NINDS grants NS49720 and NS65819, and NCI grant CA97257

1. Tumor Cell Preparation.
Cells for human brain tumor xenografts can be sourced either from tumors propagated as subcutaneous growths in athymic mice, or from cell culture. Utilization of both cell sources is discussed below, along with demonstration of a method for cell implantation.
To prepare cells from subcutaneous tumors for transfer to the intracranial compartment, excised flank tumors are placed in culture dishes, where the tissue is initially minced with a scalpel and then mechanically disrupted by repetitive pipetting to create a cell aggregate suspension1. The cell aggregate suspension is then passed through a 70 &mu;M nylon mesh filter to produce a single cell suspension suitable for intracranial injection. The cell suspension is centrifuged at 1000 rpm for 10 minutes at 4&deg;C, and the supernatant aspirated before resuspending the cell pellet in an appropriate volume of serum-free media to obtain a final working concentration (see below). For preparing established cell lines for intracranial implantation, cells are harvested by trypsinizing monolayers, or by collecting neurosphere suspension cultures, then centrifuging and resuspending the cells as indicated above 2. The number of cells injected is variable dependent on neuroanatomical location of injection. For supratentorial injections we routinely inject 3-5 x 105 cells in 3 &mu;L of serum-free media (DMEM), whereas for brainstem injections 3, as few as 5 x 104 cells are injected in 0.5 &mu;L. Injecting larger volumes than recommended can result in tumor cell reflux through the needle tract, with resultant exophytic (Figure 1), rather than intracranial tumor growth. After withdrawing sample for intracranial injection, the remaining cell suspension should be placed in ice, with contents mixed frequently to maintain appropriate concentration while completing intracranial tumor establishment among the members of an injection series.
2. Tumor Cell Implantation
Note, all procedures described below have been reviewed and approved by the Institutional Animal Use and Care Committee at University of California San Francisco.
<ol>
	<li>The surgical area should be prepared by spraying all surfaces with a disinfectant, such as 2% chlorhexidine solution. After using the disinfectant, the following supplies should be placed in the surgical area:
	<ul>
		<li>Heating pad to maintain mouse body temperature</li>
		<li>Two small Petri dishes; one containing 3% hydrogen peroxide, and one containing 2% chlorhexidine</li>
		<li>Sterile gauze and cotton swabs</li>
		<li>Sterile disposable scalpels</li>
		<li>Autoclaved surgical stapler</li>
	</ul>
	</li>
	<li>For anesthesia an injected anesthetic should be used; typically a ketamine-xylazine mixture.</li>
	<li>Once a mouse is anesthetized, the scalp is prepared by swabbing several times with a piece of sterile gauze dipped in the chlorhexidine solution. Eye ointment should be applied to maintain adequate moisture during the procedure. Using a sterile scalpel, complete a sagittal incision over the parieto-occipital bone, approximately 1cm long. The exposed skull surface is then cleaned using a cotton swab soaked in a 3% hydrogen peroxide solution. The bregma should be apparent at this point (see video).</li>
	<li>The coordinates for injection of tumor cells will vary according to the desired site for tumor establishment. The following represents the procedure we use for intracerebral tumor establishment2 in a neuroanatomical location at which many brain tumor patients experience tumor development. Other locations of interest in brain tumor research include the pons, for anatomic modeling of brainstem tumors3, and subdural injections for modeling the location of meningeal tumors4. Prior to tumor cell injection, use a sterile 25 gauge sharp needle to puncture the skull at 2 mm to the right of the bregma and 1 mm anterior to the coronal suture, thereby creating an opening for the injection of tumor cells (see video). This procedure works well for both mice and rats (22 gauge needle for rats).</li>
	<li>Prior to drawing cells into the syringe, mix the contents of the cell suspension by tapping with your finger. Load the syringe with the desired amount of cell suspension, being careful to avoid creating air bubbles. The outside of syringe should then be cleaned with an alcohol swab to wipe the exterior free of any adherent cells, which will help prevent extracranial tumor establishment and growth (Figure 1A). To ensure that the appropriate injection depth is achieved, use a scalpel to cut 3mm off the pointed end of a P20 pipetteman tip. This section of the tip can be fitted over the syringe and will act to limit the injection depth, and will additionally ensure that the tip of the syringe needle is 3mm from the underside of the skull. Place the syringe perpendicular to the skull and in the hole previously created, and slowly inject the cell suspension (a 3&mu;L suspension should be injected over a 1 minute period). An inappropriate angle of syringe insertion can result in intraventricular injection of cells and subsequent spinal dissemination (Fig. 1B: Right) Upon completing injection, leave the needle in place for another minute, then slowly withdraw (see video): these steps will help reduce tumor cell reflux.</li>
	<li>As an alternative to the unassisted or free-hand approach to intracranial tumor cell implantation5, one can use a small animal stereotactic frame (panel F of schematic overview), which generally promotes more consistent injection location, but at the expense of substantial amounts of procedural time. In our experience, two surgical staff can inject as many as 60 mice/hour when using the free-hand approach, whereas maximal injection rate with a small animal stereotactic frame is approximately 15 mice/hour. Procedural expediency is an important consideration when injecting large series of mice, and helps reduce the time in which tumor cells, to be injected, are left on ice.</li>
	<li>Clean the skull with 3% hydrogen peroxide and dry using a sterile dry cotton swab. Apply sterile bone wax to the hole. Using a forceps, draw the scalp together over the skull and staple to close. For optimal healing, the scalp should be stapled with the dermis of each side of the scalp against each other (underside against underside). The stapled scalp should be cleaned using Chlorhexidine solution, and buprenorphine then administered by subcutaneous injection for post-operative pain relief.</li>
	<li>Monitor all mice post-operatively until they become ambulant and retain normal activity. Typically, recovery time is around 30 minutes.</li>
	</ol>
3. Bioluminescence Monitoring of Tumor Growth
<ol>
	<li>Background. Bioluminescence imaging (BLI) is based on the oxidation of luciferin [d-(-)-2-(60-hydroxy-20-benzothiazolyl)-thiazone-4-carboxylic acid] in the presence of oxygen and adenosine triphosphate (ATP). This reaction is catalyzed by the enzyme luciferase, which converts chemical energy into photons with resultant emission of light. Human cells can be modified to express luciferase (see below), with only luciferase-expressing cells capable of emitting light in the presence of the luciferin substrate. There is minimal background luminescence from animals hosts treated with luciferin, such that there is a very favorable signal-to-noise ratio for detecting luminescence emissions from luciferase-modified tumor cells, allowing highly sensitive monitoring of tumor growth and response to therapy in vivo. Moreover, luciferase and its substrate, luciferin, have been shown to be non-toxic to mammalian cells, and we have observed no functional differences between cells expressing luciferase relative to corresponding unmodified cells. Luciferin readily crosses cell membranes and the blood-brain barrier after intraperitoneal (i.p.) or intravenous (i.v.) injection in mice, thereby allowing imaging of every compartment that contains luciferase-modified cells. The level of photon emission and the spectrum of emitted light from luciferase-modified cells is adequate to penetrate tissues of small research animals, such as mice and rats, and can be detected externally with low-light imaging cameras. The noninvasive nature of bioluminescence imaging allows repeated monitoring of tumor growth and response to therapy in individual animal subjects.</li>
	<li>Implanted cell sources should be stably modified for firefly luciferase expression. Such cell sources can be purchased, or produced in individual laboratories using lentivirus that have been constructed for constitutive expression of luciferase. We strongly recommend the use of cells modified with luciferase encoding lentivirus, rather than plasmid, to ensure stable cellular expression of luciferase in vivo, which is necessary for quantitative luminescence imaging to provide accurate indication of changes in tumor burden for individual animal subjects that are repeatedly imaged during the course of an experiment.</li>
	<li>BLI monitoring. We recommend conducting quantitative bioluminescence imaging (qBLI) 1x-2x weekly, beginning one week following tumor cell injection. Our qBLI is conducted using the IVIS Lumina imaging station (Caliper Life Sciences), and our results indicate similar data are obtained in using the IVIS Lumina, the IVIS 150, or the IVIS 200 imaging station. In preparation for imaging, mice are simultaneously anesthetized with Ketamine/Xylazine and administered luciferin (D-Luciferin potassium salt, 150 mg/kg, Caliper Life Sciences) via intraperitoneal injection, with mice imaged 12 minutes after injection. The pharmacokinetics of luciferin indicate that the time between administration of luciferin and determination of cell luminescence should be between 10-15 minutes post injection of luciferin, in order to obtain maximum luminescence emission and greatest sensitivity of detection. The length of time selected within the 10-15 minute time interval should be maintained as constant among the animals that are being imaged. It is extremely important to maintain consistency in length of time between injection of luciferin and obtaining BLI readings. Regions of interest encompassing the intracranial area of signal are defined using Living Image software (Figure 2), and the total photons/s/sr/cm2 (photons per second per steradian per square cm) are recorded (see video).</li>
	<li>Data analysis. Whereas tumor growth and therapy response are monitored in individual animals, we highly recommend treatment groups of at least 8 for increasing the statistical certainty of conclusions regarding tumor response, or lack thereof, to therapy. With regard to presenting qBLI results, luminescence readings are converted to normalized values by dividing each mouse s luminescence, obtained during and subsequent to completion of therapeutic regimen, with its corresponding maximal pretreatment luminescence reading6,7. For survival analysis, the Kaplan-Meier estimator8 is used, and from which survival curves are generated, and median survival values determined. Differences between survival curves are compared using a log-rank test9.</li>
	<li>Retrieval of brain for subsequent analysis of treated and untreated tumor. Upon animal euthanasia, the brain of the mouse should be quickly excised (see video), and either placed in formalin for subsequent analysis of tumor morphology and immunohistochemistry, or should be mounted in OCT for specimen freezing.</li>
	</ol>
4. Representative Results
In the example shown in Figure 3A, mice receiving intracranial tumor cell injection were monitored for intracranial luminescence until successive mean luminescence values indicated progressive tumor growth, and at which time therapy was initiated (gray arrow beginning at day 34: erlotinib administered daily at 150 mg/kg until required euthanasia). Luminescence values for each mouse are set to a normalized value of 1 at time of initiating therapy, with subsequent luminescence readings for each mouse normalized to its final pretreatment imaging value. As an example, a mouse with a final pre-treatment luminescence reading of 2.0 x 107 photons/sec at day 34, whose luminescence had increased to 6.0 x 107 photons/sec at day 38, would have a day 38 normalized luminescence value of 3.0. Mean normalized bioluminescence and corresponding standard error for control and treatment groups are plotted for each imaging time point. In this example, a significant difference in mean normalized luminescence is apparent at the first imaging time point subsequent to the initiation of therapy (day 38), with the difference in mean group luminescence showing further increase at subsequent time points. In most instances, anti-tumor activity of therapy, as indicated by qBLI, is accompanied by a corresponding significant difference in survival (i.e., p &lt; 0.05), as is the case here (Figure 3B). Panels 3C and 3D show adjacent hematoxylin &amp; eosin and anti-EGFR stained sections of mouse brain obtained at time of euthanasia, following placement of resected brain in formalin and subsequent paraffin-embedding for sectioning.
<img src="/files/ftp_upload/1986/1986fig1.jpg" alt="Figure 1" /> Figure 1. Indications of intracranial injection errors. A) Exophytic (extracranial) tumor growth (red circle) can be caused by too large an injection volume, residual cell suspension attached to the syringe, or from withdrawing the syringe too quickly after injecting the tumor cells. B) Injecting tumor cells into the ventricles can cause spinal dissemination of tumor (mouse to the right), in contrast to properly injected tumor cells, the signal for which stays localized to the injection site (mouse to the left).
<img src="/files/ftp_upload/1986/1986fig2.jpg" alt="Figure 2" /> Figure 2. Heat map image representations of bioluminescence intensity for representative mice from control (left) and treatment (right) groups of a therapy response experiment. The Living Image software can be set to define regions of interest (red circles), or instrument operators can define regions of interest manually. For using images such as these for figure construction, we recommend that the instrument operator shows heat map images using the same bioluminescence heat map range (upper portion of figure), to provide visual representation of extent of bioluminescence difference between animal subjects.
<img src="/files/ftp_upload/1986/1986fig3.jpg" alt="Figure 3" /> Figure 3. Bioluminescence, survival, and tumor tissue analysis from an experiment in which therapeutic response is evident. A) Plot of mean bioluminescence readings for control and treatment group mice, with standard error indicated for each imaging point. B) Survival plot for same mice; p-value determined through use of log-rank test 7. C) H&amp;E stained section of mouse brain with tumor. D) EGFR stained section. E and F) Magnifications of indicated areas from panels C and D, respectively, with panel E showing negative staining for tumor suppressor protein PTEN.

<ol>
	<li>Giannini, C., Sarkaria, J. N., Saito, A., Uhm, J. H., Galanis, E., Carlson, B. L., Schroeder, M. A., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+Tumor+EGFR+and+PDGFRA+Gene+Amplifications+Retained+in+an+Invasive+Intracranial+Xenograft+Model+of+GBM.">Patient Tumor EGFR and PDGFRA Gene Amplifications Retained in an Invasive Intracranial Xenograft Model of GBM.</a> Neuro-Oncol. 7, 164-176 (2005).</li><li>Ozawa, T., Wang, J., Hu, L. J., Bollen, A. W., Lamborn, K. R., Deen, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+glioblastomas+as+xenografts+in+the+brains+of+athymic+rats.">Growth of human glioblastomas as xenografts in the brains of athymic rats.</a> In Vivo. 1, 55-60 (2002).</li><li>Hashizume, R., Ozawa, T., Dinca, E. B., Banerjee, A., Prados, M. D., James, C. D., Gupta, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+brainstem+glioma+xenograft+model+enabled+for+bioluminescence+imaging.">A human brainstem glioma xenograft model enabled for bioluminescence imaging.</a> J Neurooncol. , (2009).</li><li>Baia, G. S., Dinca, E. B., Ozawa, T., Kimura, E. T., McDermott, M. W., James, C. D., VandenBerg, S. R., Lal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+skull+base+model+of+malignant+meningioma.">An orthotopic skull base model of malignant meningioma.</a> Brain Pathol. 2, 172-179 (2007).</li><li>Ozawa, T., James, C. D., Van Meir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Brain+Tumor+Cell+and+Tumor+Tissue+Transplantation+Models.">Human Brain Tumor Cell and Tumor Tissue Transplantation Models.</a> CNS Cancer: Models, Markers, Prognostic Factors, Targets, and Therapeutic Approaches. , 147-162 (2009).</li><li>Sarkaria, J. N., Yang, L., Grogan, P. T., Kitange, G. J., Carlson, B. L., Schroeder, M. A., Galanis, E., Giannini, C., Wu, W., Dinca, E. B., James, C. D. <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+Molecular+Characteristics+Correlated+with+Glioblastoma+Sensitivity+to+EGFR+Kinase+Inhibition+Through+Use+of+an+Intracranial+Xenograft+Test+Panel.">Identification of Molecular Characteristics Correlated with Glioblastoma Sensitivity to EGFR Kinase Inhibition Through Use of an Intracranial Xenograft Test Panel.</a> Mol. Cancer Ther. 6, 1167-1174 (2007).</li><li>Dinca, E. B., Sarkaria, J. N., Schroeder, M. A., Carlson, B. L., Voicu, R., Gupta, N., Berger, M. S., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Monitoring+of+Intracranial+Glioblastoma+Xenograft+Response+to+Primary+and+Salvage+Temozolomide+Therapy.">Bioluminescence Monitoring of Intracranial Glioblastoma Xenograft Response to Primary and Salvage Temozolomide Therapy.</a> J. Neurosurg. 107, 610-616 (2007).</li><li>Kaplan, E. L., Meier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-parametric+estimation+from+incomplete+observations.">Non-parametric estimation from incomplete observations.</a> J. Am. Stat. Assoc. 53, 457-481 (1958).</li><li>Peto, R., Peto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymptotically+efficient+rank+invariant+procedures.">Asymptotically efficient rank invariant procedures.</a> J. R. Stat. Soc. Ser. A. Stat. Soc. 135, 185-207 (1972).</li><li>Camphausen, K., Purow, B., Sproull, M., Scott, T., Ozawa, T., Deen, D. F., Tofilon, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+in+vivo+growth+on+human+glioma+cell+line+gene+expression:+convergent+profiles+under+orthotopic+conditions.">Influence of in vivo growth on human glioma cell line gene expression: convergent profiles under orthotopic conditions.</a> Proc. Natl. Acad. Sci. U. S. A.. 102, 8287-8292 (2005).</li></ol>

No conflicts of interest declared.

Orthotopic (intracranial) brain tumor xenograft establishment provides an appropriate microenvironment10 for modeling CNS cancer to be tested for therapeutic response. This type of modeling additionally provides information regarding therapeutic access to brain and brain tumor, which is critically important to determining whether an experimental agent should be advanced to clinical trial evaluation in patients. Because the amount of intracranial xenograft tumor can not be directly measured, such as by calipers, longitudinal monitoring of intracranial tumor growth and response to therapy requires non-invasive imaging, with our experience indicating bioluminescence imaging as the most practical approach for experiments whose primary objective is assessing extent of tumor response to therapy. When the results of bioluminescence imaging are combined with animal subject survival analysis, the two data sets provide a powerful and reliable approach for evaluating experimental therapeutic efficacy.
Finally, it is critically important that intracranial brain tumor xenografts are harvested from euthanized animal subjects in order to assess morphologic and molecular effects of therapy, and for this we prefer whole brain resection at time of euthanasia, with preservation of resected brain for subsequent analysis.
Whereas the preceding presentation has been made specific to brain tumor research, the concepts are certainly generalizeable to other human cancers that are amenable to orthotopic modeling in rodents.

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Disposable Scapels</td>
			<td>&nbsp;</td>
			<td>Feather</td>
			<td>2975</td>
			<td>No.21</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>&nbsp;</td>
			<td>Dunlap</td>
			<td>HP950</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>22028563</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>23-400-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2% Chlorhexidine</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>NC9756995</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3% Hydrogen Peroxide</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>H312P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25g Needle</td>
			<td>&nbsp;</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10ul Hamilton Sharp Microsyringe</td>
			<td>&nbsp;</td>
			<td>Hamilton</td>
			<td>20734</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Skin Stapler and Staples</td>
			<td>&nbsp;</td>
			<td>Stoelting</td>
			<td>59020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic Frame</td>
			<td>&nbsp;</td>
			<td>Stoeling</td>
			<td>51725</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Living Image Software</td>
			<td>&nbsp;</td>
			<td>Caliper Life Science</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>&nbsp;</td>
			<td>Gold Biotechnology</td>
			<td>LUCK-1G</td>
			<td>Potassium Salt</td>
		</tr>
		<tr>
			<td>Xenogen Lumina</td>
			<td>&nbsp;</td>
			<td>Caliper Life Science</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Watch this Scientific Journal Video about Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging at JoVE.com

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

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39.1K Views.  University of California, San Francisco - UCSF. Luciferasi modificati xenotrapianti di tumori al cervello umano può essere stabilita intracranially in topi atimici, con successivo monitoraggio della crescita tumorale e la risposta alla terapia con l'imaging bioluminescenza. In combinazione con l'analisi di sopravvivenza, il monitoraggio bioluminescenza è uno strumento di ricerca indispensabile per test pre-clinici di terapie che sono considerati per il trattamento dei tumori cerebrali.

Video: Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Kinematic Analysis Using 3D Motion Capture of Drinking Task in People With and Without Upper-extremity Impairments

Kinematic analysis is a powerful method for objective assessment of upper extremity movements in a three-dimensional (3D) space. Three-dimensional motion capture with an optoelectronic camera system is considered as golden standard for kinematic movement analysis and is increasingly used as outcome measure to evaluate the movement performance and quality after an injury or disease involving upper extremity movements. This article describes a standardized protocol for kinematic analysis of drinking task applied in individuals with upper extremity impairments after stroke. The drinking task incorporates reaching, grasping and lifting a cup from a table to take a drink, placing the cup back, and moving the hand back to the edge of the table. The sitting position is standardized to the individual&#39;s body size and the task is performed in a comfortable self-paced speed and compensatory movements are not constrained. The intention is to keep the task natural and close to a real-life situation to improve the ecological validity of the protocol. A 5-camera motion capture system is used to gather 3D coordinate positions from 9 retroreflective markers positioned on anatomical landmarks of the arm, trunk, and face. A simple single marker placement is used to ensure the feasibility of the protocol in clinical settings. Custom-made Matlab software provides automated and fast analyses of movement data. Temporal kinematics of movement time, velocity, peak velocity, time of peak velocity, and smoothness (number of movement units) along with spatial angular kinematics of shoulder and elbow joint as well as trunk movements are calculated. The drinking task is a valid assessment for individuals with moderate and mild upper extremity impairment. The construct, discriminative and concurrent validity along with responsiveness (sensitivity to change) of the kinematic variables obtained from the drinking task have been established.

This protocol describes an objective method to evaluate the movement performance and sensorimotor function of the upper extremity applied to individuals with stroke and healthy controls. A standardized test procedure, kinematic analysis and outcome variables for three-dimensional motion capture of drinking task are provided.

Kinematic analysis is a powerful method for objective assessment of upper extremity movements in a three-dimensional (3D) space. Three-dimensional motion ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...