This work was supported by the Key R&#38;D project of Sichuan Provincial Science and Technology Plan (2022YFS0438), the National Natural Science Foundation of China (82104533), the China Postdoctoral Science Foundation (2020M683273), and the Science &#38; Technology Department of Sichuan Province (2021YJ0175).

The animal protocol was reviewed and approved by the Management Committee from Chengdu University of Traditional Chinese Medicine (Record No. 2021-11). Male Sprague Dawley (SD) rats (260-300 g, 8-10 weeks old) were used for the present study. The rats were kept in an animal chamber and were free to drink and eat during the experiment.
1. Solution preparation
<ol>
	<li>Prepare physiological salt solution (PSS) by dissolving 118 mM of NaCl, 4.7 mM of K+, 2.5 mM of CaCl2, 1.2 mM of KH2PO4, 1.2 mM of MgCl2&#8729;6H2O, 25 mM of NaHCO3, 11 mM of D-glucose, and 5 mM of HEPES (see Table of Materials).</li>
	<li>Prepare high K+ salt solution by dissolving 58 mM of NaCl, 60 mM of K+, 2.5 mM of CaCl2, 1.2 mM of KH2PO4, 1.2 mM of MgCl2&#8729;6H2O, 25 mM of NaHCO3, 11 mM of D-glucose, and 5 mM of HEPES.</li>
	<li>Saturate the above two solutions and bubble with a mixed gas of 95% O2 and 5% CO2. Meanwhile, maintain the pH values of the solution between 7.38 and 7.42 with 2 mM NaOH. 
	NOTE: For details information on solution preparation, please see reference15.</li>
</ol>
2. Rat coronary artery dissection
<ol>
	<li>Anesthetize the rat by inhalation of 2% isoflurane. Confirm deep anesthesia by toe pinch and, if needed, administer additional anesthetics. Then immediately open the thoracic cavity to expose the heart on the portable operating table following a previously published report12.</li>
	<li>After dissociating and removing the heart, drain the residual blood from all the heart chambers by mildly squeezing with medical plastic forceps. Quickly place the pre-processed heart in a Petri dish containing 95% O2 + 5% CO2 saturated PSS at 4 &#176;C, having a pH value of 7.40.</li>
	<li>To accurately identify the anatomical position of the coronary arteries, adjust the posture of the isolated heart under the light microscope according to the schematic diagram (Figure 2A). 
	NOTE: On the frontal view, the right auricle and the pulmonary artery were on the upper left and the upper right, respectively.
	<ol>
		<li>Cut the left and right ventricular cavities along the interventricular septum from the root of the pulmonary artery with surgical scissors and tweezers (Figure 2B).</li>
	</ol>
	</li>
	<li>To dissociate the left and right coronary arteries from the myocardial tissue, dissect the right ventricle under an optical anatomic microscope to thoroughly expose the right coronary artery branch. Then identify the position of the left coronary artery by rotating the heart tissue 45&#176; clockwise (Figure 2D).</li>
	<li>After removing the surrounding sticky myocardial tissue, explicitly discern the pulsing left (about 5 mm) and right (about 5 mm) coronary arteries. Separate the coronary arteries in the middle immediately and completely immerse in PSS at 4 &#176;C. Acquire an arterial ring of about 2 mm by vertically cutting the detached artery with anatomical scissors to record the vascular tension under different stimuli (Figure 2E).</li>
</ol>
3. Suspension and fixation of arterial ring
NOTE: For details on this step, please see reference14.
<ol>
	<li>Prepare two 2 cm stainless steel wires (see Table of Materials) and pre-soak in 4 &#176;C PSS solution saturated with 95% O2 + 5% CO2. Pass both the wires parallelly through the arterial ring along with the vessel&#39;s direction under an optical anatomical microscope and with wires of equal length exposed at both ends of the vascular cavity.</li>
	<li>Fix the arterial ring with the steel wire front and back in the bath of the wire micrograph filled with bubbling PSS with 95% O2 + 5% CO2. Rotate the horizontal screw knob for an appropriate front and rear spacing so that the two wires are horizontal and the arterial ring is in a natural state of relaxation.</li>
	<li>After installing the DMT bath on the thermostatic apparatus, open the data acquisition software (see Table of Materials) to ensure that the corresponding path signal was recorded. Set the following parameters: eyepiece calibration (mm/div): 0.36; target pressure (kPa): 13.3; IC1/IC100: 0.9; online averaging time: 2 s; delay time: 60 s. The steps of arterial ring fixation are shown in Figure 3.</li>
</ol>
4. Standardization of vascular tension in rat arterial ring
NOTE: For different cavity samples, optimal initial tension was necessary for vessels to maintain exceptional activity in vitro. For details, please see reference15.
<ol>
	<li>Achieve the optimal initial tension of the arterial ring by applying a reasonable tension along the diameter of the vessel. 
	NOTE: Based on the previous study16, the maximal agonist-induced tension was accomplished at the factor k value of 0.90 with the initial stretch tension of 1.16 &#177; 0.04 mN/mm (reference values for different vessel samples: k value, 0.90-0.95; initial tension, 1.16-1.52 mN/mm).</li>
	<li>At this point, set the displayed vascular tension value to zero. Afterward, apply a 3 mN pull stimulus to the arterial ring by rotating the spiral axis of the bath.</li>
	<li>After incubation for 1 h in oxygen-saturated PSS buffer at 37 &#176;C, pH 7.40, set the tension value to 0 mN again on the tension control panel of the wire micrograph. The setting process of the initial tension of the arterial ring is shown in Figure 4.</li>
</ol>
5. Reactivity detection of coronary artery ring
<ol>
	<li>Perform the contractile activity of the coronary artery ring with the wire myograph technique14, and validate in three separate operations by stimulating with 60 mM of K+ solution for 10 min each.</li>
	<li>After each stimulation, flush the bath with oxygen-saturated PSS until vascular tone returns to its initial state. 
	NOTE: Only when the tension fluctuation of the three parallel measurements was less than 10%, and the amplitude of each contraction was greater than 1 mN/mm, qualified and highly active arterial rings could be used for further experiments. The activity verification of the rat coronary ring is shown in Figure 5.</li>
</ol>
6. Post-surgical treatment
<ol>
	<li>After surgery, euthanize the animals following institutionally approved protocols. 
	NOTE: For the present study, the animals were euthanized by inhaling excess isoflurane.</li>
</ol>

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The authors have nothing to disclose.

The disturbance of coronary microcirculation, which involves a wide range of patients with CAD, has been gradually recognized and concerned the basis for adequate myocardium perfusion. Considering the serious complications of sudden coronary heart disease and cardiovascular disease, timely drug prevention and treatment are extremely important for a clinical individual with CAD17. Inevitably, the secrecy of coronary artery anatomy and the complexity of its physiological structure have severely rest...

Coronary artery disease (CAD) has been widely recognized and concerned as a typical and representative cardiovascular disease, being the leading cause of death in both developed and developing countries1,2. As a blood and oxygen supply route for normal cardiac physiological function, circulating blood enters and nourishes the heart through two main coronary arteries and a blood vascular network on the surface of the myocardium3,4. Cholesterol and fat deposits in the coronary arteries cut off the heart&#39;s blood supply and the violent inflammatory response of the vascular system, causing atherosclerosis, stable angina, unstable angina, myocardial infarction, or sudden cardiac death5,6. In response to pathological stenosis of the coronary arteries, compensatory accelerated physiological heartbeat satisfies the blood supply of the heart itself or vital organs of the body by increasing the output of the left ventricle7. If prolonged coronary stenosis is not relieved in time, extensive new blood vessels may develop in certain areas of the heart8. At present, the clinical treatment of CAD often adopts drug thrombolysis or surgical mechanical thrombolysis and an exogenous bionic vascular bypass with frequent medication and great surgical disability9. Therefore, the functional investigation of coronary artery physiological activity is still an urgent breakthrough for cardiovascular diseases10.
There are no available technical means to detect coronary physiological activity, except for wireless telemetry systems, which can dynamically record in vivo coronary pressure, vascular tension, blood oxygen saturation, and pH values11. Therefore, considering coronary arteries&#39; textural secrecy and complexity, accurate identification and isolation of coronary arteries are undoubtedly the best choices for exploring multiple mechanisms of CAD in vitro4.
A series multi myograph system, in particular a wire micrograph microvascular tension detector (see Table of Materials), is a very mature marketable device for recording in vitro tissue tension changes of small vascular, lymphatic, and bronchial tubes with the characteristics of high precision and continuous dynamic recording12. The said system has been extensively employed to record in vitro tissue tension characteristics of cavity structures with diameters of 60 &#181;m to 10 mm. The continuous heating features of the platform of the wire micrograph largely offset the stimulation of the adverse external environment. Meanwhile, the constant inputs of the gas mixture and the pH values allow us to obtain more accurate vascular tension data in a similar physiological state13. However, considering the complexity of anatomical localization of rat coronary arteries (Figure 1), its isolation has been perplexing and limiting the mechanism&#39;s exploration of diversified cardiovascular disease and drug development. Therefore, the present protocol introduces the anatomical location and separation process of the rat coronary artery in detail, followed by tension measurement on the platform of the wire micrograph14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Apigenin</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>150731</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A501330</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A610219</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Xiya Reagent Co., Ltd., Shandong, China</td>
			<td>S3872</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100395</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100781</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart Professional version 8.3&#160;</td>
			<td>ADInstruments, Australia</td>
			<td>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100288</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi myograph system&#160;</td>
			<td>Danish Myo Technology, Aarhus, Denmark</td>
			<td>620M</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100241</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100865</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel wires</td>
			<td>Danish Myo Technology, Aarhus, Denmark</td>
			<td>400447</td>
			<td></td>
		</tr>
		<tr>
			<td>U46619</td>
			<td>Sigma, USA</td>
			<td>D8174</td>
			<td></td>
		</tr>
	</tbody>
</table>

standardized rat coronary ring preparation and real-time recording of dynamic tension changes along vessel diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

Anatomically positioned, rat coronary arteries distributed and hidden deep in myocardial tissue were not easily recognized. By comparing the coronary arteries of humans (Figure 1A) and rats (Figure 1B), rapid and accurate separation of rat coronary arteries was conducted according to the sampling process in Figure 2. After precisely locating the right auricle, pulmonary artery, and apex from the front under an optical microscope, th...

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Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, ...

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3.0K Views.  Chengdu University of Traditional Chinese Medicine. The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

Video: Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

X-ray Dose Reduction through Adaptive Exposure in Fluoroscopic Imaging

X-ray fluoroscopy is widely used for image guidance during cardiac intervention. However, radiation dose in these procedures can be high, and this is a significant concern, particularly in pediatric applications. Pediatrics procedures are in general much more complex than those performed on adults and thus are on average four to eight times longer1. Furthermore, children can undergo up to 10 fluoroscopic procedures by the age of 10, and have been shown to have a three-fold higher risk of developing fatal cancer throughout their life than the general population2,3.
We have shown that radiation dose can be significantly reduced in adult cardiac procedures by using our scanning beam digital x-ray (SBDX) system4-- a fluoroscopic imaging system that employs an inverse imaging geometry5,6 (Figure 1, Movie 1 and Figure 2). Instead of a single focal spot and an extended detector as used in conventional systems, our approach utilizes an extended X-ray source with multiple focal spots focused on a small detector. Our X-ray source consists of a scanning electron beam sequentially illuminating up to 9,000 focal spot positions. Each focal spot projects a small portion of the imaging volume onto the detector. In contrast to a conventional system where the final image is directly projected onto the detector, the SBDX uses a dedicated algorithm to reconstruct the final image from the 9,000 detector images.
For pediatric applications, dose savings with the SBDX system are expected to be smaller than in adult procedures. However, the SBDX system allows for additional dose savings by implementing an electronic adaptive exposure technique. Key to this method is the multi-beam scanning technique of the SBDX system: rather than exposing every part of the image with the same radiation dose, we can dynamically vary the exposure depending on the opacity of the region exposed. Therefore, we can significantly reduce exposure in radiolucent areas and maintain exposure in more opaque regions. In our current implementation, the adaptive exposure requires user interaction (Figure 3). However, in the future, the adaptive exposure will be real time and fully automatic.
We have performed experiments with an anthropomorphic phantom and compared measured radiation dose with and without adaptive exposure using a dose area product (DAP) meter. In the experiment presented here, we find a dose reduction of 30%.

We are developing a dynamic adaptive exposure technique using our scanning beam digital X-ray system. Rather than exposing an object uniformly, the exposure is adapted depending on the opacity of the object. Here we show an experiment on an anthropomorphic phantom that resulted in a dose saving of 30%.

X-ray fluoroscopy is widely used for image guidance during cardiac intervention. However, radiation dose in these procedures can be high, and this is a ...

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for erectile dysfunction. However, the methods used vary between laboratories, and pitfalls still exist. The goal of this study was to describe a surgical technique that would provide a reliable and reproducible model. By exposing the ischiocavernosus muscle at its point of insertion on the ischial tuberosity, the penile crus could be cannulated with minimal dissection and injury to the structures involved in erectile function. Repeated stimulation of the CN, without the need for lifting and drying, was achieved by using a 125 &#181;m bipolar silver electrode and biocompatible silicon glue to isolate the electrode-nerve complex. This method prevents neuropraxia by reducing stretching and drying the nerve and provides complete isolation of the nerve, negating electrical leakage and preventing stimulation of alternative pathways.

This study describes a simplified surgical procedure and technique for performing cavernous nerve stimulation with the isolation of the nerve-electrode complex using silicone glue and intracavernous pressure measurement.

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

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X-ray Dose Reduction through Adaptive Exposure in Fluoroscopic Imaging

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Movement Retraining using Real-time Feedback of Performance

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Real-time Cytotoxicity Assays in Human Whole Blood

Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice