PP5 or perfluorodecalin (image to right) was used as a possible component of artificial blood, or called otherwise, blood substitute(s), but never really attained full research or understanding necessary to determine whether it was a safe substitute for blood or not. Some of the early electron microscopy, which would have benefited from a huge influx of money, early on, for microscopists to examine the effects on all cell types, in vivo and in vitro, but too little was forthcoming to give it a thorough exam. It was clear however, that macrophages and the immune system were going to be big players, both because of the volume of the chemical that was infused or “breathed” and because of its alleged inert nature but odd surface tension properties.

In retrospect, looking at tissues which were taken from Leland Clark Jr’s lab in the 1970s, these particles (droplets) behaved strangely, seemingly dependent upon chemical properties, and of all examined ONLY one perfluorochemical had a shape in the cell other than very very round (this was perfluorodecyliodide – looking like a crystal instead of a droplet, as seen in this image from the liver after infusion of an emulsion).

Liquid in the cell in almost always is “slightly out of round” the best examples being fat droplets. They can be oval or indented or squeezed, as well as round.. but perfluorochemical droplets are ROUND. I would think this could be attributed to their heavier than water density.
Another property (mentioned in previous posts) is that they have various capacities to be re-emulsified, at least that is what i call it, within the cell. Example from the liquid breathing mouse posted yesterday (breathing silicone oil vs breathing E2) shows that the respective macrophages respond by producing different quantities, and also likely types of lysosomal enzymes, in addition to the recycling they do of the alveolar contents (that is, in recycling surfactant and other junk in the alveolar space.

So comparing silicone oil, E2 and PP5… seen here, once again, the lysosomal responses are quite different. Silicone oil didn’t encourage lysosomal production (and surfactant recycling) as much as E2, and E2 in turn caused a lesser (though still pretty dramatic) response than PP5. Actually, from notes taken from this series of experiments (Clark never did anything twice which made it really tough to keep notes) those mice breathing PP5 did not fare well, most did not survive as long as those breathing some other of the perfluorochemicals. E2 was probably the best in this regard, just due to the sheer number of samples and micrographs, this is what I remember.
So here is an electron micrograph of an alveolar space from a mouse which breathed PPF for 1 hour and was allowed to recover for 1.5 hours. (1499_5512_PP5_1hr_1.5hr recovery).

Highly rounded droplets, with very well defined lysosomal enzymes. The two non-highlighted droplets are likely to have (as the upper left already indicates) masses of lysosomal enzymes in a different plane of section. There are some smaller, and also very tiny droplets of PP5 visible in the two highlighted LE/PFC bodies. I did find some dense areas of the ER (not likely RER, but something different) that showed an unique substructure (see the inset with three small sample images (enlarged from boxes in the top figure) showing what I noticed.

Inset is enlarged and micron marker is determined from the size of an average (27nm) ribosome, and segments of the inset are all at the same enlargement from the original.

The bounding electron dense coat of proteins on the perfluorochemical droplets, whether phagocytosed from a neat liquid as inhaled from liquid breathing, or provided to the blood stream as artificial blood, it seems to be a fixture of the morphology.  I have scanned the edge of a single alveolar macrophage for evidence for “point of entry” of a droplet of E2 into said cell.  It is pretty clear even from this slightly too thick, thin section of a mouse lung (1349 4840 E2 3hr 48hr recovery) that the electron dense coat around droplets exists in lung of liquid breathing mice before they are phagocytosed by alveolar macrophages.  In this case the proteins likely to be lipid molecules derived from surfactant.

I suppose it is also likely that the slight lipophilicity of perfluorocarbons means that the surfactant lipids (such as phosphatidylcholine and phosphatidylglycerol) and the amphipathic proteins in surfactant (like SP-B) might contribute in some way to this droplet coat (as derived from surfactant already in the alveolus) reflecting its contribution shown in vitro studies suggesting an ability uptake of bacteria from the alveolar space (the leap is to E2 droplets) (ref Yang et al below)

There are at least 4 whole E2 droplets, and two cut off (left hand side of micrograph) droplets of E2 in this image. The tiny dotted periodicity were shown by lines in the droplet at the middle right, the green arrow points to the electron dense bounding layer of “some lipid and protein components from the alveolar space, likely from surfactant”, and the black arrow points to the space in the plasma membrane where the particle is likely being engulfed by the alveolar macrophage. Ribosome size is judged by the upper right corner inset with a portion of RER enlarged identically with the larger micrograph.  X marks an inclusion type that I have seen before but do not recognize. Any suggestions from the audience are welcome (millermn – ucmail dot uc dot edu).

Surfactant protein B and lipids look like a good candidates for the electron dense cover for E2 droplets — reading the article (Hawgood et al) and Yang et al  specifically referring to SP-B and from which I quote: “In no rank order these activities include  membrane binding, membrane lysis, membrane fusion, promotion of lipid adsorption to air–liquid surfaces, stabilization of monomolecular surface films, and respreading of films from collapse phases.”

SP-B has some interesting properties as referenced in many articles in PubMed which suggest that it has saposin like properties. Wikipedia identifies saposins thusly: “Saposins are small lysosomal proteins that serve as activators of various lysosomal lipid-degrading enzymes”. It is interesting that the huge lysosomal response that is induced by liquid breathing both in alveolar macrophages (and also when perfluorochemical emulsions when given IV) might be seen in the types of lysosomal bodies found, the length of time required to degrade/and/or reassociate the enzymes that appear to cover the droplets in the alveolar space, and concentrate (add more? or recycle more?  possibly be reflected in the fine granularity of the electron micrographic views of these MVB/LE/PFC organelles. The amount of re-emulsivication of phagocytosed E2 in alveolar macrophages appears to be time dependent, and would fit with the tim required to increase in production of some lysosomal enzymes and become associated with the E2 droplets.

The lysosomes in this alveolar macrophage from a mouse which breathed E2 for 3 hours and then allowed to recover for 5 days are awesome.  The macrophage has produced enzymes which re-emulsify the E2 into very small (in most cases) droplets within the lysosomal structure. In addition, the enzymes make a border, which is very difficult to distinguish from the trilaminar membrane which surrounds the lysosome proper.  I don’t know how to explain this look of a double membrane, but figure it is partly a physical interaction between the E2 droplet (and not unique to E2 but seen with many other perfluorochemicals) and its hydrophobicity, and slight lipophilicity.

The smallest spheres of E2 are down around 35nm diameter, and size can be compared to something just larger than a ribosome (@27nm). Picture on the top is the unretouched (i may have removed a scratch with the band-aid tool in photoshop but nothing else), and it is not too great an image (scanned from the 31/4×4 acetate negative), but interesting still, and providing lots of information. Image below is one where I have highlighted the E2 droplets in a single membrane bound lysosome and embossed them.

The dark structure (also rounded) in the upper right hand corner of the images is what I think is a phagolysosome that contains mostly phagocytosed surfactant lipids (some layering and myelin-look is seen within this structure) from the alveolar space. There are also two mitochondria near center top. It also seems likely that some of the surfactant engulfed by alveolar macrophages would find its way (during re-purposing or re-cycling) into such lysosomes containing E2 droplets.

Loooooking over an old negative of lung from a mouse that breathed oxygen bubbled liquid perfluorochemical  (in this case E2, which is a freon) I spotted what could be, maybe, seems almost possible, droplets of E2 in the endothelium (alveolar sac endothelium).  I selected portions of the image out and pseudocolored them for easy identification: Red cell within the capillary lumen, red, endothelium orange, lysosome/or vacuoles that might be E2 and comparison with those which are in a nearby alveolar macrophage, green, and the macrophage cytoplasm is purple. There are some reasonably large E2 droplets which are probably unequivocal (is that not an odd combination of words?) with more distinct membrane boundaries, and absolutely NO fuzziness within the inclusions but also having the distinctive “black cap” of lysosomal enzymes off to a little blip on some side or other of the droplet.   This is NOT seen in the endothelial inclusions…. I will look for less equivocal samples but the similarities between those and additional ones found in the macrophage is clear. 1351_4840_mouse_E2 breathing for 3 hours, 48hr recovery, capillary_endothelium and two macrophages.

Looking at lysosomes in the lung of mice which have breathed perfluorochemical (PFC) liquids is interesting. These are old archived micrographs from the early 1970s but have a lot of valuable information about the reactivity of perfluorochemicals in the body (the latter being touted as totally inert.  Regardless of what science says about the reactivity of PFC they do cause a mountain of effects in humans, not the least of which is an immediate uptake by the reticuloendothelial system, massive sequestering and dumping of enzymes into the resultant lysosomes, and a kind of partitioning, or re-partitioning, or re-emulsification of the PFC, depending upon whether it was breated as a neat liquid, or infused as an emulsion with other agents accompanying it.

So “case in point” is this alveolar macrophage, picture taken from the lung of a mouse which breathed E2 for 3 hours and was allowed to recover for 48 hours then lungs taken for electron microscopy (and probably chemical analysis, though I have no clue where those data are at this point 40 years down the pike).  I just know that my negative number is 1351, and that block number is 4840, magnification was 5,000 (Zeiss 10) date 2 17 1975 at 60kv.  Mouse weighed 16.85g, breathed E2 for 3 hours on 2 3 1975, and was processed on 2 5 1975, which equates to 48 hour recovery. The sample of lung was taken from the R, mid lobe.

The image below (L) is a portion of the original negative (scanned as a transparency from the film) with an enlargement (box demarcates it) on the right where I have “pseudocolored” the protein densities with purple, which surround the empty footprints where the PFC was fixed (leaving the nice round spaces) in the cytoplasm. About 6 ribosomes (@27nm diameter) are also highlighted beneath the central phagolysosome?lysosome?membrane structure to show that the size of the smallest perfluorochemical droplets is in the nanometer range, before — before what, perhaps being small enough to diffuse back into the alveolar air space and be exhaled.  I don’t believe anyone knows whether the larger or the smaller E2 droplets are more apt to move into the alveolar air space.  There is one tiny droplet which is about 27 nm nesteled between two ribosomes… go figure.  The enzyme response here is pretty amazing, and appears in section to be most often seen as spheres, but is more likely to be a longer sausage shaped lumpy body.

The bottom line of this image is that these droplets appear to migrate along the membrane systems.