All authors contributed towards the establishment and validation of the presented assay and to the draft of the manuscript.

All animal experimental procedures in the protocol follow the animal care guidelines of Technische Universit&#228;t M&#252;nchen, Germany.
1. Media/Extract Preparation
<ol>
	<li>Tissue homogenization 
	The quality of tissue homogenization is critical to the subsequent detection of neuroplastic alterations in the cultured neurons. Here, a homogenizer is recommended which enables tissue dissociation without major increase in the tissue temperature.
	<ol>
		<li>Transfer 5 mm x 5 mm x 5 mm cubes of pancreatic tissue directly from -80 &#176;C to liquid nitrogen and place in a solid phase in the tissue homogenator. The ideal homogenator would dissociate the tissue fast enough, without allowing it to defreeze.</li>
		<li>Immediately after dissociation, resuspend the solid, powder-like homogenate in 300-500 &#181;l of 0.1x phosphate-buffered saline (PBS). Here, do not use any lysis buffer (such as RIPA) since this may lyse the neurons.</li>
		<li>Centrifuge the homogenates for at least 15 min at the maximum speed of your bench centrifuge (e.g. 21,130 x g). Collect the clear supernatant which represents the tissue extract.</li>
	</ol>
	</li>
	<li>Cell line supernatants
	<ol>
		<li>Let the cells of interest reach at least 70 - 80% confluence in normal growth medium.</li>
		<li>Usually, these media contain serum components and may exert uncontrollable effects upon neuronal growth. Therefore, after reaching the desired cell density, wash your cells at least 3x with cell-culture grade PBS and place them in serum-free medium (SFM) for up to 48 hr. 
		Note: Here, consider that some cells (like pancreatic stellate cells) may need serum in their growth media in order to maintain their normal state and structure. In such cases, perform serial serum dilutions in the medium of the cell of interest in order to find the lowest possible serum content necessary for intact cell function.</li>
		<li>Measure the protein concentration of the tissue homogenate or cell supernatants via Bradford protein assay. While these values vary depending on the tissue and cell type, for pancreatic tissue extracts, one would expect a concentration range between 5-12 &#181;g/&#181;l, and for cell line supernatants between 3-8 &#181;g/&#181;l.</li>
	</ol>
	</li>
</ol>
2. Aliquot Extracts and Cell Supernatants
Depending on the concentration to be used in the assay,&#160;the final concentration of the extract or supernatant in the neuronal medium should be 100 &#181;g/ml 5,8.
Note: For an assay with neurons growing in 500 &#181;l medium in each well of a 24-well plate, one needs 50 &#181;g of protein from each extract or supernatant per well. At a typical extract concentration of around 10 &#181;g/&#181;l, one would need 5 &#181;l of extract/supernatant for each well. In every setting performed as a triplicate, this would correspond to 15 &#181;l of extract/supernatant. For different tissue or cell types, perform serial dilutions of the final extract or supernatant concentration and compare the observed neurotrophic effects between different extract/supernatant concentrations.
3. Isolation of Neurons
Once the extract/supernatant collection is finished, carry on with isolation of neurons.
<ol>
	<li>For DRG neurons, collect the cervical to lumbar DRG of newborn rats between postnatal day (P) 2-12 after decapitation and stereomicroscopic dissection of DRG (Figure 1A). In order to have sufficient DRG neurons for a 24-well plate, collect all cervical to lumbar DRG of one newborn rat (equaling 52 DRG) per plate.
	<ol>
		<li>Cut away the peripheral (neural) and central projections (roots) of DRG by means of microscissors. 
		Leaving the projections in place impedes trituration and increases the contamination risk of culture by fibroblasts.</li>
	</ol>
	</li>
	<li>For MP neurons, cut away the mesentery from the small intestine and manually and carefully strip off the seromuscular layer of the small intestine (for a detailed protocol on MP isolation, refer to Sch&#228;fer et al.9, Figure 1B). For MP, collect the plexus from two rats per 24-well-plate. 
	Note: Try to be as gentle as possible in order to avoid tears in the seromuscular layer since they impede its successful separation from the small intestine.</li>
	<li>Collect the DRG and seromuscular layer in ice-cold minimal essential medium (MEM) supplied with gentamicin (20mg in 500ml medium) and metronidazol (2.5 mg in 500 ml medium).</li>
	<li>Following collection of DRG, incubate them in Hank&#8217;s Balanced Salt Solution (HBSS) supplied with collagenase Type II for 20-30 min. For MP isolation, incubate in collagenase type between 1-3 hr, depending on the age of the animal (Figure 1C).</li>
	<li>For MP, collect the net-like MP pieces under stereomicroscope and transfer to ice-cold MEM.</li>
	<li>Then triturate the DRG and MP through syringes with decreasing diameter. 
	Note: Excessive trituration can destroy the neurons, but less the glia cells.</li>
	<li>Once the medium containing the DRG or MP has become cloudy, centrifuge the suspension at 93.9 x g for 5 min, discard the medium and resuspend in Neurobasal medium (supplemented with 100 U/ml penicillin, 100 &#956;g/ml streptomycin, 0.5mM L-glutamine and 2% B-27).</li>
	<li>Count the total number of cells (i.e. neurons AND glia) by means of a hemocytometer. 
	Note:&#160;The number of needed cells is dependent on the measurement parameter. For the quantification of neurite density, one needs denser cultures and thus a greater number of cells than for the measurement of neurite outgrowth, perikaryonal size and branching pattern of individual neurons. For neurite density measurements, use e.g. 10,000 cells (neuron+glia)/well or 1,500 neurons/well. For morphometry on individual neurons, brief (1 minute-long) trypsination of cells and seeding of 2,500 cells/well or 400 neurons/well is recommended. The typical yield of cells obtained from one rat is around 300,000-500,000 cells.</li>
	<li>Seed the cells on 13 mm coverslips which have been coated the night before and top the wells with Neurobasal medium (supplemented with 100 U/ml penicillin, 100 &#956;g/ml streptomycin, 0.5 mM L-glutamine and 2% B-27) (Figure 1D). 
	Note: Use poly-D-lysine (40 mg/m2) OR ornithine- and laminin-coated (1 mg/ml each) coated cover slips. Cells attach to poly-D-lysine extremely fast, thus making it suitable for experiments in which many cells are needed (i.e. neurite density measurements). Attachment to laminin is in general somewhat weaker, but laminin is a strong promoter of neurite outgrowth. Therefore, for measurements on neuronal branching and neurite length, prefer ornithine/laminin-coating.</li>
	<li>Allow the cells attach to the wells overnight. On the next day, prepare the extract-supplemented media (at a final extract/supernatant concentration of 100 &#181;g/ml).</li>
	<li>Aspirate the seeding medium, and perform an optional, very gentle wash with PBS, and then slowly pipette the extract/supernatant-supplemented media (Figure 1E).</li>
	<li>Let the cells grow for 48 hr. Aspirate the media and fix in 4% paraformaldehyde for immunostaining (Figure 1F).</li>
	<li>Perform double immunofluorescence staining using neuron-specific (e.g. beta III-tubulin) and glia-specific (e.g. glial fibriallary acidic protein/GFAP) markers (Figure 1G).</li>
	<li>For morphometry, use an inverted light microscope equipped with a CCD camera in combination with automated software which allows measurement of neurite density (see Table).</li>
	<li>The neurite density of neuronal cultures is measured on 4-5 representative photomicrographs at 200x magnification from 4 different regions of densest growth on each coverslip by overlaying a 50 &#181;m&#160;x 50 &#181;m grid and counting the fiber&#160;density per square measured in the intersecting fibers. Neurite outgrowth, mean number of branches per neuron, the mean branch length and perikaryonal size can be measured from randomly selected 30 solitary neurons from each coverslip by marking the neurites and perikarya (Figure 1H).</li>
</ol>

<ol>
	<li>Demir, I. E., Schafer, K. H., Tieftrunk, E., Friess, H., Ceyhan, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+plasticity+in+the+gastrointestinal+tract:+chronic+inflammation,+neurotrophic+signals,+and+hypersensitivity.">Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity.</a> Acta Neuropathol. 125, 491-509 (2013).</li><li>Vasina, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neuroplasticity+evoked+by+inflammation.">Enteric neuroplasticity evoked by inflammation.</a> Auton. Neurosci. 126-127, 264-272 (2006).</li><li>Lomax, A. E., Fernandez, E., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+enteric+nervous+system+during+intestinal+inflammation.">Plasticity of the enteric nervous system during intestinal inflammation.</a> Neurogastroenterol. Motil. 17, 4-15 (2005).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural Invasion in Pancreatic Cancer: The Past, Present and Future. 2, 1513-1527 (2010).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+in+chronic+pancreatitis+and+pancreatic+cancer+induces+neuronal+plasticity.">The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity.</a> Neurogastroenterol. Motil. 22, 480-490 (2010).</li><li>Schafer, K. H., Mestres, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GDNF-induced+neurite+outgrowth+and+neuronal+survival+in+dissociated+myenteric+plexus+cultures+of+the+rat+small+intestine+decreases+postnatally.">The GDNF-induced neurite outgrowth and neuronal survival in dissociated myenteric plexus cultures of the rat small intestine decreases postnatally.</a> Exp. Brain Res. 125, 447-452 (1999).</li><li>Schafer, K. H., Van Ginneken, C., Copray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+and+neural+stem+cells+in+the+enteric+nervous+system.">Plasticity and neural stem cells in the enteric nervous system.</a> Anat. Rec. 292, 1940-1952 (2009).</li><li>Liebl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+severity+of+neural+invasion+is+associated+with+shortened+survival+in+colon+cancer.">The severity of neural invasion is associated with shortened survival in colon cancer.</a> Clin. Cancer Res. 19, 50-61 (2012).</li><li>Sch&#228;fer, K. H., Saffrey, M. J., Burnstock, G., Mestres-Ventura, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+isolation+of+myenteric+plexus+from+the+newborn+rat+gastrointestinal+tract.">A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.</a> Brain Res. Brain Res. Protoc. 1, 109-113 (1997).</li><li>Ceyhan, G. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+and+artemin+are+paracrine+mediators+of+pancreatic+neuropathy+in+pancreatic+adenocarcinoma.">Nerve growth factor and artemin are paracrine mediators of pancreatic neuropathy in pancreatic adenocarcinoma.</a> Ann. Surg. 251, 923-931 (2010).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+plasticity+in+chronic+pancreatitis+is+mediated+via+the+neurturin/GFRalpha2+axis.">Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRalpha2 axis.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 303, 1017-1028 (2012).</li><li>Joseph, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+are+multipotent+in+culture+but+primarily+form+glia+in+the+adult+rodent+gut.">Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.</a> J. Clin. Invest. 121, 3398-3411 (2011).</li></ol>

The authors have no competing interests and no financial disclosures.

The present protocol is intended to illustrate the methodology behind the in vitro pancreatic neuroplasticity assay which was recently developed by our group to study the mechanisms of neuroplasticity in PCa and CP5. The protocol involves a three-day procedure which can easily be applied once the performer has gained sufficient experience in the isolation and culture of DRG and MP neurons. Furthermore, it represents a valuable tool for studying the concomitant reaction of enteric and DRG-associated gl...

Alterations in gastrointestinal (GI) nerve morphology and density have caught the attention of gastroenterologists and pathologists for a long time, but their relevance for the pathophysiology of GI diseases remains unknown1-3. Indeed, several highly common GI disorders such as gastritis, reflux esophagitis, colitis, diverticulitis, and appendicitis are associated with increased innervation density in inflamed tissue areas1. However, no genuine attention has so far been paid to the mechanisms and meaning of neuroplasticity in the GI tract. Do morphologically altered GI nerves differ from normal GI nerves, i.e. the normal state of the enteric nervous system, in terms of their function? What are the implications of altered neuropeptide/neurotransmitter content in plastic enteric nerves? Does peripheral neuroplasticity always entail altered signaling to the central nervous system? And where are the central projections of plastic extrinsic GI neural pathways? A long series of such key questions can easily be generated when looking at the paucity of our knowledge on the functional aspects of GI neuroplasticity.&#160;

The study of GI neuroplasticity at functional level requires valid, reproducible and still easily applicable experimental models. In an era of increasing popularity and acceptance of genetically engineered conditional mouse models (GECoMM), such in vivo settings bear the potential to elucidate previously unknown facets of GI neuroplasticity in a realistic fashion1. However, the design and production of GECoMM remains costly, labor-intensive and, especially, time-consuming. Furthermore, they require the a priori selection of the target to be conditionally modulated in the genetically altered mouse (e.g. transgenic overexpression of nerve growth factor/NGF in enteric epithelial cells). Hence, for the design of a successful GECoMM, researchers need some indicators (e.g. previous experimental data) of a worthwhile target, i.e. that the molecule of interest (here NGF) can at least be expected to exert some biologically relevant effects on GI nerves.

Such indicators can easily be derived from adequate in vitro models in which isolated cell subtypes from the complex microenvironment of an in vivo system can be selectively cocultured in a heterotypic manner4-7. The modulation of molecular targets in such a heterotypic culture setting is on average technically less cumbersome, faster, and can therefore aid in the prefiltering of worthwhile targets for verification in in vivo studies.
Recently, we presented an in vitro neuroplasticity assay which was designed to simulate the increased neural density and hypertrophy of intrapancreatic nerves in human pancreatic cancer (PCa) and chronic pancreatitis (CP) tissues. Here, neurons derived from newborn rat dorsal root ganglia (DRG) or myenteric plexus (MP) were exposed to tissue extracts from surgically resected PCa or CP tissues specimens and compared to those cultured in normal human pancreas (NP) tissue extracts5. Instead of tissue extracts, one can also use cell line supernatants to study the impact of selected cell types on neuroplasticity. When combined with a standardized morphometric measurement, the presented neuroplasticity assay allows valid and reproducible assessment of neuronal plasticity in response to different pancreatic microenvironments. Particularly, it allows the simulation of 1) morphological neuroplasticity, i.e. the changes in the neurite outgrowth, branching pattern and neuronal size, and 2) functional neuroplasticity, i.e. alterations in the excitability of peripheral neurons. Moreover, not only peripheral (i.e. enteric), but also central (e.g. DRG or second order spinal) neurons can be included in the present assay to assess their morphological and functional reaction to different GI tissue contents. In the present video tutorial, we demonstrate the technical protocol for the performance of this assay and discuss its advantages and weaknesses. Moreover, we draw attention to the applicability of the basic notion of this assay to the study of neuroplasticity in any GI organ.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Poly-D-lysine hydrobromide</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:124px;">P1149</td>
			<td style="width:328px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ornithine/laminin</td>
			<td>Sigma-Aldrich</td>
			<td>P2533/L4544</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">13mm coverslips</td>
			<td>Merck</td>
			<td></td>
			<td>For use in 24-well plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dismembranator S</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Beta-III-tubulin antibody</td>
			<td>Millipore</td>
			<td>MAB1637</td>
			<td>1:200 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP-antibody</td>
			<td>DAKO</td>
			<td>M0761</td>
			<td>1:400 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RIPA buffer + protease inhibitor</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Gibco/Life sciences</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 supplement</td>
			<td>Gibco/Life sciences</td>
			<td>17504044</td>
			<td>Quality of B-27 is known to depend on the lot number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin/Metronidazol</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimal essential medium</td>
			<td>Gibco/Life sciences</td>
			<td>31095-029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life sciences</td>
			<td>24020133</td>
			<td>Improves collagenase activity when containing Ca/Mg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase type II</td>
			<td>Worthington Biochemical</td>
			<td>CLS-2</td>
			<td>Obtain lots with at least 200U/mg activity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA 0,25%</td>
			<td>Gibco/Life sciences</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">analySIS docu software</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Morphological neuroplasticity
In the indicated age range of newborn rats (P2-12) and the seeding densities, MP and DRG neurons already build dense neuronal networks after 48 hr&#160;(Figure 2A). Comparison of neurite density between neurons cultivated in PCa, CP, and NP extracts reveals greater neurite density of DRG neurons in PCa or CP extracts than in NP extracts (Figure 2A)5. We particularly prefer MP neurons for measurements on individ...

Watch this Scientific Journal Video about Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay at JoVE.com

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

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9.7K visualizações.  Technische Universität München.  Plasticidade neuronal é uma característica cada vez mais reconhecida, mas não suficientemente entendido do gastrointestinal (GI). Aqui, no exemplo de desordens pancreáticas humanas, apresentamos um ensaio neuroplasticidade in vitro para o estudo da plasticidade neuronal no tracto gastrointestinal, a nível tanto morfológica e funcional. 

Vídeo: Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...