Figure 1.
Methylene blue dye staining of rectum and descending colon following a 1 ml (A) or 3 ml (B) simulated challenge.
Twenty minutes following dye exposure, animals were necropsied and rectum/distal colon was removed en block. Once fecal matter was gently cleared from tissue, stained mucosal tissue was photographed to assess both completeness coverage and the maximal distance of dye coverage from the anal verge.
Figure 2.
Magnetic resonance imaging to visualize contrast dye introduced intrarectally in two rhesus macaques exposed to a 1(A) or 3 ml (B) inoculum.
In both A and B, the left two images are maximum intensity projections representing a 3D reconstruction, while the right panel shows a cross-sectional image. The orientation of the image is indicated for each panel at top left with P indicating posterior towards the reader and R the right side towards reader. Gadofoveset trisodium contrast dye was administered intrarectally and 20 minutes later animals were returned to a recumbent position and MRI was performed. (A) Although highly variable between animals, the 1 ml exposure shown here shows robust inoculum penetration including within the distal descending colon. Panel B represents a 3 ml exposure with robust and thorough dye coverage in the rectum and in the descending colon. Bladder compression of the distal descending colon results in a thin layer of dye between these two sites, which, though difficult to see, likely coats the compressed mucosa as noted by the short arrow in the far right panel. Also present in this panel is apparent mixing of feces with the contrast agent represented by the long arrow. Panel C represents cross-sectional image showing solid fecal matter blocking contrast from contacting the mucosa. Although background contrast was detected in the small intestines due to animal diet, contrast dye in the rectum and descending colon is clearly visible. These images were selected to represent the variable outcomes of the 5 animals tested by MRI for luminal exposure.
Figure 3.
Methylene blue dye staining of female genital tract following a 2 ml inoculum challenge.
Twenty minutes following dye exposure, animals were necropsied and the entire female genital tract was extracted en block and dissected. The tissue was photographed to assess both completeness of dye coverage and length of distance from the vaginal introitus. Overall there was complete dye staining in the vaginal vault, but stain was not detected in the cervix, uterus, or ovaries.
Figure 4.
Magnetic resonance imaging to visualize contrast dye introduced intravaginally.
2-sectional image. The orientation of all images is posterior towards the reader. Panel A shows a wider view of the animal including the entire abdomen while panel B highlights the pelvis at greater magnification. Panel C is a 2D image demonstrating the inability to detect contrast penetration into the cervix/uterus despite the high degree of anatomic resolution that can be achieved by MRI. Although significant background contrast was detected in the small intestines due to minerals present in the animal diet, contrast dye in the vagina is clearly visible. These images were selected to represent the highly consistent vaginal exposure in 3 animals examined by MRI.
Figure 5.
Lymphatic draining patterns following submucosal rectal injections of methylene blue dye.
Up to 50 μl of methylene blue was injected submucosally in the rectum prior to euthanasia. During necropsy, stained lymph nodes were identified, photographed and successively reinjected (intranodaly) to identify the next stained lymph node in the draining chain. (A) An illustration of the various pelvic lymph nodes and rectal injection sites for orientation and reference. Each colored box represents the site of each photograph in panels B-D. Panel B shows evidence of dye staining within the internal iliac lymph nodes. Panel C shows evidence of dye staining within the common iliac lymph nodes following internal iliac dye injection, interestingly an anatomic variation in the lymphatic pathway in this animal resulted in dye crossing the midline and enhancing a left common iliac node as well. Panel D shows evidence of dye staining within the para-aortic lymph node following common iliac dye injection. All images are representative of the 5 animals examined by methylene blue dye.
Figure 6.
Magnetic resonance imaging to visualize contrast dye following submucosal rectal injections.
50-G5DOTA dendrimer was injected submucosally at two sites within the rectum followed immediately by MR imaging. Within minutes of injection, draining internal iliac lymph nodes were clearly visible. This 3D rendering image is shown in posterior (A) and right view (B) and is representative of the 4 animals examined by MRI.
Table 1.
Summary of lymph node staining following submucosal injection of Gd-G5-DOTA dendrimer and MR imaging.
Figure 7.
Lymphatic draining patterns following submucosal descending colon injections of methylene blue dye.
Up to 50 μl of methylene blue was injected submucosally in the colon following necropsy. Stained lymph nodes were identified, photographed and successively reinjected (intranodaly) to identify the next stained lymph node in the draining chain. (A) An illustration of the various colonic lymph nodes and injection sites for orientation and reference. Each colored box represents the site of each photograph in panels B-E. Panel B shows evidence of dye staining within the para-colonic lymph nodes. Panel C shows evidence of dye staining within the left colic lymph node following para-colonic lymph node injection, note adjacent non-draining lymph nodes. Panel D shows evidence of dye staining within the inferior mesenteric lymph node following left colic lymph node dye injection. Panel E shows evidence of dye staining within the para-aortic lymph node following inferior mesenteric node dye injection. Panels D and E also demonstrate partial staining of an inferior mesenteric lymph node with half completely enhanced by dye and half completely devoid of dye uptake. All images are representative of the 5 animals examined by methylene blue dye.
Figure 8.
Magnetic resonance imaging to visualize contrast dye following submucosal descending colon and rectal injections.
50 μl of Gd-G5DOTA dendrimer was injected submucosally at two sites within the descending colon (A) followed immediately by MR imaging. Within minutes of injection, draining para-colonic and left colic LN were clearly visible. After several minutes, inferior mesenteric LN and para-aortic LN were detectable. After several imagining sessions, 50 μl of Gd-G5DOTA dendrimer was subsequently injected submucosally at two sites within the rectum of the same animal (B) followed immediately by MR imaging. Within minutes of the second injection, draining internal iliac and common iliac LN were detected. Para-aortic LN demonstrated further enhancement showing the confluence of both routes of drainage. These 3D rendering images are shown only in posterior view and are representative of the 3 animals examined by MRI for colonic lymphatic dissemination.
Figure 9.
Lymphatic draining patterns following submucosal vaginal injections of methylene blue dye.
Up to 50 μl of methylene blue was injected submucosally in the vagina prior to euthanasia. During necropsy, stained lymph nodes were identified, photographed and successively reinjected (intranodaly) to identify the next stained lymph node in the draining chain. (A) An illustration of the various pelvic lymph nodes and vaginal injection sites for orientation and reference. Each colored box represents the site of each photograph in panels B and C. Panel B shows evidence of dye staining within the internal iliac lymph nodes. Panel C shows evidence of dye staining within the common iliac lymph node and para-aortic LN following internal iliac dye injection. All images are representative of the 2 animals examined by methylene blue dye.
Figure 10.
Magnetic resonance imaging to visualize contrast dye following submucosal vaginal injections.
50 μl of Gd-G5DOTA dendrimer was injected submucosally at two sites within the vagina followed immediately by MR imaging. Within minutes of injection, draining internal iliac LN and upstream common iliac LN were clearly visible. This 3D rendering image is shown only in posterior view and is representative of the 2 animals examined by MRI.
Figure 11.
In situ hybridization positive cells within the draining internal iliac LN 3 days following vaginal challenge with SIVmac.
A. Low magnification of an internal iliac lymph node section showing three productively infected cells with lymphocytic morphology (arrows). B. High magnification of a productively infected cell with lymphocytic morphology (arrow) in the paracortical T cell zone adjacent to a trabecular sinus. C. High magnification of four cells harboring SIV vRNA with a dendritic cell morphology (arrows) within the capsular sinus, which is consistent with cells (putatively DCs) bearing SIV immigrating into the internal iliac lymph node.
Figure 12.
Cartoon illustration representing the two distinct pathways for lymphatic dissemination from the sites of mucosal exposure.
The anatomic position and commonly used nomenclature for all relevant lymphatic nodes are shown. The two distinct lymphatic dissemination pathways are highlighted by arrows with rectal/vaginal dissemination pathway occurs via the internal iliac, then the common iliac followed by the para-aortic chain. The colonic dissemination pathway includes the para-clonic LN, the left colic LN, inferior mesenteric LN, and the para-aortic system.