Name: a simple guide screw method for intracranial xenograft studies in mice
Uploaded: 2011-09-26T13:00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

The authors would like to thank Verlene Henry and Lindsay Holmes for assistance in developing this model. This work was partially funded by the James S. McDonnell Foundation (#220020173).

1. Cell lines
<ol>
	<li>U87MG glioma cells are cultured in large tissue culture flasks with DMEM-F12 supplemented with 5% fetal bovine serum (FBS).</li>
	<li>Cells are harvested by washing flasks twice with warm Phosphate Buffered Saline (PBS) and incubating them at 37&deg;C for 5 minutes with 10 ml of PBS containing 0.25% trypsin and 0.05% EDTA. Once cells are lifted, they are placed into a 50 ml tube containing 10 ml of culture media and centrifuged (300 X g for 4 min).</li>
	<li>Following the wash, cells are resuspended at a concentration of 10 x 106/ml in culture media, which allowed for an inoculation of 50,000 cells/5 &mu;l.</li>
	<li>Cells are kept on ice until intracranial injection.</li>
</ol>
2. Guide screw intracranial bolting
This procedure can be carried out several days prior to the injection of cells. All procedures described here have been carried out under strict sterile conditions.
<ol>
	<li>Mice (BALB/c nu/nu female; 5-6 weeks; approximately 18g) are numbered for identification purposes, weighed and anesthetised with an intraperitoneal (IP) injection of a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg).</li>
	<li>The skin is wiped down with an iodine solution and a small incision (2-3 mm) is made along the right side of the midline and anterior to the interaural line. This exposes the coronal and sagittal sutures of the skull. The bregma is positioned at the junction of these two sutures.</li>
	<li>The guide screw entry point is then marked at a point 2.5 mm lateral and 1 mm anterior to the bregma. This point is located directly above the caudate nucleus1.</li>
	<li>A 1 x 1 mm deep hole is drilled with a sterile hand held twist drill through the skull to the dura.</li>
	<li>A sterilised guide screw that will extend 1.6 mm below the skulls surface into the dura is then bolted into the hole with a screw driver until flush with the skull (Figure 1A).</li>
	<li>A sterile stylet or screw dummy is then placed into the central hole of the guide screw to close the opening (Figure 1B).</li>
	<li>The wound is closed with Vetbond Tissue Adhesive (n-butyl cyanoacrylate) and the mice are given an intraperitoneal injection of reversine (small animals) (0.1 ml/kg) and carprofen (5 mg/kg/100 &mu;l) for analgesia. Mice are then allowed to recover on a warming mat (36&deg;C) which can take up to 20 minutes. Mice are frequently monitored and observed during this recovery time until they are fully conscious.</li>
</ol>
3. Intracranial cellular engraftment
<ol>
	<li>A sterile cuffed Hamilton syringe is prepared by applying a small plastic ring to the needle tip allowing only 2 mm of the needle to extend below the guide screw outlet (Figure 1B). The final inoculation point is therefore 3.5 mm below the skull surface.</li>
	<li>Four days following the guide screw surgery, the mice are again anesthetized as above (2.1) and a small incision is made over the guide screw to remove the stylet.</li>
	<li>The cuffed Hamilton syringe is then filled with 5 &mu;l of well mixed cells taking precaution not to create or draw up any air bubbles.</li>
	<li>The syringe is secured to the perfusion pump and the needle is inserted into the guide screw (Figure 1C). The cells are then infused at a rate of 30 &mu;l per hour.</li>
	<li>The automated apparatus (Figure 1D) allowed us to inject up to 10 animals at a time at a constant flow rate. Cells can also be injected manually by using the cuffed hamilton syringe or alternatively the syringe can be cuffed by a 200 &mu;l pipette tip that has been trimmed by 3 mm to allow the needle tip to extend beyond the guide screw by 2 mm. The injection must then be performed at a constant steady pace.</li>
	<li>When infusion is complete, the syringe is carefully removed and the stylet is replaced into the guide screw. The wound is glued closed and the mice are given recovery medications as before (2.7) and allowed to recover on a warming mat.</li>
</ol>
4. Therapeutic challenge
<ol>
	<li>Four days after the U87MG cell inoculation, mice are weighed and randomly divided into control and treatment groups.</li>
	<li>The control group is given an intra-peritoneal (IP) injection of PBS (100 &mu;l), while the treatment group is given an IP injection of AMG102 (100 &mu;g in 100 &mu;l of PBS).</li>
	<li>Injections are repeated every second day for 14 days, a total of 6 injections (Figure 2).</li>
	<li>After the final injection, mice are monitored daily and weighed every second day.</li>
	<li>When mice began to display significant neurological dysfunctions (balance disturbance, paralysis), dehydration, or more than 10% weight loss or moribund, they were humanely euthanised. These humane end-point days were then recorded on the Kaplan-Meier survival curve.</li>
</ol>
5. Evaluation of therapeutic efficiency
<ol>
	<li>The Kaplan-Meier survival curve was generated using GraphPad Prism 4.03 for Windows; GraphPad Software, San Diego California USA, <a href="http://www.graphpad.com">www.graphpad.com</a>3. Deaths or euthanizations were marked as end-point events and scored as 1. Animals that remained alive at the end of the experiment were recorded as non-events and scored as 0.</li>
</ol>
6. Representative results:
Control animals began to show signs of neurological disturbance and weight loss 23 days after the initial inoculation of cells. This was significantly delayed in the AMG102 treated group to 35 days. Indeed by day 35, 77% (n = 7/9) of the control group had been euthanized. The Kaplan-Meier survival curve clearly demonstrates the significant increase in survival in response to AMG102 treatment (Figure 3). By day 70, 88% of control mice had been euthanized (n = 8/9) compared to 37% (n = 3/8) of AMG102 treated group. Histological analysis of the brains confirmed that all 9 animals in the control group developed tumors compared to only 3 of the 8 AMG102 treated mice (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/3157/3157fig1.jpg" /> 
Figure 1: Image (A) shows intracranial guide screw positioned in an area located directly above the caudate nucleus. The cuffed hamilton syringe (pre-loaded with cells) is then placed inside the guide screw so that 2 mm of needle extends beyond the guide and the cells are injected 3.5 mm inside the caudate nucleus. The syringe is later removed and replaced by a stylet (B). The automatic infusion device (C) can inject up to 10 animals at a time. Shown here are five simultaneous injections (D).
<img alt="Figure 2" src="/files/ftp_upload/3157/3157fig2.jpg" /> 
Figure 2: Schematic of study protocol. Cells were injected on day 0 with treatment commencing on day 4. Control animals received an injection of PBS (100 &mu;l) IP while the treatment group received an injection of AMG102 (100 &mu;g/100 &mu;l) IP. These injections were given every second day for 10 days. A total of 6 injections were given. Animals were then monitored for 70 days for signs of neurological and physical disturbances.
<img alt="Figure 3" src="/files/ftp_upload/3157/3157fig3.jpg" /> 
Figure 3: Kaplan-Meier survival curve comparing control and AMG102 treated mice. Significant survival was obtained following treatment with AMG102 (p=0.005).
<img alt="Figure 4" src="/files/ftp_upload/3157/3157fig4.jpg" /> 
Figure 4: Microphotographs of coronal brain sections demonstrating tumor development in the control animals (A) and no brain tumor development in the AMG102 treated animals (B). Scale bar is 1 mm.

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

This work was supported by a Research Grant from Amgen Inc.

The intracranial guide screw method presented here enables the rapid and reproducible establishment of intracranial xenografts and in the hands of a trained animal technician, this procedure is very easily performed without the need for a stereotactic device. Our method combines the use of a guide screw, to accurately locate the region of brain most suitable for in vivo tumor development, and an automated infusion pump that allows for the simultaneous inoculation of up to 10 animals at a time. The guide screw augmentation procedure takes less than 5 minutes to perform. The automated infusion of cells at 30 &mu;l/hour takes just 10 minutes for a 5 &mu;l volume. This ensures accurate dosing at a constant rate and reduces the risk of cell expulsion into the extra-cranial area due to increased intracranial pressure. We have also performed this procedure with 10 &mu;l volumes and have not observed any extra-cranial tumor growth due to cellular reflux. As the automated infusion pump also allows several animals to be inoculated simultaneously we can inject up to 60 animals per hour compared to a stereotactic procedure which is limited to approximately 15 animals per hour. 
The most critical step in this procedure is the location of guide screw in the skull as its position determines the ultimate location of the tumor bulk. If the guide screw is too central, the cells will be injected into a region between the two brain hemispheres whereas if the guide is placed to far forward, the cells will not be injected into the brain at all. It is therefore essential to be as accurate as possible in locating the guide 2.5 mm lateral and 1 mm anterior to the bregma1. In this study we used a steel guide screw, although plastic guide screws are also available for those studies that require brain imaging such as MRI or PET6. Even if the automated infusion pump is cost prohibitive for some groups, cells can still be injected manually more quickly than stereotactic approaches. Overall, this rapid, accurate and reproducible method can be used to study glioma, medulloblastoma and brain stem tumors7. This procedure has been performed on over 500 mice between our two laboratories with deaths due to unexpected anesthetic reactions (&lt;1%). Mice that were bolted with the guide screw and subsequently infused with cells did not die or suffer any neurological complications as a result of the procedure. Animals have survived past 100 days without any complications. Although we preferred to use a hairless mouse, an immuno-compromised mouse with hair like the SCID would be just as appropriate provided the skull hair was removed prior to the surgery to keep the site clean and to prevent hair interfering with guide screw.
There are several novel therapeutics currently being developed for the treatment of glioma8-10. Recently we showed that subcutaneous U87MG glioma xenografts can be inhibited by treatment with AMG1023; a humanized antibody directed to HGF currently undergoing clinical evaluation in glioma5. We used the method describe here to establish intracranial U87MG tumors in the brains of nude mice. Our study clearly shows the utility of the method and demonstrates that AMG102 significantly inhibits the growth of orthotopic U87MG xenografts. We will continue to use this model to determine what other therapeutics can be used in combination with AMG102 to obtain more effective inhibition of tumor growth.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Bovogen</td>
			<td>SFBS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin		</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Balb/c nu/nu mice	</td>
			<td>ARC Perth </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screw Holder		</td>
			<td>Plastics One</td>
			<td>SD-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Drill Holder</td>
			<td>Plastics One</td>
			<td>	DH-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Drill Bit</td>
			<td>Plastics One</td>
			<td>D#60</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screw guide (metal)</td>
			<td>Plastics One</td>
			<td>C212SG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screw Dummy (stylus)	 </td>
			<td>Plastics One</td>
			<td>	C212SD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton Syringe (10 &mu;l 26g cemented needle)</td>
			<td>Grace Division Discovery Science</td>
			<td>80075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHD 22/2000 infusion pump		</td>
			<td>Harvard Apparatus</td>
			<td>70-2003	</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Vetbond		</td>
			<td>3M </td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermo pad		</td>
			<td>Harvard Apparatus</td>
			<td>340390</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a simple guide screw method for intracranial xenograft studies in mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

a-simple-guide-screw-method-for-intracranial-xenograft-studies-in-mice

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Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...