Interstitial spaces are continuous across tissue and organ boundaries in humans

Odise Cenaj, Douglas H. R. Allison, R Imam, Briana Zeck, Lilly M. Drohan, Luis Chiriboga, Jessica Llewellyn, Cheng Z Liu, Young Nyun Park, Rebecca G. Wells, Neil D. Theise

Preprint posted on August 07, 2020

We are not a set of discrete organs, but a continuum.

Selected by Mariana De Niz

Categories: cell biology, physiology


There are “reticular networks” made up of collagens, elastin, glycosaminoglycans, and other extracellular matrix (ECM) components surrounding, within, and between organs. These networks have biological and mechanical roles in defining the architecture and physiology of organs.

It has been shown that ECM networks extend beyond the confines of single organs to involve neighboring structures, including thoracic, abdominal and pelvic organs with their vasculature and surrounding fibrous, adventitial sheaths, creating structural continuity across organ boundaries. Animal experiments have shown that the connective tissue network is continuous throughout the body, and that the connective tissue of nerves creates structural continuity between the nervous system and other tissues.

The authors recently described fluid that flows through fibrous tissue coverings of nerves and blood vessels, however, it remained unclear whether the interstitial spaces are continuous through the body, or discontinuous and confined within individual organs. In the present study Cenaj and colleagues investigated interstitial space continuity.

Figure 1. Interstitial spaces are continuous across tissue and organ boundaries in humans


Key findings and developments

In their work, Cenaj et al used two orthogonal approaches. One, studying movement of non-biological particles across tissue compartments within colon and skin, and into adjacent fascia. The other, studying hyaluronic acid, a macromolecular component of interstitial spaces.

Approach 1: movement of non-biological particles across tissue compartments

For this approach, the authors used tattoo pigment and colloidal silver. The authors found that the particles were localized both intracellularly (within the cytoplasm of macrophages and interstitial lining cells), and extracellularly, within interstitial spaces between collagen bundles of the collagenous network of the dermis and  subcutaneous fascia. Silver particles were observed in similar locations, as well as the adnexa, perivascular adventitia and perineurium in the dermis.

Colon resection specimens with endoscopically-injected tattoos also showed pigment particles distant from the original submucosal injection site. Besides the colonic submucosa, particles were also at the muscularis propria and the mesenteric fascia.

Upon measuring the diameter of extracellular tattoo pigment particles, as function of the depth of their location in the bowel, the authors found that particles in deep mesenteric interstitial spaces were significantly smaller than those in more superficial compartments. This suggests that the particles are carried via fluid flow, rather than via cells (eg. macrophages), as the latter would have instead resulted in an even distribution of sizes regardless of distance from the inoculation point.

Approach 2: Continuity of hyaluronic acid across interstitial spaces and organ boundaries.

HA is found in interstitial spaces throughout the body at all stages of development. Staining with HA binding protein showed that non-vascular spaces unstained by H&E are in fact not empty spaces, but are filled with HA. This includes interstitial spaces between cells. Thus, the authors argue that HA could serve as a surrogate marker for most interstitial spaces. The authors demonstrated continuity between various interstitial spaces in the colon, the skin and the liver.

Tumour movement through interstitial spaces

Finally, the authors observed movement of different tumour types through interstitial spaces. These included cholangiocarcinoma in the liver, colon adenocarcinoma, and malignant melanoma.

Altogether, the authors suggest that there is a broad and interconnected network of interstitial fluid-filled channels throughout the body, including the structural coverings of nerves and vessels. This has important implications for our understanding of physiological and pathological processes, including cell trafficking, and the spread of tumours and infectious pathogens.


What I like about this preprint

I think a conventional view of physiology involves the view of organs and tissues as separate from each other. I like this work a) because it is out-of-the box in its approach and questions asked to understand the interconnectivity in a full organism and b) because these findings have important implications for our understanding of pathology and physiology and should guide important considerations in future experimentation in these areas.


1. Cenaj O, et al, Interstitial spaces are continuous across tissue and organ boundaries in humans, bioRxiv, 2020

2.Mall FP, A study of the structural unit of the liver, American Journal of Anatomy 1906; 5(3):227-308

3. Benias P, et al, Structure and distribution of an unrecognized interstitium in human tissues.  Scientific Reports 2018; 8: 4947-4945.

4. Armer JM & Stewart BR, Post-breast cancer lymphedema: incidence increases from 12 to 30 to 60 months. Lymphology. 2010;43(3):118–127

5. Raghavan P, et al, Human Recombinant Hyaluronidase Injections For Upper Limb Muscle Stiffness in Individuals With Cerebral Injury: A Case Series. EBioMedicine. 2016 Jul;9:306-313.

6.Miteva DO, et al, Transmural flow modulates cell and fluid transport functions of lymphatic endothelium. Circ Res. 2010 Mar 19;106(5):920-31

7.Seneviratne SL, et al, Mastcell disorders in Ehlers-Danlos syndrome.  Semin Med Genet. 2017 Mar;175(1):226-236.




Posted on: 26th September 2020


Read preprint (1 votes)

Author's response

Rebecca G. Wells and Neil D. Theise shared

Open questions 

  1. You mention throughout your work, observations performed in humans, but also work done in different animals. Do you know how and if interstitial space continuity differs between species? If so, evolutionarily what are the advantages of this continuity?

Studies by Franklin Mall from 1906 show the continuity between the interstitial spaces of the cat liver portal tract stroma (the “space of Mall”),2  akin to our work suggesting interstitial space continuity. Beyond that, to our knowledge, there is little published data. We welcome communication from your readers regarding important literature that bears meaningfully on these questions.

In the absence of evidence to suggest differences in species, we think this is likely to be a conserved structure, at least in mammalian tissues.  Interstitial pathways for cell and molecular signalling between distant sites in the body could significantly enrich the potential to keep the body’s different regions in homeostasis, in complementary ways to those of the nervous and cardiovascular systems.


  1. Following from the previous question, is there pathology known to arise from defects in IS interconnectivity?

As we noted in our first paper3 (Supplemental Figure 2), mechanical compression of the interstitial space during bowel obstruction, such as from hernias, torsion and volvulus, can lead to marked expansion of interstitial spaces and associated edema.3  Another example of site specific edema, associated with considerable and troubling symptoms including pain, immobility, and infection, is lymphedema following some cancer (surgical or radiologic) treatments. For example, breast cancer-related lymphedema of the arm is a common complication after treatment.4   Such edema is a static fluid overload of the interstitial spaces of the dermis and musculoskeletal fascia of the involved limb.  Whether it arises purely from lymphatic obstruction (“backing up” into the interstitial spaces drained by the lymphatics) or also through a component of direct interstitial injury (particularly from radiation) merits exploration.  Obstruction of the biliary tree by gallstones leads to edema of the extrahepatic and intrahepatic biliary tree, one of the conditions that predisposes to increased risk of bacterial superinfections (“ascending cholangitis”).

The group led by Antonio Stecco has suggested that altered HA content (both molecular forms and amount) in regions of the muscular fascia within paralyzed or immobilized limbs in patients with central nervous system injuries such as strokes, tumors, cerebral palsy, etc may be important in post-injury spasticity.6,7  Injection of human recombinant hyaluronidase into the affected fascia relieves the spasticity.7  Thus, both internal and external causes of obstruction could lead to changes in the function of the interstitial spaces.


  1. In your experiment analysing particle size relative to distance of inoculation site, you explore two hypotheses: one that envisions transport of particles within cells (eg. macrophages) into lymph nodes, and one that envisions transport via fluid in the interstitial space. What is the role of the lymph nodes and lymphatics in particle distribution, and how does it relate to the interstitial space (i.e. the interconnection between them). Can this not be a third form of distribution?

Fluid from interstitial spaces may move into the cardiovascular system from the small spaces (<10 microns) around capillaries within tissues.  We have not studied possible passage of small (nano-size) particles between these compartments.  However, as we demonstrate, 1,3 there is connectivity from larger interstitial spaces of fibrous tissues such as dermis and submucosa into subjacent fascia  and also into the lymphatics (indeed, it should be remembered that lymph derives from interstitial fluid7).  Particles small enough to pass through the mix of proteoglycans/HA within the interstitial spaces can be seen distant from injection sites.1  They may also be carried from the injection site within macrophages, both into the lymphatic drainage to the regional lymph nodes3  and into subjacent fascial layers.


  1. You mention in your conclusion, the relevance of the interconnectivity between organs, including vessels and nerves. Can you expand further on the implications of the involvement of vessels and nerves?

The continuity of interstitial spaces of the skin and viscera with those of the fibrous covering (adventitia) of arteries and veins entering and leaving the tissues and those of the fibrous sheaths of nerves travelling from central to peripheral nervous systems (perineurium) implies that, structurally, there is continuity is not just between layers within organs or between neighbouring organs or tissues, but throughout the body, following the adventitia of the entire vascular tree and perineurium of the entire peripheral nervous system.  Functionally, this implies the possibility of cell and molecular signalling between distant sites. These could involve, for example, the microbiome (e.g. routes for gut-brain and gut-lung axes’ communication) and pathologic processes such cancer cell and infectious spread (e.g. necrotizing fasciitis).


  1. You explore cell movement in pathology (ie metastasis). How does the interstitial space contribute to cell migration in homeostasis, and what is therefore, its key role? What are the key processes you would like to study, based on the findings you present in this work?

We have demonstrated the importance of macrophage trafficking within these spaces in engulfment and partial clearance of injected particulate matter.  Otherwise, we are interested in the fibroblast-like (vimentin+, CD34+) cells that line the supporting collagen bundle lattice of the spaces, which we have so far studied only superficially.  Many studies of interstitial spaces show other resident cell populations, though the spaces themselves were not well defined or understood.  For example, connective tissue layers such as the dermis and submucosae are known to have dendritic cells, comparatively low levels of lymphocytes when non-diseased, and frequent mast cells.  The mast cells are intriguing because there is a postulated associated between diseases of altered collagen (such as Ehlers-Danlos syndrome) and mast cell dysfunction(s).8  Do these interstitial spaces represent a “mast cell niche” and their structure/composition regulate this component of the immune system? This is one of the questions we hope to study in the future.

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