Heat Pain Testing using the Thermostimulator TSA II
1. Introduction
<ol>
<li>This procedure uses the TSA II, a commercially available thermo-sensory analyzer that delivers heat stimuli to the skin with a thermode (Medoc Advanced Medical Systems, Durham, NC). A central unit generates and transmits the signal to the thermode. This signal is controlled by proprietary software typically stored on a laptop connected to the central unit. Also attached to the central unit is a hand-held control that allows a study subject to stop the heating of the thermode.</li>
<li>Seven sequential and identical heat stimuli are delivered, with inter-stimulus intervals of 30s. The basal thermode temperature is set at a comfortable 35&ordm;C. The rate at which the thermode heats up is set at 1&ordm;C/s, while the rate at which it cools down is set at 10&ordm;C/s. The maximum thermode temperature is set at 52&ordm;C.</li>
</ol>
2. Preparation and Training the Subjects
<ol>
<li>To prepare subjects for the heat pain test seat them comfortably in a reclining chair that provides adequate support for the head, arms and legs. The chair should also allow easy access to the examined body sites.</li>
<li>The testing facility should be at a comfortable room temperature, and provide a quite and neutral environment with no distraction.</li>
<li>Before starting the experiment, all participants should be well trained in all aspects of the heat pain testing. Training can take place any time before the experiment.</li>
<li>Interactions between study subjects and investigators should be limited to those necessary for collecting data. Joking and small talk are discouraged because they may compromise data consistency. Study subjects are informed that interactions will intentionally be somewhat monotonic and neutral during the experiments.</li>
<li>Keep the instructions clear, simple, and consistent. Emphasize that pain is subjective, and there are no right or wrong answers. It is most important to be consistent. Define the heat pain threshold as the point between being &ldquo;about to be painful&rdquo; and &ldquo;just became painful.&rdquo;</li>
<li>It is critical that a subject knows precisely what to expect during a test session. Any &ldquo;surprises&rdquo; may result in less consistent data, because confounders such as anxiety may be generated. For example, if seven heat pain stimuli are used to determine the heat pain threshold, the subject should know this.</li>
<li>At any time, the subject can stop the thermode from getting any hotter by pushing the button of a hand-held control. Being in control will reduce a subject&rsquo;s anxiety and will make the data more reliable.</li>
<li>The training session offers an opportunity to judge a study subject&rsquo;s performance. It may be best to train subjects during two to four test cycles so their individual pain thresholds vary by no more than 0.8&deg;C, and ideally by no more than 0.6&deg;C between the last two test cycles. Subjects who fail to perform consistently are typically excluded from the study.</li>
</ol>
3. Determining the Pain Threshold
<ol>
<li>To begin the pain threshold testing cycles mark the placement of the thermode on the subject&rsquo;s skin. In this experiment the same inflamed skin lesion used for microdialysis will be tested for pain to learn about biochemicals released in response to the pain testing.</li>
<li>Then, initiate each cycle by placing the thermode onto the marked skin area. Match the position of the thermode with the markings as closely as possible. Allow the temperature between the skin and the thermode to equilibrate to 35&deg;C &ndash; a temperature that safely will cause no pain. This will take about 5 seconds and will be indicated by a reading of 35&deg;C on the computer screen.</li>
<li>Before beginning, give the hand-held control to the subject.</li>
<li>Use constant and gentle pressure to keep the entire surface of the thermode in full contact with the subject&rsquo;s skin during testing.</li>
<li>Now start the pre-programmed test algorithm. The thermode will start heating up at 1&deg;C/s. As soon as the subject experiences pain, they&rsquo;ll press the hand held device.</li>
<li>After the button press on the hand-held device, the thermode temperature causing pain will automatically be registered and will start decreasing at a rate of 10&deg;C/s down to its baseline temperature of 35&deg;C.</li>
<li>The temperature probe will automatically be kept at 35&deg;C for 30 s before it starts rising again at 1&deg;C/s, until a subject again pushes the button of the hand-held device. Just before the thermode temperature is about to increase again, remind the subject that this is about to happen. This will keep the subject focused.</li>
<li>This sequence will be repeated until seven temperature recordings have been registered. Calculate the average of these temperature readings; this is the heat pain threshold.</li>
</ol>
Mechanical Pain Testing with Punctuated Pressure Probes
1. Introduction
<ol>
<li>We use custom made calibrated punctuated pressure probes, because they provide more consistent results than the traditionally used and commercially available von Frey hairs. Probes are made of identical cylindrical stainless steel wires (240 &mu;m diameter) mounted on copper rods of various weights (1, 2, 4, 8, 16, 32, 49, 64, and 81 grams) that move freely within a wider hand-held aluminum tube.</li>
<li>The same general experimental conditions outlined for heat pain testing apply to mechanical pain testing. The subjects should be well prepared and trained. It is especially important to reassure subjects that the probes are blunt and will not puncture the skin. This will help the subjects to relax and provide consistent data.</li>
</ol>
2. Determining the Mechanical Pain Threshold
<ol>
<li>To determine the mechanical pain threshold, first apply the steel wire tip of the lightest probe perpendicularly to the skin at the center of the experimentally inflamed lesion.</li>
<li>The hand-held aluminum tube is then lowered towards skin without touching it. This way, the entire weight of the copper rod inside the aluminum tube rests on the steel wire tip.</li>
<li>The study subject then indicates whether the probe causes pain by saying &ldquo;yes&rdquo; or &ldquo;no.&rdquo;</li>
<li>If the subject answers &ldquo;no,&rdquo; then either the same probe or next heavier probe will be used next. This algorithm is called an ascending stimulation sequence. Randomly determine whether the next probe is heavier or of the same weight. This will keep a subject blinded to the algorithm.</li>
<li>Apply the probes in ascending sequence until a subject answers &ldquo;yes.&rdquo; The average weight of the last two probes in this series is recorded as the first deflection point.</li>
<li>After the subject answers &ldquo;yes,&rdquo; start using a descending stimulation sequence, and use either a lighter probe or the same probe as before. Randomly determine whether the next probe is lighter or of the same weight. This will keep a subject blinded to the algorithm.</li>
<li>Apply probes in a descending sequence until the subject answers &ldquo;no.&rdquo; The average weight of the last two probes in this sequence constitute the second deflection point.</li>
<li>When the subject answers &ldquo;no,&rdquo; switch back to an ascending stimulation sequence. Alternate between ascending and descending sequences until seven deflection points are recorded. The average of these seven points is the mechanical pain threshold.</li>
</ol>

<ol>
	<li>Angst, M. S., Clark, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+Koltzenburg+et+al.:+Differential+sensitivity+of+three+experimental+pain+models+in+detecting+the+analgesic+effects+of+transdermal+fentanyl+and+buprenorphine.">Comment on Koltzenburg et al.: Differential sensitivity of three experimental pain models in detecting the analgesic effects of transdermal fentanyl and buprenorphine.</a> Pain. 126, 165-174 (2006).</li><li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation,+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: A microdialysis study.</a> Pain. , (2008).</li><li>Angst, M. S., Koppert, W., Pahl, I., Clark, J. D., Schmelz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+infusion+of+the+mu-opioid+agonist+remifentanil+in+humans+causes+hyperalgesia+during+withdrawal.">Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal.</a> Pain. 106, 49-57 (2003).</li><li>Lotsch, J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mu-opioid+agonist+remifentanil+attenuates+hyperalgesia+evoked+by+blunt+and+punctuated+stimuli+with+different+potency:+a+pharmacological+evaluation+of+the+freeze+lesion+in+humans.">The mu-opioid agonist remifentanil attenuates hyperalgesia evoked by blunt and punctuated stimuli with different potency: a pharmacological evaluation of the freeze lesion in humans.</a> Pain. 102, 151-161 (2003).</li></ol>

This video demonstrates algorithms for assessing the heat and mechanical pain threshold in inflamed skin of human volunteers. The perception of pain is subjective. Collection of adequate pain data requires an appropriate testing facility (e.g. quiet and comforting), thorough training of study participants with in-built data quality control measures (test-retest reliability), strict adherence to a standardized study protocol, and avoidance of confounding factors (e.g. stress, anxiety). Reliable subjective data on pain sensitivity, paired with interventions such as the administration of an analgesic drug and the collection of mechanistic data such as the local release of nociceptive mediators provide valuable insight into mechanisms of pain, and means to prevent or attenuate it.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>TSA-II Neuro Sensory Analyzer</td>
<td>Tool</td>
<td>Medoc Advanced Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Puntuated Pressure Probes</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>Alternatively, commercially available von Frey Hairs can be used.</td>
</tr>		</tbody>
		</table>
		</div>

determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Watch this Scientific Journal Video about Determining heat and mechanical pain threshold in inflamed skin of human subjects at JoVE.com

Determining heat and mechanical pain threshold in inflamed skin of human subjects

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

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20.8K vues.  Stanford University School of Medicine. Algorithmes d'évaluer les seuils de douleur thermique et mécanique de la peau enflammée expérimentalement des sujets de l'étude humaine sont présentés. Les deux modèles de test douleurs nociceptives examiner séparément le traitement par les deux principaux périphériques populations de fibres nerveuses de transmettre la douleur, c'est à dire, non myélinisées de fibres myélinisées C et petites fibres A-delta.

Vidéo: Determining heat and mechanical pain threshold in inflamed skin of human subjects

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...