Name: assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay
Uploaded: 2015-03-10T14:00:00
Description: Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Animal work conducted at the USGS National Wildlife Health Center was performed in accordance with the NIH Office of Laboratory Animal Welfare guidelines and under institutional animal care and use committee protocol #EP080716. Tissues from hunter-harvested animals were gifts from the Wisconsin or Michigan Departments of Natural Resources.
1. Solution Preparation
NOTE: The solutions listed below are required for preparation of the CER assay substrate in Sectio...

<ol>
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For successful completion of this protocol, attention should be paid to PrPC levels in uninfected brain tissue used for substrate preparation (step 2.1.2) and the seed to substrate ratio for conversion reactions (step 4.4.2). In our experience, brains can be extracted for use as CER substrate after a substantial period post-mortem, as long as PrPC is present by immunoblotting (Figure 4). In fact, some autolysis was observed in the brains of the bobcats used for CER studies. Neverthe...

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of fatal neurodegenerative diseases with extended incubation periods that affect a variety of animals and humans. The putative etiological agent of TSEs is comprised of a misfolded isomer of the host prion protein (PrP) that is capable of self-propagation by template-driven conversion of the normal cellular form of PrP (PrPC) into an infectious, disease-associated form (PrPTSE) that accumulates in central nervous system tissues of the infected host1. Infectious mammalian prions generally transmit from host-to-host in a species-specific manner, which has given rise to the concept of the &#8220;TSE species barrier&#8221; limiting interspecies transmission events2. The biological determinants of the prion species barrier are not well understood. Amino acid sequence similarity between the infectious PrPTSE and the host PrPC can strongly influence whether conversion takes place3-5, but remains insufficient to explain all prion transmission events observed in vivo6,7.
Thus, characterization of TSE species barriers has largely relied upon animal challenge studies: exposing na&#239;ve animals from a given species to prions from another and measuring the resulting incubation time to disease onset and attack rate as indicators of transmission efficiency. Mice expressing PrPC from heterologous species are also used for this type of study8. The costs associated with transgenic mouse production and protracted prion bioassays, as well as ethical considerations of animal use, are obstacles to experimental investigation of TSE species barriers. Assessment of the human species barrier to TSEs relies on mice engineered to express human PrPC. These mice require long incubation periods to succumb to human TSEs or modifications to the human PrPC molecule for rapid disease onset9. Incubation periods for non-human TSEs in these mice may extend beyond the normal mouse lifespan making interpretation of negative results challenging. Non-human primates have also been used as proxies for studying human species barriers, but these studies are fraught with the same challenges as other types of animal experimentation and non-human primates may not precisely recapitulate disease as it proceeds in the human host.
Animal bioassays remain the &#8220;gold standard&#8221; method for measuring the susceptibility of a species to a TSE, but the obstacles, costs and ethics of these live animal studies have compelled investigation into alternatives. A number of in vitro assays, based on assessing the conversion of host PrPC to a proteinase K (PK)-resistant state (PrPres) when seeded by PrPTSE, have been developed and used to investigate TSE species barriers10-12. Examples of in vitro assays include cell-free conversion assays, protein misfolding cyclic amplification (PMCA), and the conversion efficiency ratio (CER) assay10-14. While none of these assays take into account peripheral factors involved in species barriers after natural infection, all can be useful to identify potentially susceptible hosts for TSEs.
Here we present the protocol for the CER assay, in which two denatured PrPC substrates derived from normal brain homogenates are used in bench top prion conversion reactions (Figure 1)13. PrPC in the substrate denatured at pH 7.4 can only be converted to PrPres by PrPTSE seeded into the reaction in the absence of a species barrier14. In contrast, denaturation of PrPC in the other substrate at pH 3.5 allows it to be converted to PrPres following incubation with PrPTSE from any species and serves as a control for conversion. The ratio of conversion of PrPC to PrPres in pH 7.4 substrate relative to that of the pH 3.5 substrate provides a measure of the species barrier. We have found that the CER assay predicts known species barriers of laboratory mice to various TSEs and have used the assay in efforts to predict the species barrier of numerous mammalian species, including bighorn sheep, to chronic wasting disease (CWD) and other TSEs14,15. Investigators interested in a tool allowing rapid screening of TSE species barriers or assessment of PrPC-to-PrPres conversion will find this methodology useful.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

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.

Successful use of the CER assay is largely dependent upon the quality of the substrate pairs used in conversion reactions. For this reason, following procedures to prepare CER substrate pairs, a quality control immunoblot should be performed (Figure 2). For both substrates, assay 10-25 &#956;l by immunoblotting. PrPC should be easily detectable in each substrate and PrPC levels must be approximately the same between the two. Immunoreactivity will be mainly visible above 20 kDa, but ...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

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.

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 ...

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Research

JoVE Journal

Medicine

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

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.

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.

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

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...