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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	<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&#039;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.

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 ...

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.

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3.8K vistas.  National Defense Medical College. 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 o...