This research was supported by grants from National Defense Medical College and by a Health and Labour Sciences research grant for research on intractable diseases from the Ministry of Health, Labour and Welfare, Japan.

The experimental protocol was approved by the Animal Research Committee of National Defense Medical College (no. 16058). The animals were maintained on standard laboratory chow (CLEA Japan Inc, Tokyo, Japan). The laboratory animals were treated in accordance with National Institutes of Health guidelines.
1. Collection and separation of lymphocytes
NOTE: Since lymphocytes must be fresh and cannot be stored, they must be collected from rats for each experiment. In addition, gut-tropic lymphocytes must be collected directly from the thoracic duct where they circulate.&#160;All procedures are expected to be completed within 6 hours, and all instruments, gloves, and surfaces are clean. In order to keep the animals in a physiologically stable state, all saline and other fluids used in the experiment should be kept warm.
<ol>
	<li>Form a cannulation tube (1 mm diameter) like a heparin curve (a radius of about 5 mm) after dipping it in hot water (about 80&#8211;90 &#176;C) and fix it to a plastic board using adhesive tape for a few hours in advance.</li>
	<li>Anesthetize a male Wistar rat (8&#8211;12 weeks) with intraperitoneal injection of a mixture of Midazolam (2mg/kg), Domitor (0.15mg/kg), and Betulphar (2.5mg/kg) and cut the body hair of the abdomen using a hair clipper. Remove the hair firmly after shaving so that the hair does not enter the abdominal cavity.</li>
	<li>After confirming that the stable depth of anesthesia required for the procedure is obtained by toe pinch, make an incision with surgical scissors horizontally from midline to the left subcostal area. Be careful not to damage the blood vessels in the parietal peritoneum by watching the vessels under light.</li>
	<li>Wrap the stomach with wet gauze and move the stomach gently to the right outside of the body to reveal the retroperitoneal organs. Insert a notch between the erector muscles of the spine and the adipose tissue using surgical scissors and bluntly separate them with fingers.</li>
	<li>Carefully strip off the connective tissue around the thoracic duct. The excess connective tissue increases with age. Expose the thoracic duct from just beneath the left crus of the diaphragm caudally until a 20 mm length of the thoracic duct was visible. Gently separate adhesions between the thoracic duct and aorta using precision tweezers or wet cotton swabs.
	<ol>
		<li>Perform this process carefully because some rats have arterioles branching from the abdominal artery crossing over the thoracic duct or a short thoracic duct due to a higher position of the lymph plexus (small thoracic ducts spreading like spider webs). Avoid unnecessary contact to the thoracic duct to avoid inducing edema.</li>
	</ol>
	</li>
	<li>Apply a ligature by a string (3-0 silk) on the thoracic duct just beneath the left crus of the diaphragm. The caudal thoracic duct becomes distended.</li>
	<li>Make a hole (5 mm diameter) in the abdominal wall by stabbing the cutting edge of surgical scissors, pass the hairpin-curved cannulation tube, and ligate the tube on the iliopsoas muscle at one point. Fill the cannulation tube with normal saline containing 10 U/mL heparin.</li>
	<li>After placing a string (3-0 silk) beneath the distended thoracic duct, stab the thoracic duct with the sharply cut edge of a cannulation tube, cannulate it toward the tail about 5 mm, and ligate the thoracic duct with cannulation tube for fixation.</li>
	<li>To supply saline to prevent dehydration, create a hole (3 mm diameter) on the anterior wall of the antrum of the stomach using precision tweezers and pass the silicon tube (2 mm in diameter) to the duodenum through the pyloric ring. After stitching up a wound and maintaining the animals in Bollman&#8217;s cages, start to infuse sugar-laden saline into each rat&#8217;s duodenum from the silicon tube at a flow rate of 3 mL/h. Cover the entire cage with a paper towel to keep it warm.</li>
	<li>Fix the cannulation tube to the hole in the center of the lid of a 50-mL conical tube on ice containing 6 U/mL heparin, 10% fetal bovine serum, and RPMI 1640 medium (pH 7.4; see the table of materials), and collect thoracic duct lymphocytes (TDLs) on ice. Be careful not to contact the tip of the tube to the wall of the conical tube to prevent cannulated-tube occlusion due to fibrin clot formation inside the tube. Lymph fluid (about 20 mL) including about 1.0 x 108&#65374;109 lymphocytes can be obtained in 6 hours. To obtain the lymphocytes&#160; under complete anesthesia, plane of anesthesia is monitored and lymph flow is observed appropriately. The same anesthetics are added at the same dose about 3 hours after rats are put into Ballman&#39;s cage to obtain deep anesthesia continuously for 6 hours.</li>
</ol>
2. Lymphocyte labeling with carboxyfluorescein diacetate succinimidyl ester (CFDSE)
<ol>
	<li>Dissolve CFDSE (Table of Materials) in dimethyl sulfoxide (DMSO) to 15.6 mM (500 &#181;g of CFDSE dissolved in 60 &#181;L of DMSO).</li>
	<li>Incubate lymphocytes (1 x 108&#65374;109) in 50 mL of RPMI 1640 with 50 &#181;L of CFDSE solution for 30 min at 37 &#176;C as described previously10.</li>
</ol>
3. Experimental setup for microvascular studies
<ol>
	<li>Anesthetize recipient rats (8&#8211;12 weeks) with intraperitoneal injection of a mixture of Midazolam (2mg/kg), Domitor (0.15mg/kg), and Betulphar (2.5mg/kg) and confirm the depth of anesthesia by toe pinch. Wet the abdomen before grooming the fur to minimize hair contamination. Then open the abdomen of the recipient Wister rat via a midline incision.</li>
	<li>Put a rat on a portable stainless-steel plate (about 120 x 300 mm) with a rectangle hole around the center covered with a glass slide (24 x 50 mm). Choose about 10 cm of the ileal segment including PPs for observation.</li>
	<li>Keep the intestine as warm as possible and moist with phosphate buffered saline (PBS) warmed to 37 &#176;C. Soak gauze that is used to cover the small intestine with PBS.</li>
	<li>&#160;While giving continuous anesthesia with 2 % isoflurane (see table of materials),&#160;put the slide on the stage of the microscope and choose suitable areas for observation of the microcirculation in PPs where some HEVs are running through the serosa. PPs range in size; larger ones are suitable for observation of the microcirculation using CLSM. The small intestine is not straight in places; a straight segment at least 2 cm long without any tension is suitable for observation.</li>
	<li>Cover the adjacent intestinal segment and mesentery with absorbent cotton soaked with PBS. Place the intestine segment between two rolled cotton balls and position it as far as possible from the rat&#8217;s body to prevent it from being vibrated by the rat&#8217;s heartbeat and breathing.</li>
	<li>Using a 1 mL syringe, slowly inject (over 1 min) CFSE-labeled TDLs (1 x 108 cells) into the jugular vein of the recipient rats. A rapid intravenous injection may influence the systemic circulation.</li>
</ol>
4. Microcirculation of lymphocytes
<ol>
	<li>Continuously monitor TDLs in the microvasculature of PPs using CLSM and record on a computer for 3 hours using time-lapse photography at 30 s intervals. The depth from the serosa to the HEV of PPs is about 25 &#956;m, enabling the observation of the stroma and capillary lymph vessels to a depth of 30 &#956;m.</li>
	<li>Inject Texas Red&#8211;dextran (25 mg/kg) into the jugular vein of each recipient rat to stain the bloodstream and Hoechest 33342 (5 mg/kg) to stain the cell nuclei.</li>
	<li>(Optional) To quantify lymphocyte dynamics, define lymphocytes adhering to HEVs more than 30 seconds as &#8220;adhesive lymphocytes&#8221; and lymphocytes emigrating from HEVs to stroma as &#8216;&#8216;migrating lymphocytes&#8221;. Then calculate the average percentage of migration (migrating lymphocytes / adhesive lymphocytes + migrating lymphocytes) per field of vision (approximately 0.3 mm2).</li>
</ol>

<ol>
	<li>Miura, S., Hokari, R., Tsuzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunity+in+gut+and+lymphoid+cell+trafficking.">Mucosal immunity in gut and lymphoid cell trafficking.</a> Annals of Vascular Diseases. 5, 275-281 (2012).</li><li>Stein, J. V., Nombela-Arrieta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+control+of+lymphocyte+trafficking:+a+general+overview.">Chemokine control of lymphocyte trafficking: a general overview.</a> Immunology. 116, 1-12 (2005).</li><li>Yopp, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine-1-phosphate+receptors+regulate+chemokine-driven+transendothelial+migration+of+lymph+node+but+not+splenic+T+cells.">Sphingosine-1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells.</a> The Journal of Immunology. 175, 2913-2924 (2005).</li><li>Lucke, S., Levkau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+functions+of+sphingosine-1-phosphate.">Endothelial functions of sphingosine-1-phosphate.</a> Cellular Physiology and Biochemistry. 26, 87-96 (2010).</li><li>Bollman, J. L., Cain, J. C., Grindlay, J. 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The authors have nothing to disclose.

Here we described a protocol for collecting na&#239;ve gut-tropic lymphocytes and observing their migration in rat PPs. These procedures can reveal how lymphocytes move in the microvasculature of PPs and make it possible to visually compare their dynamics under a normal or medicated condition. The direct observation of these dynamics has much merit to obtain a clue for immunological modification by some drugs, although the observational period is limited to only a few hours.
We mentioned some ...

Peyer&#8217;s patches (PPs) consist of hundreds of lymphoid follicles in the lamina propria of the small intestine. PPs are divided into follicles, the interfollicular region, and germinal centers located in the lower part of the follicles, where lymphocytes are stimulated by antigen presentation. There are no afferent lymphatic vessels, and the antigens invade the lamina propria from the intestinal lumen via the epithelial cell layer. The epithelial region covering lymphoid follicles is called the follicle-associated epithelium, within which specialized interspersed M cells uptake mucosal antigens. M cells take in antigens from the luminal side and antigens are then captured by dendritic cells and presented toward na&#239;ve lymphocytes that flow into PPs through the endothelium of high endothelial venules (HEVs)1. PPs play an important role in intestinal immunity and are related with the early stage of inflammation. Many molecular interactions involve the entrance of lymphocytes to secondary lymphoid organs (SLOs), including adhesion molecules, chemokines2,3, and sphingosine-1-phosphate4; thus, there are many expected therapeutic targets. Therefore, observing the lymphocyte dynamics within PPs enables us to catch a glimpse of the very early stage of inflammation and examine the usefulness of several promising drugs.
The method here focuses on the migration of lymphocytes in PPs, which includes several procedures (cannulation into the thoracic duct5 and collecting lymphocytes and long-term observation after the injection into collected lymphocytes). Since these procedures are complex and it was difficult to see exactly how each procedure was performed in previous reports, we mentioned here some tips to achieve a successful observation. For example, cannulation of the tubes into the thoracic duct was very difficult, and the initial success rate of cannulation was less than 50%. However, we improved the method and achieved a success rate exceeding 80%. We mentioned some other tips in this manuscript that are necessary for the successful observation to enable the quantitative evaluation of the transendothelial migration of lymphocytes under several conditions.
In previous reports, it was difficult to understand the three-dimensional changes over time, such as the intravenous injection of India ink to stain the vascular structure of PPs6, or the microscope being monofocal7. In recent years, an observational method using some photoconvertible fluorescence protein transgenic animals such as Kaede mice have clarified systematic cellular movements in vivo8. The other study clarified CD69 independent shutdown of lymphocyte egress from PPs9. We used confocal laser scanning microscopy (CLSM) because of its high analytical capability. Now we can easily obtain high-resolution images and use them to analyze lymphocyte dynamics.
In this report, we demonstrated a series of methods for evaluating lymphocyte migration in PPs. First, we showed refined methods of thoracic duct cannulation to collect lymphocytes. Second, we improved the observational methods in several ways to maintain objective organs whenever possible under microscopic observation, enabling us to obtain high-quality images for 3 hours. Third, we quantified the cellular movements of lymphocyte migration to evaluate the effects of some medications. These modified protocols will contribute to development of mucosal immunology evaluations.

<table border="0" cellpadding="0" cellspacing="0" style="width:913px;" width="913">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">A1R+</td>
			<td style="width:213px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:265px;">Comfocal Laser Scanning Microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxyfluorescein diacetate succinimidyl ester</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechest 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Wako Pure Chemical Industries</td>
			<td style="width:119px;">099-06571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium</td>
			<td>GIBCO</td>
			<td style="width:119px;">11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Texas Red&#8211;dextran</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">D1863</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopic observation of lymphocyte dynamics in rat peyer's patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Collecting lymphocytes from lymph 
To prepare the rat for thoracic duct cannulation, make an incision in the tense thoracic duct as shown in Figure 1 and then maintain the rat in a Bollman&#8217;s cage as shown in Figure 2.
When the lymphocytes are well collected, we can obtain about 20 mL/6 h lymph fluid containing about 107&#65374;108/mL lymphocytes. Of the TDLs, 70% express CD4, among whi...

Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

microscopic-observation-of-lymphocyte-dynamics-in-rat-peyer-s-patches

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3.9K Views.  National Defense Medical College. Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer’s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Video: Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

The Microscopic Transcanal Approach in Stapes Surgery Revisited

The microscopic transcanal (aka transmeatal) surgical approach was first described in the 60s, offering a minimally invasive means of reaching the external auditory canal, the middle ear, and epitympanon. Such an approach avoids a retroauricular or endaural skin incision; however, working through a narrow space needs angled microsurgical instruments and specific training in otologic surgery. The transcanal approach restricts the working space; however, it offers a binocular microscopic vision into the middle ear without extended skin incisions and thus, reducing post-operative pain and bleeding. In addition, this minimally invasive approach avoids scar tissue complications, hypoesthesia of the auricle, and potential protrusion of the pinna. Despite its numerous advantages, this method is still not routinely performed by otologic surgeons. Since this minimally invasive technique is more challenging, there is a need for extensive training in order for it to be widely adopted by otologic surgeons. This article provides step-by-step surgical instructions for stapes surgery and reports possible indications, pitfalls, and limitations using this microscopic transcanal technique.

This article describes the microscopic transcanal technique for stapes surgery, providing step-by-step surgical instructions for familiarizing surgeons with this approach.

The microscopic transcanal (aka transmeatal) surgical approach was first described in the 60s, offering a minimally invasive means of reaching the ...

Microscopic Replantation of Penile Glans Amputation Due to Circumcision

Circumcision using a disposable stapler is becoming quite popular in China. However, improper surgical procedures also bring the risk of penile glans amputation, which is a very rare iatrogenic genital injury. Such complication is conventionally treated by simple hemostasis to achieve self-healing, early gross replantation, or delayed plastic surgery. However, these may lead to obvious unfavorable outcomes such as amputated glans loss, necrosis, malformation healing, or urethral orifice stenosis. In the present study, we adopted microscopic replantation as an emergency approach to achieve the precise anastomoses and anatomic reconstruction of penile glans. The goal of this protocol is to present a detailed emergency management strategy with meticulous surgical skills for the penile glans amputation. The postoperative results showed that the original shape of the glans was perfectly restored with satisfactory cosmetic appearance. The micturition function was completely restored to normal without any obvious complications. There was also no significant reduction in the sensation of amputated glans area. Hence, early meticulous microscopic replantation as soon as possible is an ideal emergency management strategy for the penile glans amputation due to circumcision.

The present protocol describes the emergency management of microscopic replantation of penile glans amputation due to circumcision.

Circumcision using a disposable stapler is becoming quite popular in China. However, improper surgical procedures also bring the risk of penile glans ...

Microscopic Cyst Resection for the Treatment of Patients Diagnosed with Epididymal Cyst

Epididymal cysts mostly occur in men aged 20-40 years old. Previous reports have covered concerns about postoperative complexes, including postoperative asoedema, hematoma, sustaining pain, and seminal tract obstruction in patients who have undertaken nonmicroscopic epididymal cyst resection or epididymal resection. Nonmicroscopic epididymal cyst surgery is suggested for patients with childbirth plans as a precaution. The treatment of male epididymal cysts via microtechnology is obviously a beneficial option; we took the lead in carrying out microscopic epididymal exploration and cyst resection surgery in China. From September 2017 to April 2021, 41 young and middle-aged male patients diagnosed with epididymal cysts underwent microtechnology treatment in a program titled &#34;microscopic epididymal exploration and cystectomy&#34;. The postoperative follow-up lasted for 3-50 months. The results confirmed that, as microscopic manipulation largely improved visualization of the subtle tissue structures of the epididymis, the cyst could be clearly dissected apart and completely removed intact under the microscope. Bleeding during the operation was significantly reduced (2-3 mL) and wound drainage was not required. According to follow-up data, microscopic treatment significantly reduced the incidence of postoperative scrotal hematoma, edema, and long-term postoperative pain, thereby promising a higher surgical success rate as well as recurrence prevention. Besides, preliminary experience and reflection suggest that microscopic epididymal exploration and cystectomy provide efficient preservation of the epididymal patency through refined treatment, while a better prognosis can be achieved. We recommend that surgery be carried out before the epididymal cyst develops to 0.8 cm in diameter, for fear that a larger epididymal cyst (&#62;0.9 cm in diameter) could cause the complete destruction of all tubules of the ipsilateral epididymis - a more severe case with damage to the testicular output network.

Here, we present a protocol to treat patients diagnosed with an epididymal cyst using microscopic cyst resection. Based on microscopic manipulation, a largely improved visualization of the subtle tissue structures of the epididymis was obtained, so the cyst could be clearly dissected apart and completely removed intact.

Epididymal cysts mostly occur in men aged 20-40 years old. Previous reports have covered concerns about postoperative complexes, including postoperative ...