Name: microscopic observation of lymphocyte dynamics in rat peyer's patches
Uploaded: 2020-06-25T15:00:00
Description: Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

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 addit...

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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.

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	<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>
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			<td height="21" style="height:21px;">Hoechest 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">H3570</td>
			<td></td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Wako Pure Chemical Industries</td>
			<td style="width:119px;">099-06571</td>
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			<td height="21" style="height:21px;">RPMI 1640 medium</td>
			<td>GIBCO</td>
			<td style="width:119px;">11875093</td>
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			<td height="21" style="height:21px;">Texas Red&#8211;dextran</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">D1863</td>
			<td></td>
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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 ...

This protocol makes it possible to perform in vivo observation of lymphocyte dynamics in rat Peyer's patches for long periods of time. It can be used to explore the effects of various tracks on lymphocytes in vivo. Demonstrating the procedure will be Kazuki Horiuchi, a medical doctor from my laboratory.Begin by forming a cannulation tube and dipping it in hot water, then bend and fix it to a plastic board with adhesive tape. After anesthetizing a male Wistar rat and removing the hair on its abdomen, make a horizontal incision with surgical scissors from the midline to the left subcostal area, taking care not to damage the blood vessels in the parietal peritoneum. Wrap the intestine with wet gauze and move it gently to the outside of the body to reveal the retroperitoneal organs.Insert a notch between the erector muscles of the spine and the adipose tissue with surgical scissors, then bluntly separate them with fingers. Carefully strip off the connective tissue around the thoracic duct. Expose the thoracic duct from just beneath the left crus of the diaphragm caudally until 20 millimeters of the duct is visible.Gently separate adhesions between the thoracic duct and aorta using precision tweezers or wet cotton swabs. Apply a ligature on the thoracic duct just beneath the left crus of the diaphragm causing the caudal thoracic duct to become distended. Make a five millimeter hole in the abdominal wall by stabbing it with the surgical scissors, pass the hairpin-curved cannulation tube into the hole and ligate the tube on the iliopsoas muscle.Fill the cannulation tube with normal saline containing 10 units per milliliter heparin. After placing a string beneath the distended thoracic duct, stab the duct with a sharply cut edge of the cannulation tube, cannulate it approximately five millimeters toward the tail and ligate the thoracic duct with the cannulation tube. To prevent dehydration, create a three millimeter hole in the anterior wall of the antrum of the stomach using precision tweezers and pass a two millimeter silicon tube to the duodenum through the pyloric ring.After closing the wound, maintain the animals in Bollman's cages and start to infuse sugar-laden saline into the rat's duodenum from the silicon tube at a flow rate of three milliliters per hour. Cover the entire cage with a paper towel to keep it warm. To collect the thoracic duct lymphocytes, fix the cannulation tube to the hole in the center of the lid of a 15 milliliter conical tube on ice containing six units per milliliter heparin, 10%fetal bovine serum and RPMI 1640 medium.Dissolve CFDSE and dimethyl sulfoxide to 15.6 millimolar and incubate one times 10 to the ninth lymphocytes in 15 milliliters of RPMI 1640 with 50 microliters of CFDSE solution for 30 minutes at 37 degrees Celsius. Under continuous anesthesia with 2%isoflurane, placed the Wistar rat on a portable stainless steel plate with a rectangle hole around the center covered with a glass slide and open the abdomen of the recipient Wistar rat via a midline incision. Select approximately 10 centimeters of the ileal segment with Peyer's patches for observation.Keep the intestine moist and as warm as possible by soaking the gauze that is used to cover the small intestine with PBS warmed to 37 degrees Celsius. Put the slide on the stage of the microscope and choose suitable areas for observation of the microcirculation in Peyer's patches where some high endothelial venules are running through the serosa. 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's body to prevent it from being vibrated by the rat's heartbeat and breathing. Use a one milliliter syringe to slowly inject the CFSE-labeled thoracic duct lymphocytes into the jugular vein of the recipient rats from the venous catheter. Continuously monitor thoracic duct lymphocytes in the microvasculature of the Peyer's patches using CLSM and perform time-lapse imaging for three hours at 32nd intervals.Inject 25 milligrams per kilogram Texas Red-dextran into the jugular vein of the recipient rat to stain the bloodstream and five milligrams per kilogram Hoechest 33342 to stain the cell nuclei. To quantify the lymphocyte dynamics, define lymphocytes adhering to the high endothelial venules for more than 30 seconds as adhesive lymphocytes and those immigrating from the high endothelial venules to stroma as migrating lymphocytes. Then calculate the average percentage of migration in each field of view.This protocol was used to collect naive gut tropic lymphocytes and observe their migration in rat Peyer's patches. Microscopic images of the Peyer's patches are shown here. The nuclei of vascular endothelial cells are stained blue.The blood plasma is red, and the blood flow is detected by the flow of unstained cells. Gut tropic lymphocytes stained green can be seen adhering to high endothelial venules. When performing this protocol, keep in mind that the preparation to create the optimal environment for cannulation is important, including the exposure of the thoracic duct and adjustment of the positional relationship between the thoracic duct and the catheter running parallel.Observing the lymphocytes dynamics in the Peyer's patches under several conditions may be are foundations for future research of gut immunity.

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

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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 ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.

Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...

Securing the Rat in the Rat Jacket for Electroacupuncture

Wear a pair of gloves before proceeding to fit and secure the rat in the custom made rat jacket. Then using one hand hold the rat jacket's middle and back portion on the table. Using the other hand, gently hold the rat by its tail to pick it up from the feeding cage and place it into the jacket as the rat moves forward into the jacket and its head reaches the headgear point.Using both hands, carefully pull out its left and right upper limbs from the holes in the jacket. Securely wrap the tying ropes from the opening end of the jacket around the rat's trunk, and tie them tightly at the opposite end to adequately restrain its upper limbs. Tie the rope loose enough to prevent hypoxia or local limb ischemia, and tight enough to prevent the rat's escape.In a similar manner, secure its lower limbs.