Structure and Distribution of an Unrecognized Interstitium in Human Tissues

Abstract

Confocal laser endomicroscopy (pCLE) provides real-time histologic imaging of human tissues at a depth of 60-70 μm during endoscopy. pCLE of the extrahepatic bile duct after fluorescein injection demonstrated a reticular pattern within fluorescein-filled sinuses that had no known anatomical correlate. Freezing biopsy tissue before fixation preserved the anatomy of this structure, demonstrating that it is part of the submucosa and a previously unappreciated fluid-filled interstitial space, draining to lymph nodes and supported by a complex network of thick collagen bundles. These bundles are intermittently lined on one side by fibroblast-like cells that stain with endothelial markers and vimentin, although there is a highly unusual and extensive unlined interface between the matrix proteins of the bundles and the surrounding fluid. We observed similar structures in numerous tissues that are subject to intermittent or rhythmic compression, including the submucosae of the entire gastrointestinal tract and urinary bladder, the dermis, the peri-bronchial and peri-arterial soft tissues, and fascia. These anatomic structures may be important in cancer metastasis, edema, fibrosis, and mechanical functioning of many or all tissues and organs. In sum, we describe the anatomy and histology of a previously unrecognized, though widespread, macroscopic, fluid-filled space within and between tissues, a novel expansion and specification of the concept of the human interstitium.

Conflict of interest statement

Dr. Theise has received travel sponsorship for attendance at a Mauna Kea Technology sponsored scientific conference. Dr. Benias has received sponsorship for attendance at a Mauna Kea Technology sponsored scientific conference. Dr Carr-Locke has received royalties from US Endoscopy and Telemed Systems, has been a consultant for Boston Scientific Endoscopy, Cook Medical, EndoChoice, Mauna Kea Technologies, and Olympus Corporation, and holds a patent with ValenTx.

Figures

Figure 1

Identification of bile duct reticular pattern and demonstration of submucosal space. (A,B) pCLE of bile duct after fluorescein injection shows a reticular pattern at a depth of 60–70 μm. Scale bar, 20 μm. (C–E) Bile duct tissue removed at the time of Whipple surgery was frozen and ex vivo pCLE performed, demonstrating persistence of the reticular pattern. Scale bar, 20 μm. (F) Unstained frozen tissue of submucosa of a bile duct imaged by fluorescent microscopy, showing the reticular pattern in this layer of bile duct wall. The “bright” spaces are now dark (fluoresceinated fluid drained in processing and the tissue structures remained stained with residual fluorescein). (G) Masson trichrome of fresh-frozen bile duct shows that the dark bands are collagen bundles (blue) (left). The upper right shows Masson trichrome of a normally processed/fixed bile duct from the same patient, with collapse of spaces and apparent adherence of collagen bundles to each other. Lower right shows the fixed specimen stained with H&E; the thin spaces between collagen layers (arrows) reflect normally fluid-filled spaces that are almost completely collapsed. (H) Frozen (top) and fixed (bottom) bile ducts immunostained with antibodies against CD34 (left, brown) and D2-40 (right, brown) show cells lining the collagen bundles; note that bundles often seem to have a lining cell on one side, but not the other (20×, DAB, hematoxylin). (I) Schematic of the fluid-filled space supported by a network of collagen bundles lined on one side with cells. Illustration by Jill Gregory. Printed with permission from Mount Sinai Health System, licenced under CC-BY-ND. (https://creativecommons.org/licenses/by-nd/4.0/legalcode).

Figure 2

Structural evaluation of the interstitial space. (A) Transmission electron microscopy shows collagen bundles (asterisks) that are composed of well-organized collagen fibrils. Some collagen bundles have a single flat cell along one side (arrowheads). Scale bar, 1 μm. (B) Higher magnification shows that cells (arrowhead) lack features of endothelium or other types of cells and have no basement membrane. Scale bar, 1 μm. (C) Second harmonics generation imaging shows that the bundles are fibrillar collagen (dark blue). Cyan-colored fibers are from autofluorescence and are likely elastin, as shown by similar autofluorescence in the elastic lamina of a nearby artery (inset) (40×). (D) Elastic van Gieson stain shows elastin fibers (black) running along collagen bundles (pink) (40×).

Figure 3

An interstitial space is found in the dermis and submucosae and other fibroconnective tissues throughout the body. (A) Skin stained with H&E (upper left 10×, upper right 40×) shows the same structures as identified in the extrahepatic bile duct. Immunostain for CD34 (lower left, brown DAB, light blue hematoxylin counterstain, 40×) highlights that the lining cells are intermittent and often on one side of the collagen bundles, but not the other. pCLE applied to the skin in vivo following this histologic observation confirms that the histologic appearance predicts the in vivo reticular pattern when pCLE is applied to the skin. (B) Schematic showing location of identical histologic structures seen in fibroconnective tissues throughout the body (see Supplemental Fig. 1 for histology images). Illustration by Jill Gregory. Printed with permission from Mount Sinai Health System, licenced under CC-BY-ND. (https://creativecommons.org/licenses/by-nd/4.0/legalcode).

Figure 4

Continuity between interstitium and draining lymphatics. (A–C) Colon tissue with submucosal tattoo. (A) Black pigment endoscopically injected into submucosa of colonic wall before resection of colonic malignancy (H&E, 10×). (B) Black pigment is present in macrophages in the spaces between collagen bundles (H&E, 40×). (C) Pigment-containing macrophages are present in mesenteric lymph nodes draining the tattooed colon, showing that the interstitial space functionally communicates with lymphatic drainage of the colon (H&E, 20×). Typical images from 4 independent samples evaluated. (D–F) Stage T2 gastric carcinoma, poorly differentiated. (D) Gastric carcinoma present at the mucosal surface (arrows) invades into the submucosa (arrow heads); deeper invasion and lymphovascular invasion were not seen (H&E, 4×). (E) Poorly differentiated tumor cells infiltrate, singly and in very small clusters, through the interstitial space of the gastric submucosa, isolating pre-existing collagen bundles (H&E, 40×). (F) Metastatic carcinoma in draining mesenteric lymph nodes of the gastric resection specimen; no other metastases were identified clinically or histologically (H&E, 20×). (G–I) Stage T2 malignant melanoma of the skin of the left arm. (G) Malignant melanoma (dark blue) invading into the dermis; lymphovascular invasion not identified (H&E, 4×). (H) Malignant melanoma cells infiltrate, singly and in very small clusters, through the interstitial space of the dermis, isolating pre-existing collagen bundles (H&E, 40×). (I) Metastatic malignant melanoma in draining axillary lymph nodes; no other metastases were identified clinically or histologically (H&E, 10×).