The authors would like to thank the staff from the MRI facilities at the Semmes-Murphey Clinic, Memphis, Tennessee as well as the Neurosurgical department at The University of Tennessee Health Science Center in Memphis, Tennessee.

1. Preparation of Gel Phantoms and Dye
<ol>
	<li>Prepare 0.2% agarose gel by dissolving 2 g of 0.1% agarose powder in 1,000 ml of deionized water. Stir the solution for approximately 1 min to insure proper mixing; and immediately microwave the solution in 3 min intervals for 9 min or until clear, stirring between intervals.</li>
	<li>While the agarose gel is liquid, pour the solution into 5 cm x 5 cm x 5 cm containers. Allow space at the top of the container to add water and allow the agarose gel to cool and settle.</li>
	<li>Once the agarose gel has solidified (approximately 1-2 hr), add 1 cm of water to the top of the gel and refrigerate. It is best to use the gel within 24-48 hr of mixing, but it can be stored for up to a week refrigerated10.</li>
	<li>Prepare a radio-contrast dye in a 60 ml syringe consisting of 50 ml of 0.017% bromophenol blue dye (BPB), and 2 mM of gadoteridol radio-contrast media.
	<ol>
		<li>Combine 8.5 mg of BPB dye to 50 ml deionized water to create a 0.017% BPB solution.</li>
		<li>Add 0.2 ml of stock 0.5 M gadoteridol to the 50 ml 0.017% BPB solution to create a 2 mM gadoteridol solution.</li>
	</ol>
	</li>
</ol>
2. Preparation of Infusion System
<ol>
	<li>Syringe pump infusion system (preferred method): For syringe pump preparation, attach the infusion catheter directly to the syringe through the pressure sensor, reducing the dead volume of the infusion line. The purge function of the syringe pump may be used to clear the line of air using a bolus greater than the priming volume of the catheter at a rate of 10 &#956;l/min.</li>
	<li>Tube pump infusion system (alternate method): Connect the syringe containing the radio-contrast dye to the infusion pump. Attach the pressure sensor to the pump outlet with the transducer attached to the IV monitor. Attach a 16 G infusion catheter to the open end of the pressure sensor. Note: The tip of the 16 G infusion catheter has an inner diameter of 0.2 mm and an outer diameter of 0.35 mm. The tip is made of fused silica and the tip length is 3 mm. It increases to approximately 0.75 mm and continues for 15 mm, the catheter then steps up in a tapered fashion to 1.6 mm or 16 G.</li>
	<li>Prepare for infusion by purging the system for approximately 15 min at 16.667 &#956;l/min to remove any air bubbles. Do not exceed the 16.667 &#956;l/min flow rate, as the machine will cease infusion due to high line pressure. Following attaching the infusion catheter to the line exiting the infusion pump, purge lines of air by using the "Bolus" function on the infusion pump.</li>
	<li>Attach the infusion catheter mount and trajectory frame to the gel phantom container (5 cm x 5 cm x 5 cm) and place in the MRI.</li>
</ol>
3. CED Gel Infusion and MR Scanning
<ol>
	<li>Zero the pressure value (mmHg) recorded by the IV monitor before beginning the infusion.</li>
	<li>Insert the infusion catheter into the agarose gel with the infusion pump running at the lowest flow rate possible, in this case 1.667 &#956;l/min.</li>
	<li>Begin the MR scan, using the parameters listed in Table 1, and continue infusing at a rate of 1.667 &#956;l/min. Infuse the gel at a constant rate until the total volume infused reaches 60 &#956;l (approximately 38 min).</li>
	<li>Scan the gel continuously in 3 min and 50 sec intervals. Record the pressure readings every 60 sec. Once the volume infused reaches 60 &#956;l, turn off the infusion pump; and complete MR scanning while continuing to record pressure readings.</li>
</ol>
4. MR Data Analysis
<ol>
	<li>For analyzing the MR images, use an appropriate DICOM viewer with ROI segmentation functionality.</li>
	<li>Select the correct frame in each scan marked by the cross section of the catheter as seen in Figure 1.</li>
	<li>Using the "ROI - rectangle" tool, select the largest portion of the gel that does not include any portion of the infusion site. The software will output a mean pixel density with standard deviation. Find the value that corresponds to three standard deviations from the mean. This value is used as the threshold for determining when contrast is present with a confidence of 99.7%.</li>
	<li>Using the "ROI - circle" tool, encircle the infusion site with a large enough circle and give this a unique name.</li>
	<li>Select the circle and using the "ROI - set pixel values to" tool, input threshold value found in step 4.3 into "if current value is larger than:" box and checkmark this line only. Then in "to this new value:" box, enter a large value (25,000). Reset the pixel density to select the area encompassed by the threshold previously defined.</li>
	<li>Next, using the "ROI - grow region (2D/3D segmentation)" tool, select 2D growing region, confidence algorithm with initial radius parameter = 2, and brush ROI. Click inside of the infusion site for the software to compute the total area of this region.</li>
	<li>Assuming a spherical infusion cloud, calculate the volume of diffusion from the area via the following equation: V= 4/3&#960;(&#8730;(Area/&#960;))3</li>
</ol>

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The authors declare that they have no competing financial interests.

The critical steps for ensuring the success of the infusion are: purging the infusion line of air, mixing the agarose gel, analyzing the MR data, using small inner catheter diameters, using stepped catheter designs to minimize backflow, and minimizing the pressure felt by the gel or tissue into which the drug is being infused. As previously stated, the main detriment to the success of the infusion is infusion line air. Correctly and thoroughly purging the infusion line of air is critical to ensure no air enters the infusion. Equally as important is mixing of the agarose gel. Improper synthesis of the gel could lead to large variations in concentration and consistency, which in turn will cause fluctuations in infusate distribution. Consistent MR data analysis is key for accurate and objective measurements of Vd and Vd/Vi ratios. Precisely following the steps outlined in the protocol provides a consistent method for analyzing the MR data.
However, these techniques are not without limitations. When inserting the catheter, the agarose gel may fracture or lacerate unpredictably10. This unpredictable nature of the agarose gel could cause changes in the gel-catheter interface making it dissimilar from human brain tissue and unusable. Additional limitations may arise from assuming a spherical infusion cloud for calculation of the Vd. While spherical infusion clouds were common with infusions into agarose gel, attempting to reproduce the infusion in vivo may yield different results. Brain tissue is more anisotropic, more heterogeneous, and contains more regional anatomical boundaries than agarose gel and therefore will cause variance in infusion cloud morphology10. It is important to note that these Vd were approximated grossly, without taking into account the presence of magnetic susceptibility artifacts, which may decrease the signal intensity of the infusate cloud. For the purpose of this manuscript we made the assumption of anisotropic diffusion and we understand that this is an estimate. Additional modifications to the current technique may exist to improve consistency and accuracy of the results. Modifications may include additional purging of the lines to further prevent air in the infusion or using software to more accurately calculate a 3D volume to avoid assuming a spherical infusate cloud. Methods of computer modeling as described by Linninger et al. may be used to more accurately predict and measure the CED infusion cloud volume24,25.
Compared to existing methods that require cadaveric or animal brain tissue, agarose gel provides a more easily accessible model for CED testing. The translucent nature of agarose gel also offers real-time visualization of the infusion. This real-time visualization gives the trainee the ability to see reflux or air bubbles in the infusion before it is detected by the MR, allowing for quick correction and modification. For future applications, agarose gel provides the accessible models needed for future testing, research, and training in CED.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a broad spectrum of neurological disorders including malignant brain tumors, epilepsy, metabolic disorders, neurodegenerative diseases (such as Parkinson disease)1, stroke, and trauma2. CED employs positive pressure bulk flow for the distribution of a drug or other infusate. CED provides safe, reliable, and homogenous delivery of molecular weight compounds, ranging from low to high, at clinically relevant volumes3. Traditional drug delivery to brain tissue is severely restricted by the blood-brain barrier4. Formed by the tight junctions between endothelial cells that make up the capillaries in the brain, the blood-brain barrier blocks polar and high molecular weight molecules from entering the parenchyma of the brain. Direct intraparenchymal brain infusion via CED can overcome the limitations of previous therapeutic drug delivery modalities and allows the use of therapeutic agents that would not cross the blood-brain barrier, and therefore have been previously unavailable as viable treatment options5.
Researchers from the US National Institutes of Health (NIH) described CED in the early 1990s as a means of achieving greater therapeutic drug concentrations than by diffusion alone6-8. The first methods of CED involved implanting one or more catheters into the brain, connecting an infusion pump to the catheter, and pumping the therapeutic agents directly into the targeted region. The increased distribution fraction and relatively stable concentration is reported to occur as the positive pressure created by the infusion pump causes the tissues to dilate and allow for permeation of the drug9.
The fundamental technique for CED remains largely the same as it was first described. Advances in catheter design10, infusion technique11, line pressure monitoring2, and real time MRI monitoring to correct for brain shift12,13, optimize multiple collinear infusions14, and monitor for infusate loss15 have increased the safety and efficacy of the treatment10. Additional importance has been placed on the catheter design and infusion strategy including flow rate. Successful CED, with limited catheter reflux and tissue damage, has been correlated with catheter design and infusion rate. The use of a catheter with a narrow diameter and a low infusion rate to limit backflow along the brain-catheter interface as well as limit damage at the catheter tip16. MR imaging provides visual confirmation of the correct location for infusion catheter placement, and thus drug delivery, while also allowing for correction of infusion reflux or aberrant delivery17. MR images can also be used to approximate and track the volumes of distribution (Vd) of the infused drug. The Vd is calculated using an MR imaging signal intensity value greater than three standard deviations above the mean from the surrounding non-infused gel as a threshold for segmentation18. The Vd is a useful measurement for CED because it represents the volume of the drug distributed in the brain. Along with the volume infused (Vi), a ratio can be generated (Vd/Vi) quantifying the volume covered by the infused drug.
Agarose gel phantoms mimic several crucial mechanical properties of the human brain important for understanding CED such as: Vd, gel-catheter interactions, poroelastic properties, and infusion cloud morphology10. Mixtures of 0.2% agarose gel have been shown to mimic in vivo changes in local pore fraction caused by gel dilation due to CED. A similar pore fraction to human brain promotes similar interactions and accurate measurements of Vd19. Additionally, similar concentrations of agarose gels such as 0.6% and 0.8% have shown similar infusion pressure profiles to the brain20. Further, the translucent agarose gels provide the advantage of real-time visualization of catheter placement and infusion reflux. Agarose gel phantoms are relatively inexpensive to produce. The cost of the agarose gel phantoms may be key to future widespread training throughout neurological surgery. Due to these properties, agarose gels provide a useful surrogate, replicating many of the key attributes of human brain infusions without the use of brain tissue.
As stated above, image-guided CED into agarose gel models provides a beneficial in vitro method for testing, research, and training.&#160; The purpose of this article is to describe how to recreate agarose gel phantoms, to outline appropriate CED testing and analysis protocols, and to address common errors faced during CED infusions for the treatment of neurological disease.

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Prohance</td>
			<td style="width:71px;">Bracco</td>
			<td style="width:119px;"></td>
			<td style="width:195px;">Gadoteridol radio contrast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Bromophenol Blue Dye</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-0404</td>
			<td>Dye for infusate visualization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Agarose Gel Powder</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-3101EDU</td>
			<td>Agarose powder for creating gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Veris MR Vital Signs Monitor</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pressure monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">16 Gauge SmartFlow Catheter</td>
			<td style="width:71px;">SurgiVision</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Continuum MR Infusion System</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">SMART Frame MRI Guided Trajectory Frame</td>
			<td style="width:71px;">ClearPoint</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Osirix Imaging Software and DICOM Viewer</td>
			<td style="width:71px;">Osirix Imaging Software</td>
			<td style="width:119px;"></td>
			<td>OsiriX 32-bit DICOM Viewer</td>
		</tr>
	</tbody>
</table>

image-guided convection-enhanced delivery into agarose gel models of the brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Interpreting and analyzing CED infusions involve several important factors such as distribution fraction and infusate reflux. The distribution fraction calculation depends heavily upon the calculation of the Vd. Therefore accurate interpretation of the MR images is critical. We propose a semi-automated method for reliably reproducing these measurements as listed above. These methods objectively determine the cross sectional area of the infusate cloud and an approximate radius. While variable, in agarose gel the infusion cloud often proved spherical. &#160;Assuming a spherical infusate cloud, this radius can be used to determine the Vd for the CED infusion. Vd/Vi for the agarose gel infusion can then be calculated with the measured volume infused. Agarose gel at 0.2% concentration has proven a reasonable representation of brain tissue with a Vd/Vi ratio of 5.0&#160;10, falling in between measured Vd/Vi ratios of brain tissue ranging from 3.1 to 5.2&#160;21,22.
The pressure measurements taken during the CED infusion are also important for ensuring the infusion remains stable and constant. Detected pressure spikes may indicate errors in the infusion such as air bubbles or blockages in the catheter. The pressure profile of the infusion is expected to initially peak before decreasing to a relatively stable plateau for the duration of the infusion20.
The main detriment to the success of the infusion is air in the infusion line. Air alters the measurement of the infusion pressure as well as the volume of the dye being infused. It may also cause local tissue disruption and affect the distribution of the infusate. A successful study was performed using coaxial catheters that yielded parameters to minimize or eliminate the effects produced by air escape into the infusion site23. From our study, we identify a need for future investigations into proper methods of CED infusions using single cannula catheters such as the SmartFlow catheter to equally minimize or eliminate the presence of air.
A key parameter for identifying the presence of air in the infusion line is infusion pressure. As shown by the infusion pressure (mmHg) line in Figure 2, there is a spike in the infusion line pressure at the same time that air is introduced into the catheter line. Comparing the pressure readings to the MR image timestamps, a pressure spike may indicate the presence of an air bubble prior to MR image confirmation. This suggests pressure may be a potential warning marker for detecting and preventing the inappropriate delivery of air in vivo. There was time between the initial spike in pressure and when the air was actually delivered into the gel. That is important to note since air should not be infused into the brain during an actual procedure. If increased pressure was observed in an actual case there might be ample time to keep air from reaching the infusion site within the brain.
Once the air reaches the catheter tip, growth of the air bubble can be seen in the MR images as shown in Figure 3, panels A-F. The air bubble causes enlargement and irregularity of the dye acorn and also alters the measurement of Vd. Thus, it is important to identify and validate a method to prepare the system that consistently ensures it is devoid of air prior to placing the catheter, making certain air does not impair the infusion. One way to prevent air entering the catheter may be to begin the infusion prior to insertion of the catheter into the agarose gel.&#160;
Backflow of the infusate along the catheter-gel interface can adversely affect the infusion by allowing the infusate to exit the target. While backflow can occur at any point during an infusion, there is an increased incidence of backflow at the start of the infusion and when increasing the infusion rate10. Backflow has also been associated with the presence of air bubbles, catheter insertion technique, and catheter design, though back flow may still occur despite controlling for these variables23. To minimize backflow, a stepped, reflux resistant catheter was used, and the infusion rate was held constant and as low as possible (1.667 &#956;l/min). One can also avoid unnecessary backflow by preventing pressure spikes. Along with catheter diameter, initial infusion pressure spikes (IIPS) (associated with the expulsion of an end-catheter occlusion) have been shown to increase the probability that backflow will occur. Therefore, a &#8220;trickle&#8221; technique was used where the infusion was initiated at the minimum rate just prior to insertion. Porous membrane catheters, as well as valve tip catheter designs, have been proposed to mitigate end port occlusions and associated IIPS.
<img alt="Table 1" fo:content-width="6in" src="/files/ftp_upload/51466/51466table1highres.jpg" width="500" /> 
	Table 1.&#160;Imaging parameters and values used for the MR scan of the infusion.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51466/51466fig1highres.jpg" width="500" /> 
	Figure 1.&#160;Panel 1 showing a picture of the catheter mount and agarose gel alongside panel 2 containing a MR image of the agarose gel showing a cross section of infusion catheter. Labels as follows: the MR visible trajectory guide can be seen by labels A and B, the infusion catheter by label C, the water on top of the agarose gel by label D, pooling contrast agent at the gel water interface by label E, the agarose gel by label F, and the infusion cloud by label G.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51466/51466fig2highres.jpg" width="500" /> 
	Figure 2.&#160;Graph demonstrating the effects of air on the CED infusion. Air was noted in the infusion line 15 min into the infusion. At 17 min a spike in the pressure was recorded, as shown by the green line. The air bubble also has a drastic effect on the Vd and Vd/Vi ratio as seen by the blue and brown dashed lines respectively. Upon air entering the line, the Vd spiked from approximately 5 to 9 &#956;l; while the Vi remained linear. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51466/51466fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51466/51466fig3highres.jpg" width="500" /> 
	Figure 3.&#160;Magnetic resonance images showing growth of the infusion cloud and enclosed air bubble. The first image shows the gel prior to insertion of the catheter, the second image shows the insertion of the catheter after beginning the infusion, and the subsequent time lapse shown is in approximately 4-min intervals. The air bubble distorts the true volume of the infusate cloud and prevents accurate measurement of Vd. Air was seen entering the infusion catheter immediately prior to the MR scan. Panels A-F correspond to points A-F in Figure 2, demonstrating the progression of the infusion.

Watch this Scientific Journal Video about Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain at JoVE.com

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

image-guided-convection-enhanced-delivery-into-agarose-gel-models

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K Views.  University of Tennessee Health Science Center. Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Video: Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...