I am posting this not really as a huge project, but just as a reminder to self, and to those interested that sometimes data are lost and forgotten but can still have meaning. The study mentioned here was just part of a very large study which was conducted by a good friend (late, actually many years passed on) who was very careful in what she did, and didn’t press conclusions or publications beyond their real findings. She was also a good friend.  Dr. Martha Radike (who did these inhalation experiments at the University of Cincinnati, College of Medicine, Department of Environmental Health, back in the 1980s.  The test animals  (160 male Hartley guinea pigs) represented significant effort.  I did some of the electron microscopy but never completed a manuscript on the animals (and they represented only 18 out of the sum total of the experiment.

My interest in the guinea pigs was revitalized while doing a bigger review of over 1400 micrographs looking specifically for a granule of surfactant protein in alveolar type II cells.  So this study I brought up out of the archives for that purpose and found a summary, which I have posted HERE as a pdf for anyone who is interested.  vinyl_chloride_vit_C_guinea_pig_lung

Here is an enlarged image from that pdf.

Electron micrographs of portions of alveolar type II cells: Two micrographs on the left are from my research, guinea pig on the left, ferret in the middle. The image on the right comes from a publication in 1973 by Stephens, Freeman, Stara and Coffin which had to do with exposure of beagle dogs to ozone.  The claim that ozone increased these protein bodies (which for all intent and purposes match the protein body granules that I have seen in my animals), which also increased with age, particularly.  I attribute these to some kind of immune response to environemetal exposure, and/or immune disturbance (as in the case of the guinea pigs, maybe an entire room of guinea pigs in the experiment were infected with some agent that increased the proteins in lung for innate immune response.  Regardless, the point of this image is to demonstrate electron micrographs from these three species, of alveolar type II cells, showing what stress can produce in the way of organellar responses (if one can call this granule, an organelle — which begs the issue of calling Birbeck granules in Langerhans cells… organelles–again in which case these might be SP-A granules haha).  I have marked the approximate 100 nm bar in all three images, and made a sample ribosome size in each as well for suggesting magnification.  The periodicity found in the RER of the beagle electron micrograph I think was stated to be less than 100 nm… (76 nm or something instead).. you can choose.  What I have called 100 nm is nominal.

There is a book called Diagnostic Ultrastructural Pathology – A text atlas of case studies. Volume 1 ( Ann M. Dvorak, Rita A. Monahan-Earley) which on page 218 posts figure 182 (reproduced here – I thank them). It shows clearly that, while they were interested in lamellar body ultrastructure before and after ozone exposure, you can be sure that the most important structure for me is the “cisternal body” that flattened and layered protein structure that I see in the upper right-ish corner of their micrograph.  Guinea pig must have some very robust mechamism for responding to environment, producing proteins (presumably some surfactant proteins) and in this case, after ozone they do not seem to become more prominent (according to this chapter in the book) as they do in dog (see previous post), but actually become less conspicuous.

Dimensions and position within the cell are classic “cisternal body” which I really wish I could rename SP-granule.  Red arrow points to the RER granule.

There was a group of scientists in california (I could not remember which research institute) that kindly sent me some plastic-embedded tissues of dog lung, since I had found a paper someone there that described layered protein granules in alveolar type II cells like I had seen in ferret and guinea pig and had ask them for some tissue. I could not (after 35 years, remember their names).  HERE IT IS!  awesome, though at least one of the researchers is deceased (J. Stara).  I am taking some of their electron micrographs, cropping, editing contrast (only) and placing them next to the ones I found in guinea pig and ferret.  Perfect matches.  This publication is so old (1973) that there is no way that surfactant proteins could have been implicated as possible contributing factors to these granules that appeared in their ozone treated beagle dogs.  The surfactant proteins had not yet been described.  Ozone caused what was described (for lack of background information on overall type II cell function in those days) “metabolic alteration ….. this toxic agent”, which of course is a statement which NOW in 2016 in light of decades of research on surfactant and lung function looks patronizing.

Their RER measurements made this “approximately 754 A (75 nm) is not that far off from what was calculated by my own measurements 100nm (microscopes differ in magnification tables, and so neither is likely to be exact).

Because of their reporting of small, sometimes stacked “bar like” structures such as I have seen in untreated dogs (and looking a lot like what I have seen in rabbit type II cells).  it is likely that an “excessive” amount of surfactant protein is produced and maybe stored in a multi-meric configuration) within the RER awaiting utilization in assembly within lamellar bodies or multivesicular structures. Stevens et al, 1973, mention (yippie) that there is a dense line which runs parallel to the length of the RER in these infrequently seen structures.

Ribosomes in the beagle exposed to ozone that border the RER profiles on the growing ends of the layered protein do look to be in similar positions with regard to banding of the granule to those found in ferret. My inclination is to think that there is a ribosomes on either side of the central band in each period (beagle on the left, ferret on the right – where central band is not really visible in this view) and a ribosome at the point of the darkest band (counted on two sides if there is only one period but counted only one one side if there are multiple periods) making each periodicity have three ribosomes, not counting the dark band of the adjacent period. One on a dark outside band, two on either side of the inner lighter central band. In the figure below, see comparative diagrams. the red lines with black arrows and text indicate what they think is 75 nm and I think is 100 nm (right) (give or take). Ribosomes are red, distance arrows are black, inset is enlarged from white box on bottom electron micrograph, dog on left (from Stephens et al, 1973, top image of figure 3, used without permission), ferret on right, my studies. Ribosomes are the bar marker (25-30 nm each). It is important to note that not all RER cisternae in their exposed animals showed periodicity…. just like those in guinea pigs… which I attribute to tangential sectioning and multiple intersections of different “growing ends”, and also the sudden transition from flattened cisternae of RER to the layered granule.