Abstract
I hated to write this letter, and I have the utmost respect for the lab, but the article “Vacuolation of Sensory Ganglion Neuron Cytoplasm in Rats with Long-term Exposure to Organophosphates,” authored by T. Rogers-Cortone et al., deserves some response and an additional viewpoint, as many people are going to look at the article and begin to diagnose this change (large vacuoles in dorsal root ganglia) as a test article effect. If for no other reason, perhaps this letter will spark some discussion, since I would also like to know exactly what these vacuoles are. As we used to say in veterinary school, “They don’t have what they have, they have what we know.” So now that people are going to know it (the vacuoles in the sensory neurons), I am afraid they are going to start to diagnose it and interpret these vacuoles as a compound effect. That interpretation would be, I believe, incorrect in the vast majority of studies and possibly also in the study the authors report.
Although I cannot deny that the reported large vacuoles may well, in the authors’ study, be related to long-term organophosphate administration, I can verify that vacuoles identical to the ones described in this manuscript are very common findings in a variety of laboratory species, including rats, dogs, and monkeys.
Morphologically, the vacuoles are always the same, regardless of the species, time frame (i.e., fourteen-day study, twenty-eight-day study, six-month study, one-year study), or fixation method, including very well-perfused animals. In any given study, they may be more prevalent in the controls or the high-dose group or any other group. In short, these vacuoles are highly suspicious as being either artifacts of preparation or spontaneous lesions unrelated to treatment, except when presented with overwhelming evidence in the form of absolutely convincing data. To date and after reviewing at least over one hundred studies with similar vacuoles, I have never seen the convincing data.
The authors describe the ultrastructural appearance of these vacuoles but do not propose a mechanism. I have observed thousands of these vacuoles and find them in almost every study in which I take a close look at the dorsal root ganglia (they also occur in the trigeminal ganglia, although less frequently), and again, even at the electron microscope level, they are always the same. The “vacuolated” cells are never dead and they never have other signs of degeneration.
This common occurrence in many studies, including the control groups, and the exact same appearance of the vacuoles no matter the compound, species, or the length of the study, indicate that these vacuoles are very likely artifacts or spontaneous changes. One observation I have made is when these vacuoles occur in one dorsal root ganglion, they tend to occur in many of the ganglia in the same animal, even if the animal is a control.
I know of no other types of vacuoles that have this appearance, no matter the toxin or the disease. The article reports a reference that states these vacuoles occur in approximately one in every 10,000 neurons in control animals. While preparing this letter, I took a quick look at a recent study for which I evaluated seventy-two primates; a cervical, thoracic, and lumbar dorsal root ganglion was evaluated in each animal. In that study, the incidence of these vacuoles in the three-month control group was four out of twelve ganglia, a full 33% (three ganglia in each of four control animals made for a total of twelve). Historically, that percentage may be high. I suspect the actual percentage is somewhere in the 5% range for most studies. I cannot remember a study (where I was reviewing dorsal root ganglia) in the past several years in which I have not seen them, and that has been many studies. They are always large, distinct, and membrane bound, with organelles/cytoplasm bulging into the vacuole space. The vacuoles do appear to occur predominantly at the periphery of the cell, which has led me to believe they are possibly an artifact of separation from the surrounding cells. Perhaps not; perhaps they are spontaneous changes. But I would like the authors to explain, perhaps in their response to this letter, just how they believe these vacuoles form and critically evaluate the appearance of these vacuoles with vacuoles known to develop as a degenerative change.
Lastly, the morphometric data in the study are rather flawed. You simply cannot quantify objects (in this case, vacuoles) by counting them in a single “representative” section. To quantify objects in a three-dimensional structure such as a ganglion, you must use stereologic techniques to produce believable, unbiased data. I urge Toxicologic Pathology to stop, as many other journals have, accepting this type of morphometric data.
So I invite readers and the authors to consider these points. The trouble with an article like this one is that there are going to be studies in which these common structures are most common in a test article–treated group (and the very next study is just as likely to have more in the control group, in my experience). When these vacuoles are reported as being actual lesions (except in studies where the data are really compelling), it requires a great deal of time and expense to prove that these structures are merely common, spontaneous, or artifactual changes or of no significance.