We wish to acknowledge a contract (WS1763368) from Pfizer Pharmaceuticals for support in this work. We would also like to thank the Saudi Arabian Ministry of Higher Education (MOHE) for their support.

1. The Exercise
<ol>
 <li>The muscle of interest for this experiment was the elbow flexors (Biceps Brachii).</li>
 <li>Muscle strength was measured for each participant to be able to give each individual an appropriate resistance. This was determined by testing each participant for their resistance maximum (RM).</li>
 <li>For testing the RM, we used a strain gauge device interfaced with a computer through a BioPac (DA-100C) bioelectric amplifier module (BioPac Systems, Goleta, CA) to measure muscle strength. The module was connected to an MP-100 analog to digital converter sampling at a frequency of 1,000 hertz per second, and at a resolution of 24 bits (Fig. 10).</li>
 <li>The strain gauge device was fixed to a bench at a 45&deg; angle. The subjects were instructed to sit behind the device and rest their elbows on the padded area, so that the exertion force is through their wrists. This was the best way to insure that the subject will not recruit any muscle other than the biceps (Fig. 11).</li>
 <li>Strength was determined on 3 occasions with each contraction being 3 seconds in duration with approximately 45 seconds separating the contractions. The average of the 3 measurements was the RM.</li>
 <li>After determining the RM for the biceps muscle of each participant, the intended session of exercise was carried out with 35% of their RM.</li>
 <li>All the subjects underwent the same exercise using appropriate weighted dumbbells to induce the muscle soreness (DOMS). This was carried out by doing 4 sets of 25 repetitions of biceps concentration curls while seated on a chair, and with the elbows supported on their thighs (Fig. 12).</li>
 <li>Each subject was given a 90 second resting period between each set. Subjects either did the full set of 25 repetitions, or were instructed to stop if they failed to steadily control the weight during the exercise.</li>
</ol>
2. Infra-Red Camera Preparation &amp; Setup
<ol>
 <li>The room where the infra-red imaging takes place was set at a constant temperature to minimize any external bias from differences in room temperature, which could lead to false thermal readings. For the purpose of this experiment we had a temperature controlled room which was maintained at approximately 23&deg;C (+/- 0.5&deg;C).</li>
 <li>The camera was set at a distance of 1 meter away, and at a perpendicular angle to the skin being measured (Fig. 9a)*.</li>
 <li>After the required distance was set up, the subjects were advised to stand still until the image has been taken. This shouldn't take more than a couple of seconds, but it is very critical to minimize movement to insure the accuracy of the taken image.</li>
 <li>It is preferable that the room has darker colored paint, rather than lighter colors, to minimize any infra-red interference.</li>
 <li>Lighting is also critical when dealing with infra-red images, because light source that emit infra-red waves like fluorescent or tungsten lighting could give false high readings. The best lighting option would be a room equipped with uniform LED lights, as LED lights hardly produce any infra-red interference (Fig. 9b)*.</li>
</ol>
* A series of tests were done at our labs using the FLIR 660 IR Camera (Fig. 8), where we compared images of the skin at different angles (0 (perpendicular), 15, 30, 45, and 60 degrees), and at different distances (1, 2, and 5 meters) from the skin, to accurately detect the temperature of the skin. All images were compared to calibrated thermocouples, and the best correlation between the images and the thermocouple readings was at a perpendicular angle and at a distance of 1 meter away from the skin (r = 0.93). The different angles and distances caused a pixilation loss, and decreased the overall correlation between the images and the thermocouple readings.
3. Image Acquirement
<ol>
 <li>For the purpose of this experiment, the image of the exercised muscle was taken before the exercise, and at 24, and 48 hours post exercise.</li>
 <li>Body heat from sources other than the target could disrupt the thermal image and give false readings. For this reason, no one should be standing beside or behind the intended target.</li>
 <li>In this investigation, pictures of both the exercised and non-exercised arm were taken for comparison purposes. We exercised one of the arms, as was mentioned previously, and the other arm was used as a control (Fig. 4 and 5).</li>
 <li>Image numbers from the IR camera were recorded immediately on a separate spreadsheet, as it could be difficult to identify which image belongs to whom.</li>
</ol>
4. Image Processing &amp; Analyses
<ol>
 <li>The acquired IR images were processed using the "ThermoVision ExaminIR" software Version: 1.10.2.</li>
 <li>After selecting the required image for analysis, four regions of interest were identified on the acquired image of the arm using statistical boxes on the software interface (Fig.6).</li>
 <li>When the required regions across the arm have been located, the software shows the Means and Standard Deviations of the temperatures for each of the selected regions. We can then either cross compare each region individually or obtain an average temperature of the whole arm (Fig. 7).</li>
</ol>
5. Visual Analog Scale &amp; Blood Analysis
<ol>
 <li>A visual analog scale (VAS) was used to assess subjective soreness of the arm. The scale had a 10 cm (100mm) long line marked "no pain" at one end, and "extremely sore" at the opposite end. Each participant was directed to make a mark along the 10 cm line to indicate their response to soreness.</li>
 <li>The VAS's were administered to the subjects before the exercise, 24 hours after the exercise, and at 48 hours.</li>
 <li>Peripheral blood was collected from the subjects to measure myoglobin concentration levels in the blood.</li>
 <li>The blood was drawn from the subjects antecubital vein before the exercise, 30 minutes after the exercise was over, and at 48 hours.</li>
 <li>The blood was centrifuged at 4000 rpm for 10 min to separate the serum from the cells. The samples were then stored at -80&deg;C until the analyses of myoglobin was done.</li>
 <li>Myoglobin was measured using a TOSOH "AIA-360" Automated enzyme Immunoassay Analyzer (TOSOH Corp., Tokyo, Japan). The myoglobin Assay kits (Myo 025297, ST AIA-PACK Myoglobin) were used according to the manufactures instructions.</li>
</ol>
6. Representative Results
The results of IR thermal images taken during this investigation are clearly represented in figure 1. Images taken at the 3 time periods (pre-exercise, 24 hours post-exercise, and 48 hours post-exercise) for the exercised arms of the 41 subjects, showed a noticeable increase in temperature on day 2 (24 hours post-exercise) when compared to pre-exercise temperatures, and temperatures taken at 48 hours. As shown in figure 1, the average skin temperature was 32.80 +/- 1.03 &deg;C for day 1 (pre-exercise), and 33.96 +/- 1.46 &deg;C for day 2 (24 hours post-exercise), and 32.82 +/- 1.29 for day 3 (48 hours post-exercise). This difference in skin temperature from day 1 to day 2 was significant (ANOVA p &lt; 0.01).
However, for the un-exercised arm, changes amongst the 3 time periods were not evident. Figure 1 shows that the average skin temperature was 33.08 +/- 0.83 &deg;C for day 1 (pre-exercise), and 32.79 +/- 1.42 &deg;C for day 2 (24 hours post-exercise), and 33.17 +/- 0.95 for day 3 (48 hours post-exercise). This difference in skin temperature over the 3 days was not significant (ANOVA p = 0.38).
The results of the pain readings from the VAS are shown in figure 2. As seen in figure 2, the reported pain had a dramatic increase on days 2 and 3. Pain levels of the exercised muscle increased from 3.6 +/- 6.1 on day 1, to 36.3 +/- 22.8 on day 2, and 37.5 +/- 25.3 on day 3. This increase from day 1 was significant (ANOVA p &lt; 0.01).
The results of the myoglobin concentration levels are shown in figure 3. As seen in this figure, there was hardly any change between the 2 myoglobin concentrations on day 1 (pre, &amp; 30 minutes post exercise). But on day 3, the increase in myoglobin was very large. This increase on day 3 was approximately 147 nanograms per milliliter (ng/mL) of blood when compared to the first 2 concentrations on day 1. Myoglobin concentrations were 30.12 +/- 7.66 ng/mL at baseline, 31.66 +/- 11.89 ng/mL 30 minutes post exercise, and 178.96 +/- 249.51 ng/mL on day 3. This increase on day 3 was highly significant (ANOVA p &lt; 0.01).
A correlation analysis was done between the skin temperatures obtained from the IR images, and the VAS soreness levels. It was found that there was a considerable correlation between the VAS readings on day 2, and the skin temperature measurement on day 2. This correlation was significant (r = 0.312, p &lt; 0.05). However, there was no evident correlation between the VAS readings and the skin temperatures on day 3. This correlation was insignificant (r = 0.047, p = 0.77).
<img src="/files/ftp_upload/3551/3551fig1.jpg" alt="Figure 1"/> 
Figure 1. A representative graph of the differences in skin temperature in the exercised arms (Diamonds), and un-exercised arms (Squares) of the 41 subjects over the 3 day time period.
<img src="/files/ftp_upload/3551/3551fig2.jpg" alt="Figure 2"/> 
Figure 2. A representative graph of the differences in perceived muscle soreness measured with the VAS over the 3 day time period for all the 41 subjects.
<img src="/files/ftp_upload/3551/3551fig3.jpg" alt="Figure 3"/> 
Figure 3. A representative graph of the differences in myoglobin concentrations for all the 41 subjects over the 3 time periods.

<img src="/files/ftp_upload/3551/3551fig4.jpg" alt="Figure 4"/> 
Figure 4. A) a typical IR image of a subject's exercised arm before the exercise. B) an IR image of the same subjects arm 24 hours after the exercise.
<img src="/files/ftp_upload/3551/3551fig5.jpg" alt="Figure 5"/> 
Figure 5. A) a typical IR image of a subject's un-exercised arm before the exercise. B) an IR image of the same subjects arm 24 hours after the exercise.
<img src="/files/ftp_upload/3551/3551fig6.jpg" alt="Figure 6"/> 
Figure 6. An illustration of the 4 regions of interest for analyzing the thermal image of the arm.
<img src="/files/ftp_upload/3551/3551fig7.jpg" alt="Figure 7"/> 
Figure 7. Software interface for the "ThermoVision ExaminIR" showing the 4 boxes of interest on an IR image of an exercised arm. Also shown are the statistical interpretations for each box.
<img src="/files/ftp_upload/3551/3551fig8.jpg" alt="Figure 8"/> 
Figure 8. The IR thermal camera used for this investigation (FLIR 660).
<img src="/files/ftp_upload/3551/3551fig9.jpg" alt="Figure 9"/> 
Figure 9. A) The Setup of the IR Camera 1 meter away from the subjects arm. B) The LED lights used in the lab where the images were taken.
<img src="/files/ftp_upload/3551/3551fig10.jpg" alt="Figure 10"/> 
Figure 10. A) The BioPac Modules used for measuring the muscle strength. B) The strain gauge device fixed to a 45&deg; angled bench and hooked to the BioPac system.
<img src="/files/ftp_upload/3551/3551fig11.jpg" alt="Figure 11"/> 
Figure 11. A typical subject exerting force on the strain gauge device.
<img src="/files/ftp_upload/3551/3551fig12.jpg" alt="Figure 12"/> 
Figure 12. A subject undergoing the exercise protocol for inducing the muscle soreness.

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No conflicts of interests declared.

The primary purpose of this investigation was to assess the usefulness of thermal IR imaging in detecting and measuring muscle soreness after strenuous exercise, and our results suggest that IR imaging could be a valid technique for detecting DOMS, especially within the first 24 hours of exercising. This is not surprising, as Pennes18 provided a very detailed model of heat flow from muscle to skin in limbs. This model predicts that heat in deeper tissues such as muscles can be dissipated into blood and into th...

<table border="1">
<tbody>
 <tr>
 <td>Name of the Device</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Infra-Red Thermal Camera</td>
 <td>FLIR</td>
 <td>FLIR SC660</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Thermal Infra-Red Analysis Software</td>
 <td>ThermoVision ExaminIR</td>
 <td>Software Version 1.10.2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bioelectric Amplifier Module</td>
 <td>BioPac</td>
 <td>DA100C</td>
 <td>The DA100C provides variable gain settings, and adjustable voltage references.</td>
 </tr>
 <tr>
 <td>Analog to Digital Converter Module</td>
 <td>BioPac</td>
 <td>MP100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Automated enzyme Immunoassay Analyzer</td>
 <td>TOSOH</td>
 <td>AIA -360</td>
 <td>This device was used to analyze the blood samples, and obtain the myoglobin readings.</td>
 </tr>
 </tbody>
</table>

the use of thermal infra-red imaging to detect delayed onset muscle soreness

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically inactive for prolonged periods of time, and begin with an unexpected bout of exercise1-4, but can also occur in athletes who exercise beyond their normal limits of training5. The symptoms associated with this painful phenomenon can range from slight muscle tenderness, to severe debilitating pain1,3,5. The intensity of these symptoms and the related discomfort increases within the first 24 hours following the termination of the exercise, and peaks between 24 to 72 hours post exercise1,3. For this reason, DOMS is one of the most common recurrent forms of sports injury that can affect an individual&#x2019;s performance, and become intimidating for many1,4.
For the last 3 decades, the DOMS phenomenon has gained a considerable amount of interest amongst researchers and specialists in exercise physiology, sports, and rehabilitation fields6. There has been a variety of published studies investigating this painful occurrence in regards to its underlying mechanisms, treatment interventions, and preventive strategies1-5,7-12. However, it is evident from the literature that DOMS is not an easy pathology to quantify, as there is a wide amount of variability between the measurement tools and methods used to quantify this condition6. It is obvious that no agreement has been made on one best evaluation measure for DOMS, which makes it difficult to verify whether a specific intervention really helps in decreasing the symptoms associated with this type of soreness or not. Thus, DOMS can be seen as somewhat ambiguous, because many studies depend on measuring soreness using a visual analog scale (VAS)10,13-15, which is a subjective rather than an objective measure. Even though needle biopsies of the muscle, and blood levels of myofibre proteins might be considered a gold standard to some6, large variations in some of these blood proteins have been documented 6,16, in addition to the high risks sometimes associated with invasive techniques. 
Therefore, in the current investigation, we tested a thermal infra-red (IR) imaging technique of the skin above the exercised muscle to detect the associated muscle soreness. Infra-red thermography has been used, and found to be successful in detecting different types of diseases and infections since the 1950&#x2019;s17. But surprisingly, near to nothing has been done on DOMS and changes in skin temperature. The main purpose of this investigation was to examine changes in DOMS using this safe and non-invasive technique.

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The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

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19.1K 次观看.  Loma Linda University. 本次调查的目的是评估是否使用红外热成像摄像机是一个有效的检测和定量运动后肌肉酸痛工具。

视频: The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.