McCloskey et al. found sub-endothelial cells in sheep, which could potentially be pace-maker cells. These cells stained positively for vimentin and c-kit (McCloskey et al. 2002). The cells were named Interstitial Cajal-like cells due to their similar appearance to the interstitial cells of Cajal found in the gastro-intestinal tract.
Interstital Cajal-like cells (ICLCs) have been studied and described in various human body tissues including myocardium (S. Kostin, L. M. Popescu. 2009), resting mammary gland stroma (Gherghiceanu and Popescu 2005), uterus and fallopian tube (Popescu et al. 2007), and urinary bladder (Johnston et al. 2010) as well as in the largest part of the lymphatic system, the thoracic duct (Briggs Boedtkjer et al. 2013).
More specifically, Briggs Boedtkjer et al. showed ICLCs to be located in the outer layer of SMC (Briggs Boedtkjer et al. 2013). Popescu et al. published a table containing criteria for the ultrastructural diagnosis of ICLCs and designated it the
“platinum standard” (Popescu et al. 2007). The criteria of this table coincide with the cells found in my research that I have designated to be ICLCs (Table 3).
ICLCs were identified ultrastructurally as ramified cells whose nuclei were surrounded by a slender perinuclear body, had an indented nucleus, and contained a low amount of caveolae and long cytoplasmic processes. These processes varied in caliber, were often in close contact with SMC and contained ‘varicous’ dilations along the processes. The areas of close contact between ICLC processes and SMCs showed a dense facing with caveolae at the contact sites. A patchy basal lamina was not observed as described in research on the thoracic duct (Briggs Boedtkjer et al.
2013).
It will require further research beyond the histological structure of the ICLC to fully understand its function and interaction with surrounding cells. Understanding the exact mechanism of communication between the ICLCs and the SMCs will be essential to developing treatment option for patients where this signaling pathway is defective.
1. Location in the non-epithelial space 2. Close contact with targets nerve bundles, and/or epithelia,
and/or smooth muscle cells, and/or
4. Gap junctions With smooth muscle cells or with each other
5. Basal lamina occasionally present
6. Caveolae 2-4 % of cytoplasmic volume; ~0.5
caveolae/ µm of cell membrane length
7. Mitochondria 5-10 % of cytoplasmic volume
8. Endoplasmic reticulum About 1-2 %, either smooth or rough 9. Cytoskeleton intermediate and thin filaments, as well
as microtubules 10. Myosin thick filaments undetectable
Table 3: Interstitial tissue of the thigh lymphatic collector contains cells that meet the criteria for interstitial Cajal-like cells (Popescu et al. 2007)
(Usage of this table was kindly permitted by Sandra Maria Cretoiu (Ciontea))
5 Summary
The lymphatic vascular system is an important part of the body that still requires extensive research. Although great advances have been made over the past 20 years, many more will have to take place in order to fully understand the parts that make this system function. Gaining knowledge on the anatomy, histology, physiology and molecular biology will be important in creating cures and treatment options for patient suffering from lymphatic diseases such as lymphedema and in understanding the diseases as such.
The research that I have performed has added to the knowledge of the histological composition of the lymphatic collector. In particular, I was interested in establishing the presence of the interstitial Cajal-like cells ultra-structurally as the possible pace-maker of the lymphatic contractions, after the ICLCs had been identified in the largest lymphatic vessel, the thoracic duct.
I was able to verify that the general structure of the lymphatic collector is similar to that of the previously described thoracic duct (Briggs Boedtkjer et al. 2013) and the generally accepted structure of blood vessels, but also shows specific differences. The collectors contained a tunica interna (intima), tunica media and tunica externa (adventitia). The intima consisted of lymphatic endothelial cells. Unlike endothelial cells of larger blood vessels, however, the LECs were usually not resting on a sub-endothelial tissue layer, but were in close contact with smooth muscle cells. The tunica media was of variable thickness and contained smooth muscle cells (SMCs). I observed SMCs of light and dark cytoplasm, while both contained typical features such as dense bodies, caveolae and a basal lamina. Further research is required to
for a difference in function, similar to that of modified cardiac muscle cells with pace-maker and conducting functions in the heart.
Unlike what was observed by Boggon and Palfrey (1973) in their study of the microscopic anatomy of the human lymphatic truck, I was able to find elastic fibers amongst the SMCs of the media, and also very clearly showed the presence of vasa vasorum throughout the media.
The adventitia layer of the lymphatic collectors was mainly composed of collagen fibers organized in bundles running obliquely or circumferentially. Fibroblasts were also frequently observed. Occasionally, scattered cells such a mast cells were found.
Blood vessels and adipocytes were also seen in close approximation to the lymphatic collectors.
The discovery of ICLCs in the lymphatic collectors and their relationship to the surrounding SMCs was the main purpose of this thesis. In using the proposed criteria for ICLC identification put forth by Popescu et al. (2007), I was able to ultrastructurally identify ICLCs in the lymphatic collector. The discovery of a dense cavelar facing on the SMC membrane that was in close contact with the process of an ICLC opens up the possibility of communication via caveolae between the two cell types in order to initiate contractions. Now that the existence of ICLCs in the lymphatic collectors and lymphatic trunks has been established, it is necessary to find out how they function and communicate with SMCs, in order to learn how to influence them.
6 Appendix
6.1 List of Figures
Fig. 1: Immunofluorescence studies of epifascial lymphatic collectors from the human
thigh ... 22
Fig. 2: TEM studies of epifascial lymphatic collector endothelial cells ... 25
Fig. 3: Ultra-thin section of human epifascial lymphatic collector endothelial cell ... 26
Fig. 4: Ultra-thin section of human epifascial lymphatic collector ... 27
Fig. 5: Magnified ultra-thin section of human epifascial lymphatic collector endothelial cell ... 27
Fig. 6: Ultra-thin sections of human epifascial lymphatic collectors smooth muscle cells ... 29
Fig. 7: Ultra-thin sections of human epifascial lymphatic collector LEC/SMC and smooth muscle cell connections ... 30
Fig. 8: Ultra-thin sections of human epifascial lymphatic collector vasa vasorum ... 31
Fig. 9: TEM studies of ICLCs in the media of lymphatic collectors ... 33
Fig. 10: TEM studies of ICLC processes in the media of lymphatic collectors ... 34
Fig. 11: TEM studies of ICLC processes in the media of lymphatic collectors ... 35
Fig. 12: Ultra-thin section of human epifascial lymphatic collector media connective tissue ... 36
Fig. 13: Ultra-thin section of human epifascial lymphatic collector media containing elastin... 37
Fig. 14: Ultra-thin section of human epifascial lymphatic collector adventitia collagen ... 38
containing mast cell ... 38 Fig. 16: Ultra-thin section of human epifascial lymphatic collector adventitia ... 39 Fig. 17: Ultra-thin section of human epifascial lymphatic collector adventitia with fibrocyte ... 40
6.2 List of Tables
Table 1: Sequence for hematoxylin-eosin stain ... 14 Table 2: Contrast sequence for copper apertures ... 21 Table 3: Interstitial tissue of the thigh lymphatic collector contains cells that meet the criteria for interstitial Cajal-like cells ... 44
6.3 List of Abbreviations
ICLC Interstital Cajal-like cells KH2PO4 Monopotassium phosphate K2HPO4 Dipotassium phosphate LEC Lymphatic endothelial cellsMNA Methylnacid anhydride Na3(C6H5O7) Sodium citrate NaOH Sodium hydroxide NaH2PO4 Monosodium phosphate Na2HPO4 Disodium phosphate OsO4 Osmium tetroxide Pb(NO3)2 Lead nitrate
PFA Paraformaldehyde
PBS Phosphate-buffered saline PPB Potassium phosphate buffer SMC Smooth muscle cell
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