Name: Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window (Video) | JoVE Journal
Uploaded: 2012-11-20T12:00:00.000+00:00
Duration: 9 min 44 s
Description: Learn to image glioma initiation in vivo using a polished thin-skull cranial window. This method enables longitudinal two-photon microscopy of glioblastoma cell behavior and vascular interactions in live mice.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work is supported by The Jackson Laboratory Cancer Center Pilot Grant and The Maine Cancer Foundation.

1. Protocol
<ol>
 <li>Anesthetize the mouse with ketamine and xylazine at a dose of 0.1 mg ketamine and 0.01 mg of xylazine per 1 g of body weight. Carprofen (0.005 mg per 1 g of body weight) is used for analgesia and is administered preoperatively.</li>
 <li>All of the surgical tools, including the dental drill bit, are steam sterilized in an autoclave. If batch surgeries are to be performed, the tips of surgical instruments must be re-sterilized with a glass bead sterilizer (Fine Science Tools FST 250) before each subsequent surgery.</li>
 <li>Once the mouse has reached a surgical plane of anesthesia, assessed by absence of a response to a toe pinch, it is placed on a cotton pad that is then placed on a heated pad to maintain a body temperature of ~37 °C. The depth of anesthesia is monitored frequently during the surgery.</li>
 <li>Fur is removed from the dorsal head in a roughly triangular area ~ 3 mm caudal to the nares and extending caudally to the cervical vertebrae. Ophthalmic ointment is applied to eyes to protect them from dehydration and irritation.</li>
 <li>The mouse is placed in ventral recumbancy and the skin is disinfected with surgical iodine, 70% ethanol, and sterile swabs. A 0.8 x 1.0 cm flap of skin covering the dorsal aspect of the frontal and parietal bones of the skull is removed using fine scissors.</li>
 <li>A topical analgesic, 0.5% lidocaine, is applied locally to the periosteum and the exposed tissues at the periphery of the wound. The periosteum is removed by gently scraping the skull with a scalpel blade; this will help the cyanoacrylate glue to adhere to the bone.</li>
 <li>The cortical area of interest is outlined on the skull with a marker. The area of interest should not be located over skull suture lines, to avoid damage to underlying large vessels.</li>
 <li>A thin layer of cyanoacrylate adhesive is applied to the wound margins, to prevent the seepage of serosanguinous fluid, and to the skull except in the area of interest.</li>
 <li>Light-activated dental cement is used in our protocol for sealing the skull. Before application of dental cement, all exposed skull except for the area intended for thinning is treated with the primers and subsequently with the bonding agent from the light-activated dental cement kit. Once the bonding agent is dried, the light-activated dental cement is applied to cover the skull. To stabilize the head of the mouse for subsequent surgery and imaging, a sterile #00-90 hex nut is embedded into the dental cement at a distance from the area to be thinned. The use of light- activated dental cement allows us to prepare the skull without having to rush to finish the entire procedure. Once the dental cement preparation on the skull is completed, the dental cement is cured with visible light.</li>
 <li>After the dental cement is cured, a headpiece holder from the stereotactic device is mounted to the hex nut on the mouse head using a #00-90 screw.</li>
 <li>A drop of room-temperature, 0.9% sterile saline is applied to the skull area to be thinned. With the aid of a stereomicroscope, a high-speed micro-drill is used to thin a circular area of the skull (typically ~4-5 mm in diameter) over the region of interest. Drilling and the application of saline to the drilled area are performed intermittently during the thinning procedure to avoid thermal injury to the underlying cerebral tissue. Saline absorbs heat and also helps soften the bone.</li>
 <li>The mouse skull comprises two thin layers of compact bone, sandwiching a thick layer of spongy bone. The external layer of compact bone and most of the spongy bone are removed using the drill.</li>
 <li>After the majority of the spongy bone is removed, the remaining cavities within the spongy bone can be seen under a dissecting microscope, indicating that drilling is approaching the internal compact bone layer. At this stage, skull thickness should still be more than 50 μm. Skull thinning should be continued carefully to obtain a very thin (~20 μm) and smooth preparation.</li>
 <li>Once the desired thickness of skull is achieved (~20 μm), the skull is first polished using a custom-made silicone whip with diamond paste (6-15 μm) for approximately 10 min. The diamond-paste polishing serves two purposes. First, it further thins the skull without applying pressure on the thinned skull, thereby avoiding accidental damage to the underlying brain tissue. Second, it serves as an initial polishing step for the thinned skull before the fine-grained tin oxide is used for further skull polishing.</li>
 <li>Following the diamond-paste polishing, the thinned skull is polished with tin oxide for approximately 10 min. Once the skull is polished, the tin oxide is flushed away with saline until the thinned skull appears clean.</li>
 <li>At this step, if the cranial window is to be used for longitudinal in vivo imaging, the cleaned thin skull area is dried. A small drop of clear cyanoacrylate glue is applied to seal the cranial window. The layer of cyanoacrylate glue between the thinned skull and the coverslip should be as thin as possible.</li>
 <li>If an injection is to be performed after creation of a PoRTS cranial window, at this step, a 26-gauge syringe needle is used to create a small opening on one side of the polished thin-skull window. This opening is used for GBM cell injection.</li>
 <li>A sterile glass pipette with a tip opening approximately 50 μm in diameter is pulled using a pipette puller. This pipette will be used for GBM cell injection, and is loaded onto a micromanipulator. The pipette is loaded at a 30-degree angle so that the GBM cell injection site will be away from the final imaging site. The tip of the pipette is removed under a dissection microscope. The GBM cell suspension is front-loaded into the pipette by suction using a syringe pump.</li>
 <li>Once the injection pipette is loaded with GBM cells, the mouse is moved back under the microscope. The injection pipette is lowered towards the small opening in the cranial window and finally inserted into the brain through the opening using a micromanipulator. 1 μl of a GBM cell suspension is injected into the brain 100-200 μm from the brain surface (speed: 0.1 μl/min, duration: 10 min).</li>
 <li>The skull is dried, a small drop of clear cyanoacrylate glue is applied to the dried skull, and a piece of 3-mm size #1 coverslip is adhered to the thin and polished skull area. The space between the coverslip and adjacent dental cement is sealed with cyanoacrylate glue.</li>
 <li>After surgery, the mouse is injected subcutaneously with 1 ml warm sterile saline and provided with supplemental heat to maintain body temperature until fully recovered from anesthesia.</li>
 <li>For imaging, mice are anesthetized with ketamine and xylazine at a dosage of 0.1 mg ketamine and 0.01 mg of xylazine per 1 g of body weight. Mice are immobilized using a custom stereotactic frame under the microscope. If repetitive in vivo imaging is performed on daily basis, isofluorane will be a better option, as mice will show signs of dependence with repetitive use of ketamine.</li>
</ol>
2. Representative Results
A successful PoRTS cranial window surgery allows the cranial window to remain clear for weeks to months. Figure 1 shows the vasculature beneath a PoRTS cranial window as visualized using backscattered light. These vasculature images were used as landmarks for locating the same brain areas for repetitive imaging. To visualize both the vasculature endothelial cells and GBM cells, we crossed the B6.Cg-Tg(Tek-cre)1Ywa/J mouse, which labels vascular endothelial cells with the B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mouse, and generated mice with vascular endothelial cells labeled with the red fluorescent protein tdTomato. We then performed the PoRTS craniotomy procedure on these mice, and injected GFP-labeled GBM cells into their brains through a small opening on the side of a cranial window. We then performed in vivo two-photon imaging longitudinally on the injected mice. The laser excitation wavelength was set at 910 nm, which resulted in excitation of both GFP and tdTomato. Two-photon imaging was performed on a custom-built two-photon laser-scanning microscope with two detectors for simultaneous green/red fluorescence detection capability. Figure 2 shows an in vivo example of glioma initiation from injected GBM cells near the vasculature. The PoRTS cranial window is also an excellent choice for chronic time-lapse in vivo imaging. Figure 3 shows GFAP staining of cortical sections from a mouse without PoRTS surgery, a mouse 7 days following PoRTS surgery, and a mouse 7 days following PoRTS surgery and saline injection. It is evident from the images that the mouse without surgery and the mouse with PoRTS surgery do not have gliosis, whereas following saline injection, gliosis is observed in the brain tissue of the mouse, due to pipette penetration and saline injection in the brain. This result demonstrates that gliosis does not occur under the PoRTS cranial window, providing independent validation that the PoRTS cranial window described here does not lead to the gliosis that is typically associated with an "open skull" chronic cranial window. Figure 4 shows high-resolution imaging of dendritic spines from the same mouse on the day of surgery (Day 0) and 32 days after the PoRTS window creation (Day 32), demonstrating that the PoRTS window is also an excellent choice for high-resolution in vivo imaging.
<img alt="Vascular growth observation diagram post-injection, day-by-day progression over two weeks. " src="/files/ftp_upload/4201/4201fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Visualization of vasculature under a polished and reinforced thinned skull cranial window using backscattered light. Days represent the number of days after surgery; images were taken starting on the day of surgery immediately after completion of GBM cell injection. Scale bar: 0.5 mm. <a href="https://www.jove.com/files/ftp_upload/4201/4201fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Time-lapse confocal microscopy images of vascular growth over 13 days, fluorescence labeling." src="/files/ftp_upload/4201/4201fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Visualization of injected GBM cells and vasculature in vivo. Mice carrying both Tek-Cre and ROSA26 CAG-tdTomato were used for GBM cell injection. Vascular endothelial cells were labeled with tdTomato (red), and GBM cells were labeled with GFP (green). Images were acquired starting 24 hr after GBM cell injection. A total of 10-12 adjacent fields of view were taken and were assembled adjacent to one another, with the identified injected GBM cells in the center of the total field, to produce the final images. Days represent the number of days after surgery. Scale bar: 100 μm. <a href="https://www.jove.com/files/ftp_upload/4201/4201fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Fluorescence microscopy images of B6 mouse brain tissues; post-surgery and saline injection analysis." src="/files/ftp_upload/4201/4201fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Gliosis does not occur following PoRTS cranial window surgery. Upper panels: Mouse brain coronal section under lower magnification. Left panel: a mouse without any cranial window surgery; no GFAP activation is observed in the brain tissue. Middle panel: a mouse with a PoRTS cranial window generated on the right side of the brain; no GFAP activation is observed in the brain tissue. Right panel: a mouse with a PoRTS cranial window and saline injection from the side of the PoRTS window. GFAP activation is observed on the side of the PoRTS window, but not on the side of intact brain tissue. Scale bar: 1 mm. Lower panel: higher-power images of brain tissues in areas as indicated in the white box of the upper-panel images. Scale bar: 100 μm. <a href="https://www.jove.com/files/ftp_upload/4201/4201fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Neural network evolution, electron microscope image, Day 0 vs Day 32, microstructure changes." src="/files/ftp_upload/4201/4201fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. High-resolution imaging of dendritic spines from Thy-1 YFPH mice. Images were acquired on the day of PoRTS surgery, and 32 days after PoRTS surgery. It is apparent from the images that most spines present on day 0 are clearly visible 32 days following PoRTS surgery. Scale bar: 2 μm.

<ol><li>Paulino, A. C., Teh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+of+brain+tumors%5BTitle%5D)+AND+%22N.+Engl.+J.+Med%22%5BJournal%5D)">Treatment of brain tumors.</a> N. Engl. J. Med. 352, 2350-2353 (2005).</li>
<li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma%5BTitle%5D)+AND+%22N.+Engl.+J.+Med%22%5BJournal%5D)">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N. Engl. J. Med. 352, 987-996 (2005).</li>
<li>Singh, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+human+brain+tumour+initiating+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li>
<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hallmarks+of+cancer%3A+the+next+generation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li>
<li>Cheng, L., Bao, S., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+therapeutic+implications+of+cancer+stem+cells+in+glioblastoma%5BTitle%5D)+AND+%22Biochem.+Pharmacol%22%5BJournal%5D)">Potential therapeutic implications of cancer stem cells in glioblastoma.</a> Biochem. Pharmacol. 80, 654-665 (2010).</li>
<li>Cheng, L., Ramesh, A. V., Flesken-Nikitin, A., Choi, J., Nikitin, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+for+cancer+stem+cell+research%5BTitle%5D)+AND+%22Toxicol.+Pathol%22%5BJournal%5D)">Mouse models for cancer stem cell research.</a> Toxicol. Pathol. 38, 62-71 (2010).</li>
<li>Dirks, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+tumor+stem+cells%3A+the+cancer+stem+cell+hypothesis+writ+large%5BTitle%5D)+AND+%22Mol.+Oncol%22%5BJournal%5D)">Brain tumor stem cells: the cancer stem cell hypothesis writ large.</a> Mol. Oncol. 4, 420-430 (2010).</li>
<li>Ebben, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cancer+stem+cell+paradigm%3A+a+new+understanding+of+tumor+development+and+treatment%5BTitle%5D)+AND+%22Expert+Opin.+Ther.+Targets%22%5BJournal%5D)">The cancer stem cell paradigm: a new understanding of tumor development and treatment.</a> Expert Opin. Ther. Targets. 14, 621-632 (2010).</li>
<li>Germano, I., Swiss, V., Casaccia, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+brain+tumors%2C+neural+stem+cell%2C+and+brain+tumor+cancer+cells%3A+where+is+the+link%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">Primary brain tumors, neural stem cell, and brain tumor cancer cells: where is the link.</a> Neuropharmacology. 58, 903-910 (2010).</li>
<li>Park, D. M., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biology+of+glioma+cancer+stem+cells%5BTitle%5D)+AND+%22Mol.+Cells%22%5BJournal%5D)">Biology of glioma cancer stem cells.</a> Mol. Cells. 28, 7-12 (2009).</li>
<li>Bao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell-like+glioma+cells+promote+tumor+angiogenesis+through+vascular+endothelial+growth+factor%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.</a> Cancer Res. 66, 7843-7848 (2006).</li>
<li>Calabrese, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+perivascular+niche+for+brain+tumor+stem+cells%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">A perivascular niche for brain tumor stem cells.</a> Cancer Cell. 11, 69-82 (2007).</li>
<li>Barami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+of+neural+stem+cells+with+their+vascular+niche%3A+implications+in+the+malignant+progression+of+gliomas%5BTitle%5D)+AND+%22J.+Clin.+Neurosci%22%5BJournal%5D)">Relationship of neural stem cells with their vascular niche: implications in the malignant progression of gliomas.</a> J. Clin. Neurosci. 15, 1193-1197 (2008).</li>
<li>Gilbertson, R. J., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+a+tumour%27s+bed%3A+glioblastoma+stem+cells+and+the+vascular+niche%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">Making a tumour's bed: glioblastoma stem cells and the vascular niche.</a> Nat. Rev. Cancer. 7, 733-736 (2007).</li>
<li>Cho, R. W., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+cancer+stem+cells%5BTitle%5D)+AND+%22Curr.+Opin.+Genet.+Dev%22%5BJournal%5D)">Recent advances in cancer stem cells.</a> Curr. Opin. Genet. Dev. 18, 48-53 (2008).</li>
<li>Drew, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Chronic optical access through a polished and reinforced thinned skull.</a> Nat. Methods. 7, 981-984 (2010).</li>
</ol>

No conflicts of interest declared.

The key to a successful PoRTS cranial window is the thinning and polishing. While the initial thinning can be performed quickly, care should be taken to ensure homogenous thinning of the skull over a large area. We typically apply a thin layer of saline to the skull, and then thin the skull one pass at a time, such that the saline solution evaporates shortly after the microdrill has passed over the skull once. This allows us to slowly but homogenously further thin the skull. A steady hand under the microscope is also key...

<table><tbody><tr><td>DMEM/F12(1:1) with Sodium Pyruvate</td><td>Thermo Sci Hyclone</td><td>SH3026101</td><td> </td></tr><tr><td>B-27 Serum Free Supplement (50x)</td><td>Invitrogen</td><td>17504-044</td><td> </td></tr><tr><td>GlutaMax-1 Supplement</td><td>Invitrogen</td><td>35050061</td><td> </td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Thermo Sci Hyclone</td><td>SV30010</td><td> </td></tr><tr><td>Accutase-Enzyme Cell Detachment Medium</td><td>eBioscience</td><td>00-4555-56</td><td> </td></tr><tr><td>T25 flasks-vent cap green</td><td>Sarstedt Ltd</td><td>83.1810.502</td><td> </td></tr><tr><td>70% alcohol</td><td>JAX LAHS</td><td></td><td> </td></tr><tr><td>10% povidone-iodine topical solution</td><td>JAX LAHS</td><td></td><td> </td></tr><tr><td>Ketamine HCl</td><td>Butler Animal Health Supply</td><td>NDC# 11695-0550-1</td><td> </td></tr><tr><td>Xylazine</td><td>Akorn, Inc.</td><td>NADA# 139-236</td><td> </td></tr><tr><td>Carprofen</td><td>JAX LAHS</td><td></td><td> </td></tr><tr><td>Ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>17033-211-38</td><td> </td></tr><tr><td>0.5% Lidocaine HCl</td><td>REGENT, Inc.</td><td>NDC 0517-0625-25</td><td> </td></tr><tr><td>Cyanoacrylate glue</td><td>Henkel Corp</td><td>46551</td><td> </td></tr><tr><td>Sterile Swabs</td><td>Fisher Scientific</td><td>23-400-114</td><td> </td></tr><tr><td>Diamond paste</td><td>Widget Supply</td><td>BBE60</td><td> </td></tr><tr><td>Tin oxide</td><td>LORTONE, Inc.</td><td>591-038</td><td> </td></tr><tr><td>Liner Bond 2V</td><td>KURARAY Medical Inc.</td><td>1921-KA</td><td> </td></tr><tr><td>Clearfil AP-X</td><td>KURARAY Medical Inc.</td><td>1721-KA</td><td> </td></tr><tr><td>Saline</td><td>JAX LAHS</td><td></td><td> </td></tr><tr><td>Cover glass</td><td>Warner Instruments</td><td>64-0720</td><td> </td></tr><tr><td>Hex Nut</td><td>Small Parts, Inc.</td><td>HNX-0090-C</td><td> </td></tr><tr><td>Syringe Pump</td><td>Syringepump.com</td><td>NE-1000</td><td> </td></tr><tr><td>Two-Photon imaging system</td><td>Custom built (Any commercial system would work)</td><td></td><td> </td></tr></tbody></table>

imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

The overall goal of this procedure is to visualize initial stages of glioma initiation from single injected glioblastoma cells in vivo. This is accomplished by first thinning, an area of mouse skull down to a thickness suitable for in vivo imaging purpose. Next, the thin skull area is polished.Then GFP labeled glioblastoma cells are injected into the mouse brain and the cranial window is sealed for in vivo imaging. Finally, the injected glioblastoma cells are imaged daily to visualize glioma initiation from injected glioblastoma cells in vivo. Ultimately, results can be obtained that show in vivo glioma initiation from a single injection of glioblastoma cells and its relationship with vascular endothelial cells through dual color in vivo two photon microscopy.This method can help answer key questions in cancer stem cell field, such as the critical role of interaction between glioma stem cells and vascular endothelial cells and glioma initiation in vivo, demonstrating the procedure will be under lapier manager of JAK Surgical Services and postal in my laboratory To prepare for surgery in an autoclave, steam sterilize all surgical tools, including the dental drill bit after the mouse has been anesthetized with ketamine and xylazine and administered analgesia. Check the level of anesthesia by performing a toe pinch using a small electric shaver. Remove the fur from the dorsal head in a roughly triangular area, about three millimeters, coddle to the nares and extending coddly to the cervical vertebrae.Apply ophthalmic ointment to the eyes. Place the mouse in ventral recumbent and use surgical iodine followed by 70%ethanol. To disinfect the skin, place the animal on a cotton pad and transfer it to a heating pad.To maintain a body temperature of about 37 degrees Celsius, apply a topical analgesic to the periosteum and the exposed tissues of the wound. Using a scalpel blade, remove the periosteum by gently scraping the skull. This will help the cyanoacrylate glue adhere to the bone.Next, use a marker to outline the cortical area of interest, making sure to avoid suture lines to avoid damaging the underlying large vessels. Then apply a thin layer of cyanoacrylate adhesive to the wound margins to prevent the seepage of serosanguinous fluid and to the skull except in the area of interest before sealing the skull with light activated dental cement, treat all exposed skull except the area intended for thinning with primers and then bonding agent. Once the bonding agent has dried, cover the skull with dental cement to stabilize the mouse's head.For surgery and imaging, embed a number double zero dash 90 hex nut into the dental cement at a distance from the area to be thinned. Once the dental cement preparation is completed, use visible light to cure it. Then using a number zero zero dash 90 screw mount, a headpiece holder from the stereotactic device to the hex nut.Next, place the mouse's head under a stereo microscope and apply a drop of room temperature, 0.9%sterile saline to the skull area to be thinned. Use a high speed micro drill to thin a circular area of the skull over a region of interest. To avoid thermal injury to the underlying cerebral tissue, intermittently pause drilling and apply saline.The mouse skull comprises two thin layers of compact bone, sandwiching of thick layer of spongy bone. Use the drill to remove the top thin layer and most of the spongy bone. After the majority of the spongy bone has been removed, the remaining cavities within the spongy bone can be seen under the dissecting microscope indicating that drilling is approaching the internal compact bone layer.At this stage, skull thickness should still be more than 50 microns. Carefully continue skull thinning to obtain a very thin and smooth preparation. To further thin the skull, use a custom made silicone whip and diamond paste to polish it for about 10 minutes.Continue to polish the skull with fine-grained tin oxide for an additional 10 minutes. Use saline to flush away the tin oxide until the thin skull appears clean. To perform an injection, use a 26 gauge syringe needle to create a small opening on one side of the polished window under the microscope.Use the micro manipulator to lower the tip of the pipette through the hole in the cranial window and into the brain. 100 to 200 microns deep. Then inject one microliter of cell suspension at a rate of 0.1 microliters per minute.After removing the needle to seal the cranial window, dry the skull and apply a small drop of clear cyanoacrylate glue and a piece of three millimeter. Number one, cover slip. Use cyanoacrylate glue to seal the space between the cover slip and adjacent dental cement.After surgery, inject the mouse subcutaneously with one milliliter of warm sterile saline and provide supplemental heat to maintain body temperature until fully recovered from anesthesia. Administer subcutaneous injections of analgesic daily for five days after surgery, and observe the mouse daily to ensure that the wound is healing properly and that the mouse is eating, drinking, and behaving normally to image. After anesthetizing the mouse with ketamine and xylazine immobilize it using a custom stereotactic frame under the microscope.A successful ports cranial window surgery allows the cranial window to remain clear for weeks to months. This figure shows the vasculature between a port's cranial window as visualized using back scattered light. These vasculature images were used as landmarks for locating the same brain areas for repetitive imaging.To visualize both the vascular endothelial cells and GBM cells, mice were generated with vascular endothelial cells labeled with the red fluorescent protein TD tomato. After the port's craniotomy procedure, GFP labeled GBM cells were injected into their brains, and in vivo two photon imaging was performed.Shown. Here is an in vivo example of glioma initiation from injected GBM cells near the vasculature.This figure shows GFAP staining of cortical sections from a mouse without port surgery, a mouse seven days following port surgery, and a mouse seven days following port surgery with saline injection. It is evident from the images that the mouse without surgery and the mouse with port surgery do not have gliosis. Whereas following saline injection, gliosis is observed in the brain tissue of the mouse due to pipette penetration and saline injection in the brain.This result demonstrates that gliosis does not occur under the port's cranial window. Providing independent validation that the port's cranial window described here does not lead to the gliosis that is typically associated with an open skull chronic cranial window. Here, high resolution imaging of dendritic spines are shown from the same mouse on the day of surgery, day zero and 32 days after the port's window creation.Demonstrating that the port's window is also an excellent choice for high resolution in vivo imaging. After watching this video, it should have a good understanding of how to image glioma initiation by injecting glioblastoma cells through a polish and reinforce the cranial window.

imaging-glioma-initiation-vivo-through-polished-reinforced-thin-skull

Research

JoVE Journal

Medicine

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

This video presents a surgical procedure to implant cranial window in a mouse model for stable optical access to different regions of the 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

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.

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

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells' migratory, invasive, and proliferative behavior at a single cell level.

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in 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.

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

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.

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

Learn to image glioma initiation in vivo using a polished thin-skull cranial window. This method enables longitudinal two-photon microscopy of glioblastoma cell behavior and vascular interactions in live mice.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Explore More Videos