The section on the free jejunal flap is the result of work with Akiko Yano, MD, Kochi Medical School.

A clinical investigation of ICG imaging was performed under approval by the Ethics Committee of Kochi Medical School with informed consent from every patient. A total of 25 patients were included, who underwent reconstructive surgery using free jejunal graft following resection of cancer of the pharynx or cervical esophagus between 2011 and 2016. Regarding the US, the video records obtained in clinical practice between 2000 and 2018 have been reviewed. Ethical approval was waived on this, according to the institutional ethical review committee.
1. Transesophageal echocardiography (TEE)
NOTE: TEE, which necessitates the insertion of an esophageal probe, is suitable for making a diagnosis or monitoring in the OR or ICU where CT assessment is not available. TEE provides morphologic and kinetic information as well as the perfusion status of the intestine27,28. Although it requires expertise in visualizing the SMA, it is not so hard for experienced heart and thoracic aorta examiners. The SMA can be visualized with the TEE probe (see Table of Materials) advanced into the stomach and the transducer directed posteriorly (Figure 1A).
<ol>
	<li>Visualize the descending aorta on the short axis (scanning plane 0&#176;), then advance the probe into the stomach with the image of the aorta kept in view by rotating the probe counterclockwise with a slight anteflexion of the probe tip to keep the transducer in contact with the esophageal wall.</li>
	<li>If the image of the aorta moves downward, bend the probe tip further (Figure 1B).</li>
	<li>Use color Doppler mode to facilitate the identification of visceral branches by flow signal, and ensure that the orifice of the celiac artery appears at the 12 o&#39;clock position of the abdominal aorta (Figure 1C). It divides into two or three arteries within a few centimeters of the orifice.</li>
	<li>Advance the probe one inch further so that the SMA appears at the 12-2 o&#39;clock position. 
	NOTE: A leftward bending of the probe tip is helpful to rotate the image and depict the SMA at 12 o&#39;clock position.</li>
	<li>Ensure that the distal portion of the SMA is located between the pancreas (splenic vein) and abdominal aorta, where the left renal vein crosses behind the SMA.</li>
	<li>Rotate the scanning plane to 90&#176; to visualize the long-axis view of the aorta and visceral branches. The distal portion of the SMA can be assessed more easily (Figure 1D). 
	NOTE: Figure 1C,D show the TEE findings in a cardiovascular surgical case without mesenteric ischemia.</li>
</ol>
2. Abdominal US
NOTE: This modality is suitable for suspecting or excluding mesenteric ischemia among several patients with abdominal pain, together with physical examination. It is used for assessing the morphology and kinetics of the intestine and the blood flow in the SMA. Figure 2A shows the place of the probe (see Table of Materials) for each purpose.
<ol>
	<li>Use a convex or sector probe with a frequency range of 2 to 5 MHz to facilitate visualization and above assessment of the intestine via the abdominal wall with adequate resolution and sensitivity. 
	NOTE: Use a transducer with a frequency range between 2.5 and 5 MHz for visualizing the bowels in the abdomen with the gain setting maximal without generating background noise.</li>
	<li>Place the probe on the abdominal wall around the navel to visualize the intestine (Figure 2B). Find any acoustic window (yellow arrow) between the intestinal gas (blue dotted line).</li>
	<li>Check the size and peristaltic movement of the intestine, mucosal edema, or the presence of ascites around it. The latter indicates that intestinal necrosis takes place.</li>
	<li>For assessing the SMA flow, the probe was placed vertically above the navel level. Find the SMA, which arises from the abdominal aorta and directs caudally within a few centimeters (Figure 2C). 
	&#8203;NOTE: The US findings in Figure 2B,C was recorded in healthy individuals.</li>
</ol>
3. ICG imaging
NOTE: This modality is suitable for assessing the perfusion of the tissues in the surgical field.
<ol>
	<li>Prepare the ICG imaging system following the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Inject a total of 2.5 mg ICG (see Table of Materials) dissolved in 10 mL of distilled water (0.25 mg/mL) into the central venous line, followed by flushing with 10 mL of saline (Figure 3A).</li>
	<li>Visualize the perfused ICG into the mesenteric artery and then the intestinal tissue on display (Figure 3B). It usually appears approximately 10 to 20 s after injection. 
	&#8203;NOTE: The ICG imaging findings in Figure 3B were recorded in a reconstruction case with a free jejunal graft enrolled in the above study.</li>
</ol>
4. Near-infrared spectroscopy (NIRS)
NOTE: To solve the problem in plastic and reconstructive surgery (as mentioned in the Introduction section), this study proposed the use of the NIRS system, which has been used in cardiovascular surgery29; however, validation to confirm that rSO2 reflects the ischemic status of the jejunum was needed. When the jejunal flap was harvested, an NIRS sensor was placed on the jejunum, and changes in rSO2 were monitored when the artery and vein were clamped, and perfusion was resumed after reconstruction. In addition, rSO2 changes were observed for 3 days postoperatively with the NIRS sensor placed on the skin of the neck. The recommended procedures for assessing the rSO2 of the intestine directly in the surgical field are described here.
<ol>
	<li>Prepare the NIRS system following the manufacturer&#39;s instructions (see Table of Materials) (Figure 4A).</li>
	<li>Use an appropriate sensor for measuring the rSO2 of the tissue according to the depth of the target region to be assessed (Figure 4B). Place the sensor directly on it with light contact so as not to press excessively. 
	NOTE: This study used a sensor with a distance between the emitter and receiver of 2 cm.</li>
	<li>Check the rSO2 value indicated on display, updated every 5 s (Figure 4B).</li>
</ol>

The author has no conflicts of interest regarding this work.

Mesenteric ischemia remains an unsolved problem beyond the clinical field. To solve such a common problem, similar pathology in other organs may be helpful to take a hint. The concept of &#34;ischemic cascade&#34; was proposed for acute myocardial infarction32, and regional wall motion abnormalities (hypokinesis, akinesis, and dyskinesis) located at the early stage of the cascade have been used as an indicator of myocardial infarction instead of coronary blood flow, which cannot be assessed noninv...

Acute mesenteric ischemia can be life-threatening unless diagnosed and treated without delay1,2; however, early diagnosis followed by restoration of perfusion before progressing to bowel necrosis, preferably within 4 h, remains challenging for several reasons: (1) mesenteric ischemia is caused via multiple mechanisms and associated with several diseases managed by different specialties; (2) there are no symptoms, signs, or laboratory data specific for mesenteric ischemia; and (3) computed tomography (CT), the gold standard for diagnostic imaging, is misleading because ischemia can be present despite an opacified superior mesenteric artery (SMA)2,3,4,5.
Causes of mesenteric ischemia include embolism, thrombosis, dissection, or non-occlusive mesenteric ischemia (NOMI)3,6. Embolism is caused by a cardiogenic thrombus in patients with atrial fibrillation, dilated left ventricle, or atheroma in the aorta, which is asymptomatic until embolization. Occasionally, a thrombus is generated in the SMA or superior mesenteric vein. It has recently been shown that COVID-19 can lead to thrombus formation7. In aortic dissection, the intimal flap in the aorta occludes the orifice of the SMA, or dissection extends into the SMA, and an expanded false lumen compresses the true lumen. Because this obstruction is &#34;dynamic,&#34; mesenteric ischemia occurs even when the SMA is shown to be opacified on contrast CT. It is not uncommon for mesenteric ischemia to appear together with other critical conditions, such as stroke, myocardial infarction, or aortic rupture, thus necessitating a prompt and accurate diagnosis to prioritize treatment. In patients who undergo blood dialysis for years, the SMA is often narrowed due to calcifications, and the blood flow can be critically reduced following cardiac surgery using extracorporeal circulation or various types of stress8,9,10. NOMI can be caused by inadequate oxygen supply to the SMA due to heart failure, cardiac arrest, or hypoxemia despite a patent SMA11,12,13. Considering various etiologies and patterns of occurrence, not only blood flow in the SMA but also ischemic status in the intestinal wall must be assessed.
Another reason for delayed diagnosis is a lack of key symptoms or physical findings. Defense becomes obvious after the intestine is necrotized. Although several laboratory tests, such as C-reactive protein, lactate, citrulline, or intestinal fatty acid-binding protein, have been investigated as potential indicators of mesenteric ischemia4,14, no laboratory test has been shown to detect an early stage of mesenteric ischemia to date15. Although CT is the standard diagnostic imaging modality of mesenteric ischemia16,17,18, there can be errors in diagnosis or pitfalls in the filming technique5,19, and thus expertise is needed for an accurate diagnosis, which may necessitate the transfer of the patient to another facility. In addition, CT is not available for patients in the operating room (OR), emergency department (ER), or intensive care unit (ICU) who cannot be transferred to the Radiology Department. Allergies to contrast media, renal toxicity, or radiation exposure also limit CT as the initial diagnostic examination for every patient with abdominal pain.
Bowel ischemia is also problematic for plastic and reconstructive surgeons. During radical surgery for pharyngeal cancer, a free jejunal flap is used to reconstruct the resected pharynx. A portion of the jejunum is harvested with an artery and vein pedicle, which is anastomosed to the vessels in the cervical region, followed by anastomosis of the jejunal flap to the pharynx and esophagus. To confirm the competency of the vascular anastomosis, indocyanine (ICG) imaging was performed intraoperatively (sections 3). However, there are occasions when the flap develops necrosis within several days after surgery. Although rare, flap necrosis can be fatal unless detected and treated without delay. Thus, various attempts for detecting jejunal ischemia have been developed, such as frequent ultrasonography (US) to confirm blood flow, repeated endoscopy to verify mucosal color, or designating a sentinel portion of the jejunum to monitor perfusion, which is buried afterward by an additional surgical procedure20,21,22; however, such maneuvers are difficult for both patients and physicians. Other modalities applied to the clinical use for diagnosing bowel ischemia include optical coherence tomography23, laser speckle contrast imaging24, sidestream dark field imaging25, and incident dark field imaging26. These promising modalities are expected to become widely available through further development.
Considering the nature of mesenteric ischemia, which affects several fields in various situations, it is important to have multiple measures for detecting it. This article proposes two potential candidates for this purpose, US and near-infrared light and presents the representative findings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HyperEye Medical System</td>
			<td>Mizuho Ikakogyo Co., Ltd.</td>
			<td></td>
			<td>ICG imaging system used in Figure 3</td>
		</tr>
		<tr>
			<td>Indocyanine green&#160;</td>
			<td>Daiichi Sankyo Co., Ltd.</td>
			<td></td>
			<td>ICG used for ICG imaging in Figure 3</td>
		</tr>
		<tr>
			<td>TEE system</td>
			<td>Philips Electronics</td>
			<td>iE33</td>
			<td>TEE system used in Figure 5</td>
		</tr>
		<tr>
			<td>TOS-96, TOS-OR</td>
			<td>TOSTEC Co.</td>
			<td></td>
			<td>NIRS system used in Figure 4</td>
		</tr>
		<tr>
			<td>Ultrasonographic system</td>
			<td>Hitachi, Co.</td>
			<td>EUB-555, EUP-ES322</td>
			<td>echo system used in Figure 1</td>
		</tr>
		<tr>
			<td>Ultrasonographic system</td>
			<td>Aloka Co.</td>
			<td>SSD 5500</td>
			<td>echo system used in Figure 2</td>
		</tr>
		<tr>
			<td>Vscan</td>
			<td>GE Healthcare Co.</td>
			<td></td>
			<td>Palm-sized echo used in Figure 2</td>
		</tr>
	</tbody>
</table>

multimodality diagnosis of mesenteric ischemia

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no laboratory data specifically indicates intestinal tissue ischemic status before necrosis develops. While computed tomography is the standard for diagnostic imaging, there are several limitations: (1) repeated assessments are associated with increased radiation exposure and risk of renal damage; (2) the computed tomography findings can be misleading because necrosis occasionally occurs despite opacified mesenteric arteries; and (3) computed tomography is not necessarily available within the golden time of salvaging the intestines for those patients in the operating room or at a place far from the hospital. This article describes a challenge to overcome such limitations using ultrasonography and near-infrared light, including clinical studies. The former is capable of providing not only morphologic and kinetic information of the intestines but also perfusion of the mesenteric vessels in real-time without transferring the patient or exposing them to radiation. Transesophageal echocardiography enables precise assessment of mesenteric perfusion in the OR, ER, or ICU. Representative findings of mesenteric ischemia in seven aortic dissection cases are presented. Near-infrared imaging with indocyanine green helps visualize the perfusion of vessels and intestinal tissues although this application requires laparotomy. Findings in two cases (aortic aneurysm) are shown. Near-infrared spectroscopy demonstrates oxygen debt in the intestinal tissue as digital data and can be a candidate for early detection of mesenteric ischemia without laparotomy. The accuracy of these assessments has been confirmed by intraoperative inspections and postoperative course (prognosis).

TEE 
There were two types of findings: (1) &#34;branch type&#34; with a true compressed lumen in the SMA by an expanded false lumen without blood flow, and (2) &#34;aortic type&#34; with the intimal flap at the orifice of the SMA and lack of blood flow in the SMA (Figure 5A). The representative TEE findings of three cases with bowel necrosis caused by acute aortic dissection are shown. In one case of the former type, the true lumen in the SMA were severely compressed (<...

Watch this Scientific Journal Video about Multimodality Diagnosis of Mesenteric Ischemia at JoVE.com

Multimodality Diagnosis of Mesenteric Ischemia

This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no ...

multimodality-diagnosis-of-mesenteric-ischemia

Research

JoVE Journal

Medicine

464 Views.  Kochi Medical School. This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Video: Multimodality Diagnosis of Mesenteric Ischemia

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.