Name: microscopic observation of lymphocyte dynamics in rat peyer's patches
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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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			<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>
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			<td height="21" style="height:21px;">Carboxyfluorescein diacetate succinimidyl ester</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">C1157</td>
			<td></td>
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			<td height="21" style="height:21px;">Hoechest 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">H3570</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 ...

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