Flow of lymph from the peritoneum has been investigated in the context of peritonitis11
and peritoneal dialysis.12
Our study aims to investigate lymphatic drainage of the peritoneum in the context of the spread of cancer cells to regional lymph nodes and distant lymphatics. Stomach, colon, and ovarian cancer, and any malignancy with potential shedding of cells to the peritoneal space, may drain to a SLN with potential access to distant lymphatics. This study in the rat indicates that a specific peritoneal lymph drainage pattern exists. Although this pattern may vary among species, NIR fluorescent imaging is a means to identify peritoneal SLNs. Thus accurate nodal staging could be achieved without complete lymphadenectomy of several lymph node stations. Individual peritoneal SLNs could be identified and biopsied for staging, even before appreciable, bulky nodal disease or carcinomatosis occurs. For example, ovarian cancer with malignant cells in peritoneal washings can be staged as low as IC
depending on the primary tumor. However, involvement of distant lymph nodes is defined as stage III.13
In routine staging laparotomy, the pelvic and para-aortic lymph nodes are sampled because of the lymph drainage pattern of the ovary. However, possible SLNs of the peritoneal space are not sought, identified, or purposely resected. Consequently, there is a potential for more accurate lymph node staging of cancers with potential shedding into peritoneal space.
Knowledge of lymph node drainage patterns could also aid in cytoreductive surgery in the setting of peritoneal carcinomatosis. Survival of patients with carcinomatosis from colorectal cancer, pseudomyxoma peritonei, and peritoneal mesothelioma increased based on the completeness of disease resection.14–17
Similarly, with advanced uterine cancer, incomplete cytoreduction resulted in increased morbidity and mortality.18
Therefore, identifying and assessing lymph nodes communicating with the peritoneal space can aid in completeness of resection and possibly survival.
In the rat model, there are specific NIR-fluorescent positive and negative nodes within each lymph node group, suggesting a particular lymph node drainage pattern, not merely diffusion into all intraabdominal lymphatics. In this study, we did not document the number of positive and negative lymph nodes within a group. However this would be a feasible study since NIR fluorescent imaging has the resolution to distinguish individual positive and negative nodes in both small and large animal models. 2,8,9,19–21
Since NIR fluorescent imaging is real-time, it should be possible to perform directed dissection of individual positive lymph nodes, which would obviate the need to do a complete lymphadenectomy of a lymph node station.
In the present study, we employed novel NIR fluorescent lymph tracers to identify the lymph drainage pattern of the peritoneum. The peritoneum can be considered as one lymph space with drainage to candidate lymph nodes, namely the celiac, periportal, and superior mesenteric lymph node groups. Abernethy et al. suggested that intrathoracic, not intraabdominal, lymph nodes were the primary recipient of lymph drainage of the peritoneal space.6,22
They used Evans blue and 125
I-labeled human serum albumin as lymph tracers. These are smaller, more mobile molecules that may have passed through abdominal lymph nodes to concentrate in intrathoracic lymph nodes. In Abernathy et al.’s studies they did find some lightly blue-stained intraabdominal lymph nodes, which may have been obscured by noted peritoneal surface staining.
We used differently-sized NIR lymph tracers that have several advantages over conventional lymph tracers. First, NIR fluorescent tracers fluoresce brightly through up to 1 cm of tissue.8
The entire peritoneum and thorax can be imaged in real-time for in situ
communicating nodes, thus maximizing sensitivity and minimizing sampling error. The images can also be magnified to assist in precise and complete resection of communicating lymph nodes. Second, NIR has insignificant background staining because in vivo
tissue has minimal intrinsic NIR fluorescence. Third, and most important, the two differently-sized tracers allowed us to map the SLN with QDs and, in addition, map distant lymphatics beyond the SLN with HSA800.
In our study, the large 20-nm QDs did not pass through lymph nodes, but lodged and concentrated within the first node encountered, thus providing a more accurate identification of the SLN. Though some QD signal was found in superior mediastinal lymph nodes, these thoracic lymph nodes did not have the same convincing, bright presence as the intraabdominal lymph nodes. This suggests that the dominant drainage of the peritoneum is to intraabdominal SLN groups. It is possible that less important, but parallel, lymphatics drain the peritoneum directly to intrathoracic lymph nodes.
The optimal size of lymph tracers remains controversial, but it is thought to be in the range of 4–100 nm.23,24
Cancer cells, which are much larger than lymph tracers, may also have the capacity to alter their own lymph flow. For example, tumor burden negatively affects the ability to identify SLNs in breast cancer because of altered lymph flow.25
Therefore, study of smaller and distant lymph channels of the peritoneum was warranted. As QDs have been proven to be reliable and safe tracers for SLNs, HSA800 has proven to be a reliable tracer of lymphatic draining beyond the SLN. .2,8,9,19–21
At early time points, HSA800 identified the same candidate SLNs of the peritoneum as did QDs. Within 1 hour HSA800 identified communicating lymph channels with the thoracic duct, superior and anterior mediastinal lymph nodes and more faintly to the diaphragmatic and anterior chest wall lymphatics. Though the intraabdominal lymph node groups appear to be the SLNs of the peritoneal space, there is also communication, either in parallel or series, with diaphragmatic, thoracic duct, and chest wall lymphatics. Identification of intrathoracic lymph nodes and lymphatic channels corroborates findings of multiple studies with smaller lymph tracers.4,26
Given the large parietal and visceral surface area of the peritoneum, it is possible, therefore, that lymphatic drainage proceeds through multiple defined SLNs and lymphatics leading to the central circulation. These findings confirm previous studies of peritoneal space lymphatic drainage that also found multiple and parallel lymphatic drainage patterns.22
Multiple SLNs are also found in SLN mapping of breast and melanoma.27
In fact, the identification of multiple SLNs improved the false negative rate from 14.3% to 4.3% when only one SLN was identified.28
The different permutations found from animal to animal of celiac, periportal, and superior mesenteric lymph node groups as SLNs indicate a complex and individualized lymphatic drainage pattern of the peritoneum. Drainage patterns could be specific to individual animals, species, or disease processes. These issues remain to be clarified; however, the development of NIR fluorescent lymph node mapping makes these future investigations feasible.
The candidate SLN groups, namely the celiac, periportal, and superior mesenteric lymph node groups are the same lymph node groups receiving lymph flow from the bowel. This suggests that the visceral, not the parietal side of the peritoneal space is the significant contributor of peritoneal lymphatic drainage. To further test this hypothesis, six rats received maximal bowel resection while still preserving continuity and periportal, celiac, and superior mesenteric lymph node groups. Bowel-resected rats and sham surgery controls underwent intraperitoneal injection of QDs or HSA800 and imaging 1 hour later. After bowel resection, QDs identified peritoneal SLNs, to be intrathoracic, not intraabdominal. Furthermore, there was an absence of HSA800 in the thoracic duct and enhanced presence of HSA800 in diaphragmatic and anterior chest wall lymphatics. Therefore, lymphatics associated with the large and small intestine direct peritoneal lymph flow to intraabdominal SLNs, then to the thoracic duct, and finally to intrathoracic lymph nodes at later time points. In the absence of bowel lymphatics, the first draining lymph node group was actually intrathoracic, probably through diaphragmatic and anterior chest wall lymphatics (). Therefore the visceral bowel surfaces, not the parietal surfaces of the peritoneum, govern peritoneal flow to select intraabdominal lymph nodes, followed by the thoracic duct and eventually to thoracic lymph nodes.
Flow of Lymph from the Peritoneal Space and Changes in Flow Post-Bowel Resection
Of note, sham-surgery rats and non-operated rats did not always have QD or HSA800 uptake at the base of the mesentery. Possibly, the lymph of the peritoneum travels via subserosal lymphatics, spuriously intermixing with submucosal lymphatics of the bowel. It is possible that lymph could avoid contact with superior mesenteric lymph nodes altogether. The contribution of visceral surfaces to peritoneal space lymph drainage in humans is not fully appreciated. If our results in the rat are any indication, lymph flow to the bowel could be a novel mechanism of metastatic spread. Additionally, patients undergoing bowel resection could have an enhanced lymph flow from the peritoneal space through alternate lymph channels of the diaphragm and parietal surfaces.
In small and large animals, QDs and HSA800 have proven to be reliable and safe tracers for detection of SLNs.2,8,9,19–21
A potential limitation of QDs and HSA800 is their unknown toxicity. With QDs, the individual metals comprising the inorganic core have known toxic effects, especially at concentrations higher than those used in our study. The toxicity of these metals when complexed as salts with an organic shell is unknown. In our studies, there were no signs of acute toxicity, namely changes in heart rate or rhythm, blood pressure, or oxygen saturation. HSA800 has greater potential for immediate clinical application since it is purely organic and the product of nontoxic components. However studies directed at establishing the toxicity, if any, of these tracers must still be performed before applying the technology to humans.
In conclusion, our findings of peritoneal SLN drainage to the celiac, periportal, and superior mesenteric lymph nodes of the rat cannot be extrapolated to humans. Indeed, our findings of redirected lymph drainage to the thorax in the presence of massive bowel resection compel further studies in large animal models and hopefully humans. In our rat model, we were able to appreciate intraabdominal lymph nodes with a laparotomy and intrathoracic lymph nodes with a thoracotomy. To make this technology more applicable to the clinical setting, we are actively developing the NIR fluorescent imaging system for a thoracoscope and laparoscope. Our findings do, however, indicate that the lymph drainage of the peritoneum is complex and individualized. NIR fluorescent imaging thus has potential to contribute to patient-specific, minimally invasive investigation of SLNs of the peritoneal space.