case report--a new piezoelectric ridge expansion technique.</a> Int J Periodontics Restorative Dent. 4, 358-365 (2000).</li><li>Vercellotti, T., De Paoli, S., Nevins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+piezoelectric+bony+window+osteotomy+and+sinus+membrane+elevation:+introduction+of+a+new+technique+for+simplification+of+the+sinus+augmentation+procedure.">The piezoelectric bony window osteotomy and sinus membrane elevation: introduction of a new technique for simplification of the sinus augmentation procedure.</a> Int J Periodontics Restorative Dent. 6, 561-567 (2001).</li><li>Vercellotti, T., Podesta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthodontic+microsurgery:+a+new+surgically+guided+technique+for+dental+movement.">Orthodontic microsurgery: a new surgically guided technique for dental movement.</a> Int J Periodontics Restorative Dent. 4, 325-331 (2007).</li><li>Stubinger, S., Stricker, A., Berg, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezoelectric+surgery+in+implant+dentistry.">Piezoelectric surgery in implant dentistry.</a> Clin Cosmet Investig Dent. 7, 115-124 (2015).</li><li>Basheer, S. A., Govind, R. J., Daniel, A., Sam, G., Adarsh, V. J., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Study+of+Piezoelectric+and+Rotary+Osteotomy+Technique+for+Third+Molar+Impaction.">Comparative Study of Piezoelectric and Rotary Osteotomy Technique for Third Molar Impaction.</a> J Contemp Dent Pract. 18, 60-64 (2017).</li><li>Pavlikova, G., 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=Piezoelectric+surgery+prevents+brain+tissue+damage:+an+experimental+study+on+a+new+rat+model.">Piezoelectric surgery prevents brain tissue damage: an experimental study on a new rat model.</a> Int J Oral Maxillofac Surg. 40, 840-844 (2011).</li></ol>

There are no disclosures.

The surgery can be modified by changing the surgical tip. There are many different sizes of tip that can be applied to the handpiece. Changing the size or shape of the tip will result in different sized windows. In addition to the 4 mm tip, we also tried a 3 mm tip and found it also worked well. Even with irrigation with ice cold ACSF, we were not able to get good results with tips that were too narrow (approximately 0.25 mm) due to the concentrated vibrations causing too much heat and burning the bone leading to damage to the underlying dura. We have not tried the multitude of other tips that are available, and we anticipate that other labs may find new applications for these different tips. While the surgery is relatively straightforward, we found that there are several steps that may require troubleshooting. The first is that the vibrating tip produces a lot of turbulence in the ACSF, which diminishes visibility and produces difficulty in ascertaining bone depth during the bone thinning step of the surgery. We recommend that if it is too difficult to see how thin the bone is, take a break to dry the skull and window by applying a sterile cotton swab to the side of the bone. Do not apply the cotton swab directly to the window, since the rough surface is damaging to thin bone. After checking the depth, reapply ACSF and continue with the surgery. We also found that if there is damage to the dura, it is usually due to the surgeon putting too much pressure on the tip. Holding the handpiece more gently and applying less force will likely fix this problem. If the surgeon is forcibly scraping the tip on the bone, it will cause a break in the thin bone and damage the skull. Finally, if after thinning the bone, the underlying dura appears bruised, it is likely due to excess heat from the vibrations. Increasing the rate of ACSF flow will fix this issue.
The main limitation of PHASOR is that the handpiece can thin the bone, but cannot remove all the bone over the dura. The diamond coated tip has small rough bumps. The rubbing needed to thin the skull would abrade the dura and cause bleeding if it were used to remove all the bone. Thus, the use of forceps is required to remove the remaining thin layer. While there is ongoing debate on whether imaging through a thin skull produces less inflammation and less proliferation of microglia and reactive astrocytes, in some cases a window with no bone remaining is preferred, e.g., to provide a greater depth of imaging3.
We have optimized cranial window preparation for multiphoton imaging in mice. PHASOR is, to our knowledge, the first application of piezoelectric technique to rodent cranial window surgery for optical imaging. We find that the use of piezoelectric surgery in mice shares the advantages of increased speed and decreased adverse events also reported in humans7,8,9,10. Utilization of the piezoelectric device resulted in faster surgeries (Figure 2A) and fewer adverse events of bleeding when compared to a high-speed rotary drill (Figure 2B). We also found, within our lab, that PHASOR was easier for new surgeons to learn than traditional approaches to cranial window surgery. Advantages of speed and ease of use are likely to differ among surgeons. Thin skull preparations typically require 30-45 min to thin the bone with a drill and scalpel2, while the PHASOR approach typically requires less than 10 min.
We found that in windows prepared with PHASOR we could image calcium transients in layer 4 pyramidal neurons in motor cortex that had been transfected with adeno associated virus encoding the calcium indicator JRGECO1a (AAV9.Syn.NES-jRGECO1a.WPRE.SV40). We also found that, compared to a thin skull cranial window prepared with a rotary drill and scalpel, the imaging region was larger, while a thin skull prepared with a rotary drill and scalpel has an imaging window of 20 &#181;m diameter2. With PHASOR, we were able to quickly thin an area of 3-4 mm. This is similar to the imaging window reported with PoRTS4. This larger window retains the benefit of rapid imaging reported for thin skull windows prepared with a dental drill and scalpel. Moreover, the window prepared with PHASOR can be immediately imaged, as opposed to traditional bone removal windows that may require weeks to heal before the window can be optimally imaged3.
This technique can be applied to study alterations in blood flow in the vessels under the dura. An important future study is to compare immunoreactivity of PHASOR with other methods of cranial window. This would be important to determine if PHASOR produces less inflammation in comparison to existing methods. We hope that the piezoelectric surgical technique documented here will allow more labs to perform cranial windows and successfully use multiphoton imaging in vivo.
Make sure that the handpiece is set to vibrate on the lowest setting and that the ACSF is being irrigated at a constant rate. The ice cold ACSF must be applied constantly or it will heat up and damage the dura. We found that ACSF must be applied at a rate of at least 1 mL/min. Either use a syringe held in the hand, or use a peristaltic pump, rather than the built-in irrigation in the handpiece. The irrigation system in the handpiece ejects ACSF too forcefully and produces too much turbulence, which will significantly impair visibility.

Cranial window surgery to prepare rodents for multiphoton imaging in vivo has become an important technique in neuroscience. The removal or thinning of bone is necessary to prepare the mouse for optical imaging with a multiphoton microscope. This surgery is performed either by completely removing an area of bone to expose the underlying dura1, or by thinning a region of bone without full removal from the dura2. The thin skull approach may produce less inflammation and activation of microglia3 but provides a shallower depth of imaging, a smaller imaging window size (200 &#181;m) and a limited time period during which the window can be imaged due to bone regrowth2. The addition of a polished and reinforced glass window (PoRTS) can increase the imaging size and imaging period but is difficult to perform4.
Both current surgeries use a high speed rotary drill to thin or remove the bone from the skull. The thin skull technique also uses a scalpel after the drill to further thin the bone2. The PoRTS technique requires the extra step of high speed polishing with grit4. In a high speed rotary drill, an air powered turbine or electric motor causes the drill bit to spin at a high velocity. As rotary drills section both bone and soft tissue, there is a risk of damaging the dura and underlying blood vessels. The success of the surgery depends on the skill of the surgeon. In addition to these windows prepared with mechanical surgical methods, a chemical method of optically clearing the skull with different solutions has been developed5,6. However, since piezoelectric surgery is a mechanical method of surgery, our comparisons here will be limited to other mechanical methods.
Piezoelectric surgical devices utilize ultrasonic vibrations to break down mineralized bone without damaging underlying soft tissue, and thus offer an approach to rapidly thin a large area of bone. In a piezoelectric surgical handpiece, the turbine is replaced by a stack of ceramic disks and when current is applied, the disks vibrate at ultrasonic frequencies. The vibrations are transferred through the handpiece to diamond coated tips to cut through bone without damaging soft tissues, an advantage over rotary drills that do not discriminate between tissue types. Piezoelectric surgery was originally developed for use in human by Tomaso Vercellotti and has led to improvements in dental and cranio-maxilofacial surgery7,8,9,10,11.
Piezoelectric surgery has been used to create an osteotomy in Wistar rats and was found by magnetic resonance imaging (MRI) and histology to produce significantly less damage than a traditional dental drill12. The authors concluded that piezoelectric surgery was safe for removing the bone near soft brain tissue. Mice, however, have a thinner dura that is more easily damaged, and that study did not prepare windows for chronic optical imaging. Chronic imaging requires that blood vessels are not damaged and that blood clots do not form under the window. Damage to the dura leads to inflammation that causes the window to cloud, and activates microglia and the proliferation of reactive astrocytes. Here we have optimized piezoelectric surgery for mice to create both thin skull and full bone removal cranial windows suitable for chronic imaging. We compared this surgical technique to cranial window surgeries prepared with a high speed rotary drill.

<table>
	<tbody>
		<tr>
			<td>Piezosurgery Touch</td>
			<td>Mectron</td>
			<td>5120062</td>
			<td>Piezosurgery GP model has the same settings</td>
		</tr>
		<tr>
			<td>Circular 4mm flat piezosurgery tip (# OT11)</td>
			<td>Mectron</td>
			<td>3370019</td>
			<td>This tip was ideal for our windows but there are many other tips of different sizes availible.</td>
		</tr>
		<tr>
			<td>Stereotax frame</td>
			<td>Kopf</td>
			<td>963</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse adaptor</td>
			<td>Stoelting</td>
			<td>51625</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump for irrigation.</td>
			<td>Cole-Parmer</td>
			<td>WU-77120-42</td>
			<td>Makes it easier to irrigate and frees up the other hand to provide stability. Irrigation can be performed by hand with a syringe if necessary.</td>
		</tr>
		<tr>
			<td>Avitene Ultrafoam</td>
			<td>Bard-Davol</td>
			<td>1050020</td>
			<td>Important to stop any minor bleeding instantly.</td>
		</tr>
		<tr>
			<td>C&#38;B Metabond</td>
			<td>Parkell</td>
			<td>S380</td>
			<td>Much stronger than regular dental acrylic.</td>
		</tr>
		<tr>
			<td>Artificial cerebro spinal fluid (ACSF)</td>
			<td>Tocris</td>
			<td>3525</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube opthalmic ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>JAX</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Prairie Ultima multiphoton microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>JRGECO1a (AAV9.Syn.NES-jRGECO1a.WPRE.SV40)</td>
			<td>UPENN Vector Core</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Before proceeding with piezoelectric surgery, remove any residual periosteum from the skull. Once the skull is opaque and smooth (Figure 1a), the surgeon may begin piezoelectric surgery. When removing bone with the vibrating piezo tip, it is critical to irrigate the skull with ice cold ACSF. Proper irrigation is achieved when the bottom 1 mm of the tip is submerged in ACSF (Figure 1b). Without proper irrigation, the bone will overheat and damage the brain. After the skull has been thinned to the desired depth chosen by the surgeon, there may be some residual bleeding from blood vessels located in the bone. In order to rapidly cease all bleeding, apply a small 1 mm piece of collagen foam soaked in ACSF to the area of the window. Let the collagen sit on the skull for approximately 30 s (Figure 1c). After the collagen foam is removed, the window will appear translucent, allowing clear visualization of the blood vessels in the dura. The dura will be intact without significant bruising. (Figure 1d). If the dura appears red and inflamed, it is likely due to broken blood vessels from too much pressure applied to the tip during the surgery. To protect the new window and prepare it for chronic imaging, a glass coverslip must be placed over the area. A properly applied glass coverslip will gently sit on top of the window and will not cause any damage to the area. The glass coverslip must cover the entire window. If the glass coverslip is properly applied to the skull, the window will remain translucent under the glass. All the blood vessels in the dura will still be visible (Figure 1e). Dental acrylic must be applied around the glass coverslip to permanently adhere it to the surface of the skull. The edges of the glass coverslip must also be covered in dental acrylic (Figure 1f).
We found that piezoelectric surgery is typically much faster than a traditional cranial window, taking around 10-12 min per surgery (Figure 2A). We also found that there were less complications due to dura bruising and bleeding as observed by eye (Figure 2B). A properly prepared window will allow multiphoton imaging of fluorescent indicators in cortical neurons in vivo. We chose to image the red calcium indicator JRGECO1a in cell bodies of layer 4 cortical neurons (Figure 3A-3B). We could observe calcium transients in these cell bodies through the window prepared using PHASOR.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56172/56172fig1.jpg" /> 
Figure 1: Piezoelectric surgery. (a) The skull after it has been properly prepared for PHASOR. All the periosteal tissue has been removed, leaving a smooth and clean surface. (b) A representative image of PHASOR in progress. The bottom 1 mm of the vibrating surgical tip is submerged in ice cold ACSF. The fluid is applied to the skull at a rate of 1 mL/min. A light circular motion is applied to the skull and the bone is thinned via ultrasonic vibrations. (c) After the skull has been thinned, a 1 mm circular piece of collagen foam soaked in cold ACSF is allowed to rest on the window to stop any micro bleeds from the bone. (d) A successful window with a translucent dura displays all of the intact blood vessels. There is no visible damage or bruising to the surface of the brain. (e) A glass coverslip is placed over the window to protect the surface of the brain. (f) Dental acrylic is applied to the skull to permanently fix the glass coverslip over the window. Scale bar 1 mm in all images.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56172/56172fig2.jpg" /> 
Figure 2. Comparison of success rate in training for PHASOR or dental drill surgery. (A) Average time per surgery was lower for PHASOR than dental drill surgeries (p&#60;0.05, two tailed t test) n= 30 surgeries per group and average 10 surgeries per surgeon. (B) The percentage of successful surgeries (defined as no bleeding or visible damage to the dura as observed by the surgeon&#39;s eye through the objective set to 350X zoom) was higher for PHASOR than dental drill surgeries (n = 30 surgeries for each group, one tailed z test). Error bars show SD.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56172/56172fig3.jpg" /> 
Figure 3. Multiphoton imaging in vivo through a cranial window thinned with PHASOR. (A) Calcium transients observed with the red calcium indicator JRGECO1a were imaged in layer 4 pyramidal neurons in murine motor cortex in vivo. Scale bar = 100 &#181;m. (B) The same window but different region of layer 4 motor cortex. Headfixed mouse with 3 mm window prepared with PHASOR. Scale bar = 30 &#181;m. Imaged with a multiphoton microscope, 40X objective, 1040 nm excitation wavelength.

Watch this Scientific Journal Video about Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice at JoVE.com

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.

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 ...

piezo-high-accuracy-surgical-osteal-removal-phasor-technique-for

Research

JoVE Journal

Neuroscience

10.6K Views.  Columbia University Medical Campus. 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.

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

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, ...

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

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 ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous deoxyhemoglobin as an endogenous contrast agent to detect changes in blood-level-dependent oxygenation (BOLD effect). We combined fMRI with a novel robotic device (MR-compatible hand-induced robotic device [MR_CHIROD]) so that a person in the scanner can execute a controlled motor task, hand-squeezing, which is a very important hand movement to study in neurological motor disease. We employed parallel imaging (generalized auto-calibrating partially parallel acquisitions [GRAPPA]), which allowed higher spatial resolution resulting in increased sensitivity to BOLD. The combination of fMRI with the hand-induced robotic device allowed precise control and monitoring of the task that was executed while a participant was in the scanner; this may prove to be of utility in rehabilitation of hand motor function in patients recovering from neurological deficits (e.g., stroke). Here we outline the protocol for using the current prototype of the MR_CHIROD during an fMRI scan.

We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits.

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

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 ...