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 &deg;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 &mu;m. Skull thinning should be continued carefully to obtain a very thin (~20 &mu;m) and smooth preparation.</li>
 <li>Once the desired thickness of skull is achieved (~20 &mu;m), the skull is first polished using a custom-made silicone whip with diamond paste (6-15 &mu;m) 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 &mu;m 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 &mu;l of a GBM cell suspension is injected into the brain 100-200 &mu;m from the brain surface (speed: 0.1 &mu;l/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="Figure 1" src="/files/ftp_upload/4201/4201fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/4201/4201fig2.jpg" /> 
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 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4201/4201fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4201/4201fig3.jpg" /> 
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 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4201/4201fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4201/4201fig4.jpg" /> 
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 &mu;m.

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	<li>Paulino, A. C., Teh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+brain+tumors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+therapeutic+implications+of+cancer+stem+cells+in+glioblastoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+cancer+stem+cell+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+stem+cells:+the+cancer+stem+cell+hypothesis+writ+large.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+paradigm:+a+new+understanding+of+tumor+development+and+treatment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+brain+tumors,+neural+stem+cell,+and+brain+tumor+cancer+cells:+where+is+the+link.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+glioma+cancer+stem+cells.">Biology of glioma cancer stem cells.</a> Mol. 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Clin. Neurosci. 15, 1193-1197 (2008).</li><li>Gilbertson, R. J., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+tumour's+bed:+glioblastoma+stem+cells+and+the+vascular+niche.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+cancer+stem+cells.">Recent advances in cancer stem cells.</a> Curr. Opin. Genet. Dev. 18, 48-53 (2008).</li><li>Drew, P. 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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 border="1">
 <tr>
 <td>Name of 			reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments 			(optional)</td>
 </tr>
 <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>eBiosciences</td>
 <td>00-4555-56</td>
 <td></td>
 </tr>
 <tr>
 <td>T25 			flasks-vent cap green</td>
 <td>SARSTEDT</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>
</table>

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.

Watch this Scientific Journal Video about Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window at JoVE.com

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

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.

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 ...

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13.0K Views.  The Jackson Laboratory. 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.

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

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...