Wiley

A stereological method for obtaining estimates of the total number of neurons in five major subdivisions of the rat hippocampus is described. The new method, the optical fractionator, combines two recent developments in stereology: a three‐dimensional probe for counting neuronal nuclei, the optical disector, and a systematic uniform sampling scheme, the fractionator. The optical disector results in unbiased estimates of neuron number, i.e., estimates that are free of assumptions about neuron size and shape, are unaffected by lost caps and over‐projection, and approach the true number of neurons in an unlimited manner as the number of samples is increased. The fractionator involves sampling a known fraction of a structural component. In the case of neuron number, a zero dimensional quantity, it provides estimates that are unaffected by shrinkage before, during, and after processing of the tissue. Because the fractionator involves systematic sampling, it also results in highly efficient estimates. Typically only 100–200 neurons must be counted in an animal to obtain a precision that is compatible with experimental studies. The methodology is compared with those used in earlier works involving estimates of neuron number in the rat hippocampus and a number of new stereological methods that have particular relevance to the quantitative study of the structure of the nervous system are briefly described in an appendix.

The observed fit of bone mass to a healthy animal's typical mechanical usage indicates some mechanism or mechanisms monitor that usage and control the three longitudinal growth, bone modeling, and BMU‐based remodeling activities that directly determine bone mass. That mechanism could be named a mechanostat. Accumulated evidence suggests it includes the bone itself, plus mechanisms that transform its mechanical usage into appropriate signals, plus other mechanisms that detect those signals and then direct the above three biologic activities. In vivo studies have shown that bone strains in or above the 1500–3000 microstrain range cause bone modelling to increase cortical bone mass, while strains below the 100–300 microstrain range release BMU‐based remodeling which then removes existing cortical‐endosteal and trabecular bone. That arrangement provides a dual system in which bone modeling would adapt bone mass to gross overloading, while BMU‐based remodeling would adapt bone mass to gross underloading, and the above strain ranges would be the approximate “setpoints” of those responses.

The anatomical distribution of those mechanical usage effects are well known. If circulating agents or disease changed the effective setpoints of those responses their bone mass effects should copy the anatomical distribution of the mechanical usage effects. That seems to be the case for many agents and diseases, and several examples are discussed, including postmenopausal osteoporosis, fluoride effects, bone loss in orbit, and osteogenesis imperfecta.

The mechanostat proposal is a seminal idea which fits diverse evidence but it requires critique and experimental study.

The properties of the inorganic dye ruthenium red are presented with emphasis upon its use for electron microscopy of cells and tissues. Although commercial ruthenium red often can be used directly, it always contains various impurities and by‐products. One of these, termed ruthenium violet, can be isolated and is useful by itself. Absorption spectra of the ruthenium dyes and common impurities are given so that an assay is possible for any sample. Convenient fixative recipes containing ruthenium red or violet are provided together with constraints necessary for a reliable reaction to label extracellular acidic mucosubstances. Perfusion was not successful. The specificity of the ruthenium red reaction was evaluated by spot testing with 57 substances, and by titration with chemically defined pectins. The results indicate that ruthenium red, as a hexavalent cation, precipitates a large variety of polyanions by ionic interaction, and that its classical reaction with pectin is typical rather than specific. New data are presented regarding its reaction with phospholipids. For electron microscopy, a further reaction with OsO₄ amplifies the feeble electron density, which is the counterpart of its intense optical labeling, to a practical level resulting in strong contrast. An hypothesis is presented for the mechanism underlying this intensification.

The source of the new nuclei appearing during the growth of muscle fibers was examined in the tibialis anterior muscle of young Sherman rats (14–17 days of age) using radioautography at various intervals after a single injection of a small, non‐toxic dose of ³H‐thymidine (2 μCi/g body weight). Two techniques were employed: (1) labeled nuclei were detected in 1 μ thick radioautographs examined in the light microscope, and identified by simultaneous electron microscope examination of an adjacent section. The nuclei were then classified either as “true” muscle nuclei (within the plasmalemma of the fibers) or as belonging to “satellite cells” (which are mononucleated cells with scanty cytoplasm wedged between plasmalemma and basement membrane). (2) Muscle fibers freed by collagenase digestion were radioautographed one hour after ³H‐thymidine injection in order to determine the total number of labeled nuclei (true muscle nuclei plus those of satellite cells) per unit length of fiber.

Certain nuclei within the basement membrane of muscle fibers are labeled one hour after ³H‐thymidine and, therefore, synthesize DNA. The electron microscope demonstrates that these nuclei invariably belong to satellite cells, never to true muscle nuclei. Furthermore, the total number of labeled nuclei per unit length of fiber doubles between 1 and 24 hours; and, therefore, the labeled satellite cell nuclei undergo mitosis.

Following mitosis, half of the daughters of satellite cells are incorporated into the fibers to become true muscle nuclei. The remaining half divides again later; and half of their daughter cells are incorporated. Thus, satellite cells in young rats divide repeatedly and function as a source of true muscle nuclei.

In normal adult rats some germ cells degenerate at several vulnerable steps of spermatogenesis. These are the type A spermatogonia, midpachytene spermatocytes, primary and secondary spermatocytes which degenerate during their respective maturation divisions and step 7 and 19 spermatids. In the present study, these degenerating cells were examined under the electron microscope, and their frequency was determined in toluidine blue stained semithin sections of testes from normal, hypophysectomized (at 5.5 days after operation) and hypophysectomized rats injected with FSH and LH separately or in combination. With the exception of the step 19 spermatids, the degenerating germ cells underwent necrosis in vacuolated spaces delimited by Sertoli cells. In the case of the affected step 19 spermatids, an apical cytoplasmic process of the Sertoli cell initially ensheathed a long segment of their flagellum, and then each degenerating cell was drawn deep in the seminiferous epithelium where it was phagocytozed by the Sertoli cell. Soon after hypophysectomy the incidence of degenerating mid‐pachytene spermatocytes, step 7 and 19 spermatids which are present in stages VII or VIII of the cycle of the seminiferous epithelium, increased significantly. In contrast the number of degenerating primary or secondary spermatocytes during the meiotic divisions seen in stage XIV of the cycle or of any other germinal cell was not significantly modified. While the injection of FSH alone had no influence on the number of degenerating cells in hypophysectomized rats, injections of LH at the two doses administered (0.7 μg or 20 μg) reduced significantly the number of degenerating cells seen in stages VII‐VIII of the cycle; combined injections of FSH and LH (20 μg) reduced the number of these degenerating cells to the normal low values. Thus it appeared that the mid‐pachytene spermatocytes and the step 7 and 19 spermatids, all present in the adluminal compartment of the seminiferous epithelium in stages VII or VIII of the cycle, were more sensitive to the presence of absence of gonadotropic hormones than the other germ cells present in the seminiferous epithelium.

Major national and international critiques of the medical curriculum in the 1980s noted the following significant flaws: (1) over‐reliance on learning by rote memory, (2) insufficient exercise in analysis and synthesis/conceptualization, and (3) failure to connect the basic and clinical aspects of training. It was argued that the invention of computers and related imaging techniques called to question the traditional instruction based on the faculty‐centered didactic lecture. In the ensuing reform, which adopted case‐based, small group, problem‐based learning, time allotted to anatomical instruction was severely truncated. Many programs replaced dissection with prosections and computer‐based learning. We argue that cadaver dissection is still necessary for (1) establishing the primacy of the patient, (2) apprehension of the multidimensional body, (3) touch‐mediated perception of the cadaver/patient, (4) anatomical variability, (5) learning the basic language of medicine, (6) competence in diagnostic imaging, (7) cadaver/patient‐centered computer‐assisted learning, (8) peer group learning, (9) training for the medical specialties. Cadaver‐based anatomical education is a prerequisite of optimal training for the use of biomedical informatics. When connected to dissection, medical informatics can expedite and enhance preparation for a patient‐based medical profession. Actual dissection is equally necessary for acquisition of scientific skills and for a communicative, moral, ethical, and humanistic approach to patient care. Anat Rec (New Anat) 269:20–32, 2002. © 2002 Wiley‐Liss, Inc.

A method for rapid freezing is described in which use is made of the good heat conducting properties of silver. The freezing was accomplished by bringing the tissue in contact with a polished silver surface at the temperature of liquid nitrogen either at atmospheric or reduced pressure. Helium gas flowing over this surface prevented the condensation of water or air on the silver. After freezing the tissue was placed in a substituting solvent. The best results were obtained with 2% osmium tetroxide in acetone at −85°C. The ultrastructure of the tissue was well preserved in a narrow surface layer only.