Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOTE: This protocol was reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
1. Preparation of Modified Krebs Solution and Test Drug Aliquots
<ol>
	<li>Make 6 L of modified Krebs solution that contains (in mM): 117.9 NaCl, 4.7 KCl, 25 NaHCO3, 1.3 NaH2PO4, 1.2 MgSO4, 2.5 CaCl2, 11.1 D-glucose, 2 sodium butyrate, 20 sodium acetate, 0.004 nifedipine (to block spontaneous muscle contractions), and 0.003 indomethacin (to block synthesis of endogenous prostaglandins). Use ice-cold and warm Krebs solutions for tissue dissection and single-fiber recording, respectively.</li>
	<li>Prepare any chemical solutions (e.g., IS, capsaicin, ATP) in aliquots at desired concentrations.</li>
</ol>
2. Dissection of the Colorectum-nerve Tissue
<ol>
	<li>Anesthetize and euthanize male mice (6 - 8 weeks old, 20 - 30 g) in a CO2 chamber at a flow rate that displaces 10 - 30% of the chamber volume per minute until mice stop breathing completely as indicated by the absence of chest movement.</li>
	<li>Immediately following euthanasia, exsanguinate by cutting open the thoracic chamber, perforating the right atrium, and immersing the mouse carcass in an ample volume (~500 ml) of ice-cold Krebs (4 &#176;C) solution bubbled with carbogen (95% O2, 5% CO2).</li>
	<li>Carefully remove all viscera but the colon and pelvic organs. Transect the mouse in half across the T12 spinal segment slightly above the thoracic diaphragm and transfer the caudal half to a dissection chamber containing ice cold, bubbled Krebs solution.</li>
	<li>Under a stereomicroscope, remove the bladder and the reproductive organs by transecting at their junctions to the urethra, and remove the descending/abdominal aorta until it bifurcates into common iliac arteries. Free the PN or the LSN from their surrounding tissues by blunt dissection and follow the nerve from outside the iliac crest till its ventral entry point into the L6 and S1 vertebral column (for PN) or T13 and L1 vertebral column (for LSN).</li>
	<li>Cut the pubic symphysis and right and left acetabular joints, and remove the iliac bone. Carefully free either the PN or LSN from the attached muscle and connective tissue from close to the colorectum until where the nerve enters the vertebral column.</li>
	<li>Carefully resect the iliac bone to expose the distal colorectum. Dissect out the distal colorectum with the attached PN or LSN in continuum.</li>
	<li>Transfer the colorectum with the nerve attached to the bath compartment of the tissue chamber. Remove excessive connective tissue by further dissection, and open the colorectum longitudinally along the anti-mesenteric border.</li>
	<li>With mucosal side facing up, pin the mesenteric edge of the colorectum adjacent to the recording compartment into the silicone base of the chamber and attach the antimesenteric length of the colorectum to a rake of hooks connected to a force actuator (illustrated in Figure 1 and photographed in Figure 2A).</li>
	<li>Extend the PN or LSN into the recording compartment, which is connected to the bath compartment by a mouse hole and gate. Gently lay the nerve trunk onto a small glass mirror in the recording compartment, which provides a hydrophilic surface for the nerve to adhere to. Superfuse the bath compartment with warm (30 - 32 &#176;C), oxygenated Krebs solution and fill the recording compartment with mineral oil.</li>
</ol>
3. Single-fiber Recording and Localization of the Receptive Field
<ol>
	<li>Carefully peel back the epineurium (nerve sheath) from the PN or LSN under the stereomicroscope at high magnification (50 - 60X). Using fine forceps, tease the nerve trunk into 5 - 8 nerve bundles of ~100 &#181;m thickness.</li>
	<li>Place the platinum-iridium reference electrode in contact with the Krebs solution in the tissue chamber. Sequentially place the individual nerve bundles onto the recording electrode made of the same material.</li>
	<li>Use a soft paint brush to evoke APs from the colorectal afferents by gently stroking up and down the colorectal surface. Locate the nerve bundle(s) that innervate the colon through detectable AP (action potential) recordings. 
	NOTE: The PN and LSN also innervate the urinary bladder and other pelvic organs.</li>
	<li>Use a pair of 30 G needle tips to further split the nerve bundle into fine fascicle filaments of ~10 &#181;m thickness and place an individual filament onto the recording electrode.</li>
	<li>Place the round-tipped concentric electrode perpendicular to the mucosal surface to electrically excite afferent endings at suprathreshold stimulus intensity (10 mA magnitude, 0.5 msec duration @ 0.3 Hz), which produces a ~2 mm radius of current spread. Move the electrode systematically (~1.5 mm steps) along the length and width of the flattened colorectum to localize receptive endings.</li>
	<li>When an afferent ending is excited, adjust the electrode position to pinpoint the site of activation (receptive field, RF) that requires minimum stimulus intensity (stimulus threshold). Discard endings with a stimulus threshold &#62;3 mA19.</li>
	<li>Calculate the conduction velocity (CV) from 1) the distance between the stimulating electrode at the receptive field (RF) and the recording site and 2) the conduction delay (e.g., Figure 2B) between the stimulus artifact and the onset of the action potential. 
	CV (m/sec) = distance (mm) / conduction delay (msec).</li>
</ol>
4. Functional Classification of Mechanosensitive Colorectal Afferents
<ol>
	<li>After locating a RF by electrical stimulation, apply the following three mechanical stimuli to the RF:
	<ol>
		<li>Conduct the probing stimulus by pressing the tip of a calibrated von Frey-like nylon monofilament (0.4 and 1 g force) perpendicularly towards the RF on the flattened colorectum.</li>
		<li>Conduct the stroking stimulus by gently stroking the colorectal mucosa with a fine nylon filament strand (10 mg force) to generate a small surface shear stress at the RF.</li>
		<li>Conduct the circumferential stretch using a computer-controlled force actuator, which delivers a ramped stretch force (0 - 170 mN at 5 mN/sec) in circumferential direction along the anti-mesenteric edge of the colorectum via the rake of hooks described in step 2.8.</li>
	</ol>
	</li>
	<li>Classify afferents as serosal (respond only to blunt probing), mucosal (respond to mucosal stroking and blunt probing), muscular (respond to circumferential stretch and blunt probing) muscular/mucosal (respond to circumferential stretch, mucosal stroking and blunt probing), or MIA (not responsive to any of the three mechanical stimuli).</li>
	<li>For mesenteric afferents (only in the LSN innervation) that are difficult to activate selectively by electrical stimulation, locate their receptive endings by mechanical stroking/probing of the mesentery.</li>
	<li>For stretch-sensitive afferents (muscular and muscular-mucosal), determine the response threshold, which is defined as the force that evokes the first AP during ramped stretch.</li>
	<li>For serosal afferents, record their responses to ascending levels of punctate probing of the receptive field driven by the computer-controlled force actuator.</li>
</ol>
5. Chemical Application/Modulation of Receptive Endings
<ol>
	<li>Record a baseline response to a mechanical stimulus (i.e., response to ramped stretch, punctate probing, or mucosal stroking).</li>
	<li>Coat the bottom edge of a piece of tubing (brass or stainless steel, 10 mm high and 4 x 4 mm2 square or 4 - 5 mm diameter) with petrolatum and place it over the receptive field on the colorectum.</li>
	<li>Remove the Krebs solution inside the tubing, and expose the receptive ending for 3 - 5 min to 170 &#956;l of the solution containing the chemical(s) to be tested.</li>
	<li>Monitor the response of the afferent during chemical application (some afferents are chemosensitive).</li>
	<li>Remove the chemical solution and tubing to terminate the action of the chemical. Within 4 - 6 min, test the afferent response to the same mechanical stimulus as in the baseline response.</li>
	<li>Reapply the mechanical stimulus again after sufficient period of wash-out (&#62;15 min).</li>
</ol>
6. Recording and Discriminating AP spikes
<ol>
	<li>Digitize the electrical signals recorded from axons at 20 kHz and save the data to a computer. Monitor the signal on-line by an audio monitor.</li>
	<li>Analyze the AP spikes off-line and discriminate single units based upon principal component analysis of individual spike waveforms25. 
	NOTE: One record should contain no more than two easily discriminable active units.</li>
</ol>

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The authors have nothing to disclose.

The in vitro colorectum-nerve preparation described here has proven to be a powerful approach to study neural encoding functions of individual colorectal afferents, which nicely complements other non-functional approaches (e.g., cellular, molecular, and histological studies) on visceral sensory neurons (see review 27 for details). Neuronal mechanisms contributing to nociception and long-term colorectal hypersensitivity have been revealed and pharmacological manipulations have been performed t...

Pain and hypersensitivity are the predominant complaints of patients suffering from functional gastrointestinal disorders, including irritable bowel syndrome (IBS), which exist in the absence of apparent pathobiological cause or tissue damage. For example, IBS patients exhibit hypersensitivity, including heightened responses to rectal balloon distension and increased sensitivity during normal bowel function, as well as hypersensitivity of somatic referral (i.e., tenderness to palpation of the abdominal area)1. Because targeting colorectal afferents has proven to be effective in alleviating pain and hypersensitivity in IBS patients (e.g., intra-rectal instillation of local anesthetics2,3; oral ingestion of the guanylate cyclase-C agonist linaclotide4-6), improved understanding of the afferent innervation of the colorectum is important.
Visceral afferents, including colorectal afferents, are capable of responding to chemical/nutrient- and thermal modalities (e.g., 7-9). However, visceral afferents responding to mechanical stimuli (i.e., mechanosensitive afferents) have been the most thoroughly studied because mechanical stimuli (e.g., luminal distension, stretch) are those that generally give rise to conscious sensations, including discomfort and pain10-16. In addition, the viscera are also innervated by mechanically insensitive afferents (MIAs), commonly termed silent or sleeping nociceptors17. Under normal physiological conditions, MIAs do not respond to mechanical stimulation or have very high response thresholds18, but can become active and acquire mechanosensitivity in pathophysiological conditions and contribute to hypersensitivity.
Using the in vitro preparation and protocol described here, we developed and employed an electrical stimulus strategy to search for receptive endings, permitting unbiased identification of both mechanosensitive and MIA endings in the colorectum19. The colorectal innervation is derived from lumbar splanchnic (LSN) and pelvic nerve (PN) pathways, and includes colorectal afferents that can be categorized into five mechanosensitive classes (serosal, mucosal, muscular, muscular-mucosal, mesenteric) and one MIA class20. Using this in vitro preparation, we found that colorectal MIAs acquired mechanosensitivity (sensitize) following brief exposure of their receptive fields to an inflammatory soup (IS), which sensitized 71% of MIAs in the PN pathway and 23% of MIAs in the LSN pathway19. We also documented long-term sensitization (up to 28 days) of MIAs in the context of long-lasting behavioral visceral hypersensitivity (i.e., in mice receiving intracolonic treatments with zymosan21 or 2,4,6-trinitrobenzenesulfonic acid (TNBS)22).
Among mechanosensitive afferents, muscular and muscular-mucosal afferents are the only classes that tonically encode circumferential stretch of the colorectum (i.e., are stretch-sensitive) and subserve the encoding of noxious colorectal distension23,24. Using a computer-controlled force actuator, we applied a standard, homogeneous, and reproducible ramped stretch in the circumferential direction of the flattened colorectal tissue and further categorized stretch-sensitive afferents as low-threshold and high-threshold23. In addition, the time course of sensitization of stretch-sensitive afferents after intracolonic zymosan21 or TNBS22 treatment corresponds to the onset, persistence, and/or recovery of behavioral visceral hypersensitivity, suggesting a role of stretch-sensitive colorectal afferents in visceral pain and hypersensitivity.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

The setup is illustrated in Figure 1. It includes a custom-made tissue chamber that houses the colorectum in a silicone-lined bath compartment and the attached nerve in a contiguous mineral oil-filled compartment. The two-compartment chamber was machined from a solid block of acrylic plastic by a CNC machine; the bottom of both compartments was subsequently lined with firm silicone to allow easy pin down of the colorectal tissue. Extracellular APs from teased nerve fascicles are recorded using a low-nois...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

9.5K Views.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Video: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).