implantation of cranial imaging window in mouse model: a surgical procedure to implant glass-based coverslip for stable optical access to regions of murine brain

Implantation of Cranial Imaging Window in Mouse Model: A Surgical Procedure to Implant Glass-based Coverslip for Stable Optical Access to Regions of Murine Brain

After confirming a lack of response to toe pinch in an anesthetized adult mouse, mount the mouse on a stereotactic frame and secure the head with a nose clamp and two ear bars.
Use a heating lamp to preserve the body temperature and apply ointment to the animal&#39;s eyes. Using sharp scissors, shave the fur on the skull from the eyes to the base of the skull, and use 70% ethanol to sterilize the exposed skin.
Cut the skin in a circular manner, and use a cotton swab to scrape away the exposed periosteum. Treat the surgical area with a drop of 1% lidocaine and a 1:100,000 concentration of epinephrine for 5 minutes before removing the excess solution with a cotton swab.
Use cyanoacrylate glue to adhere the edges of the skin to the skull, and place the stereotactic frame under a dissection stereomicroscope with a 4X magnification.
Next, carefully drill a superficial 5-millimeter diameter circular groove over the right parietal bone and apply a drop of cortex buffer to the groove.
Use thin forceps to lift the bone flap to visualize the brain surface, and use curved tapered, very fine point forceps to remove the dura mater. If bleeding occurs, use an absorbable gelatin sponge to achieve hemostasis.
To prepare the cranial imaging window, replace the cortex buffer with a drop of silicone oil to the craniotomy site to avoid air bubbles under the window.
Seal the exposed brain with a 6-millimeter coverslip and apply cyanoacrylate glue between the coverslip and the skull. Then, use fine tweezers to gently press the coverslip against the skull.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tumor Cell Implantation and Cranial Imaging Window Preparation
<ol>
	<li>Surgical Preparation
	<ol>
		<li>Use adult mice (&#62;6 weeks old) of any strain or gender. Sedate a mouse by injecting fluanisone [neuroleptic] + fentanyl [opioid]) (0.4 mL/kg) + benzodiazepine sedative (2 mg/kg) at a dose of 1:1:2 in sterile water. Assess the animal&#39;s sedation state by toe pinch. 
		NOTE: In this protocol, both female and male C57BL/6 mice were used due to the same genetic background of the tumor cell line used in these experiments (GL261). The mouse will stay fully sedated for 1.5 h. Alternatively, use inhalation anesthesia such as isoflurane (1.5%&#8211;2% isoflurane/O2&#160;mixture).</li>
		<li>Mount the mouse on a stereotactic frame and secure the head using a nose clamp and two ear bars.</li>
		<li>Use a heating lamp to preserve body temperature.&#160;Heating lamp should be used with caution, alternatively water recirculating heating pads can be used.</li>
		<li>Apply eye ointment to protect the mouse&#39;s corneas from drying.</li>
		<li>Use sharp scissors to shave the fur on the skull (dorsal area from the mouse&#39;s eyes to the base of its skull) and disinfect&#160;the exposed skin with 70% ethanol.</li>
		<li>Cut the skin in a circular manner with sharp scissors and scrape away the periosteum underneath with a cotton swab. Apply a drop of lidocaine 1% + epinephrine 1:100,000 for 5 min and remove the excess with a cotton swab.</li>
		<li>Glue the edges of the skin to the skull with cyanoacrylate glue.</li>
		<li>Place the stereotactic frame under a dissection stereo microscope with 4x magnification.</li>
		<li>Visualize the skull through the dissection microscope and drill a circular groove of 5 mm in diameter over the right parietal bone. Perform this step carefully and only superficially, avoiding any pressure on the skull.</li>
		<li>Apply a drop of cortex buffer (125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES buffer, 2 mM MgSO4, and 2 mM CaCl2&#160;[pH 7.4]) and lift the bone flap using thin forceps.</li>
		<li>For the following steps, keep the brain surface covered with cortex buffer unless indicated otherwise.</li>
		<li>Under the dissection microscope, visualize the brain surface and remove the dura mater using curved, tapered, very fine point tweezers. If bleeding occurs at this stage, use an absorbable gelatin sponge to stop it.</li>
	</ol>
	</li>
</ol>
2. Cranial Imaging Window Preparation
<ol>
	<li>Remove the cortex buffer and place a drop of silicone oil on the craniotomy site to avoid air bubbles under the window.</li>
	<li>Seal the exposed brain with a 6 mm coverslip. Apply cyanoacrylate glue between the coverslip and the skull. Gently press the coverslip against the skull with the help of fine tweezers to ensure minimal distance between the brain and the coverslip.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25G x 16 mm hypodermic needles&#160;</td>
			<td>BD Microlance&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable gelatin sponge&#160;</td>
			<td>Pfizer&#160;</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips round 6 mm&#160;&#160;</td>
			<td>VWR international</td>
			<td></td>
			<td>Gelfoam</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue&#160;</td>
			<td>Pattex&#160;</td>
			<td>631-0168</td>
			<td>Pattex Ultra gel</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Vertex Dental&#160;</td>
			<td></td>
			<td>Vertex Self-Curing</td>
		</tr>
		<tr>
			<td>Drill&#160;</td>
			<td>Dremel&#160;</td>
			<td></td>
			<td>Dremel 3000 (dental drill may be more convenient) + 105 Engraving Cutter</td>
		</tr>
		<tr>
			<td>Fine curved Tweezers&#160;</td>
			<td>Dumont&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Oil&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>AGT508</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Stoelting&#160;</td>
			<td>181838</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical stereo microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Lab standard stereotaxic, rat and mouse</td>
		</tr>
		<tr>
			<td>Opthalmic ointment&#160;</td>
			<td>Kela Veterinaria&#160;</td>
			<td></td>
			<td>Duodrops veter kela 10 m</td>
		</tr>
		<tr>
			<td>Xylocaine (Lidocaine 1% + Epinephrine 1:100,000) Local anesthetic&#160;</td>
			<td>Astrazeneca</td>
			<td></td>
			<td>Xylocaine (Lidocaine 1% + Epinephrine 1:100,000)</td>
		</tr>
		<tr>
			<td>Hypnorm&#160;&#160;</td>
			<td>VetaPharma Ltd</td>
			<td></td>
			<td>Hypnorm (Fentanyl citrate 0.315 mg/mL + Fluanison 10 mg/mL)</td>
		</tr>
		<tr>
			<td>Midazolam&#160;</td>
			<td>Actavis&#160;</td>
			<td></td>
			<td>Midazolam Actavis 5 mg/mL</td>
		</tr>
	</tbody>
</table>

Source: Alieva, M. et al. <a href="https://www.jove.com/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.</a>&#160;J. Vis. Exp.&#160; (2019)
This video presents a surgical procedure to implant cranial window in a mouse model for stable optical access to different regions of the brain.

Source: Alieva, M. et al. Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.&#160;J. Vis. Exp.&#160; ...

Cranial window implantation is a surgical procedure wherein an exposed region of the brain is sealed with a coverslip. The implanted imaging window facilitates access to the brain for long-term high resolution in vivo imaging.&#160;&#160;
To begin, stabilize an anesthetized mouse in a stereotactic unit.
Maintain its body temperature using a heating pad.
Next, shave the mouse&#8217;s scalp and sterilize the exposed skin.
Make a circular incision and scrape off the underlying periosteum - a fibrous connective tissue layer - from the skull surface.
Subsequently, apply a suitable vasoconstrictor and an anesthetic solution over the surgical area to minimize bleeding in the following steps.&#160;
Secure the skin edges to the skull with cyanoacrylate glue.
Then, under a dissection microscope, visualize the skull.
Drill a shallow groove over the region of interest until the bone flap becomes loose. Treat the groove with cortex buffer to prevent tissue dehydration.
Gently lift the bone flap, creating a bone window to access the brain underneath.&#160;
Now, remove the outermost membrane layer surrounding the brain - the dura mater. Clear the buffer and apply silicone oil to prevent air bubbles under the window.
Mount a coverslip over the exposed area using glue, ensuring minimal distance between the brain and the coverslip.&#160;
The mouse with the implanted cranial window is ready for brain imaging.

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Implantation of Cranial Imaging Window in Mouse Model: A Surgical Procedure to Implant Glass-based Coverslip for Stable Optical Access to Regions of Murine Brain

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