Factors restricting maximal preservation of neuronal glycogen after perfusion fixation with dimethyl sulfoxide and iodoacetic acid in Bouin's solution. Histochemical observations in the brain of the Netherlands dwarf rabbit.

Thirty seconds after an initial intracardial epinephrine injection, deeply anesthetized animals are perfused consecutively with saline, Bouin's and 100% ethanol solutions, each containing 1% or 5% DMSO (Me2SO) and 0.01 M iodoacetic acid. In the Netherlands dwarf rabbit and the guinea pig, a maximal preservation of dimedone PAS-stainable, saliva-digestible glycogen is achieved, without signs of polarization of glycogen, in many neuronal and neuroglial cells occupying either brain stem nuclei or occasionally narrow perivascular zones. Tentatively, these results are ascribed to a combined effect of (a) the alleged capacity of DMSO to accelerate fixation and to suppress activation of adenylate cyclase and (b) the rapid action of Bouin's solution so that the glycogen particles become instantaneously enclosed in situ in a skeleton of coagulated proteinaceous elements. The paradoxical over-all reduction in preservation of neuronal and astrocytic glycogen may be associated either with a demonstrable loss of the fixative into the peripheral vasculature, because of contrary actions of DMSO and epinephrine, or with a transvascular passage of epinephrine resulting in neuronal glycogenolysis where the blood-brain barrier is absent or affected by DMSO. Other defects are the occurrence of myriad pericapillary foci of inadequate tissue preservation, rare petechial hemorrhages, post mortem fat emboli, and ubiquitous Buscaino plaques. Despite these adverse results preventing utilization of this technique in systematic histochemical investigations on neuronal glycogen, remarkable qualitative characteristics such as the neurons' capacity to store glycogen throughout their perikarya have been revealed.

Argentophil neuronal perikarya and neurofibrils induced by postmortem trauma and hypertonic perfusates.

Argentophil neuronal perikarya and perikaryal neurofibrils similar to those illustrated in Ramón y Cajal's classical studies have in the present investigation been found to be manifestations of the chromophil neuron. Conclusive evidence of such association was obtained by silver impregnation with the Bodian technique of sections previously stained with cresyl violet. Regardless of the fixative used, silver-impregnated neurofibrils were evident when (1) normal tissues were fixed by immersion or unsuccessfully fixed by perfusion, (2) normal tissues were exposed and touched after death but before perfusion with the fixative, or (3) flow of perfusates was compromised by the effect of an experimental procedure, as well as when (4) a hypertonic saline solution was used in the first perfusate. These cytologic peculiarities were still discernible after 24 h of postmortem autolysis following a delay in removal of the brain or in immersion of the exposed brain in the fixative. After immersion fixation, argentophilia and chromophilia occurred ubiquitously in the brain of the newborn guinea pig; however, argentophil neurofibrils were noted in the absence of chromophil neurons in the brain stem of the newborn rat, rabbit and cat. After fixation by perfusion, perikaryal neurofibrils were not impregnated in either newborn or old animals or in animals with facial nerve transection. Affinity for Congo red or birefringency, exhibited by neurons with marked neurofilbrillary changes in human senile brain atrophy, were absent in the present material. On the basis of the current light-microscopic observations, it is concluded that argentophilia of neuronal perikarya and perikaryal neurofibrils is another manifestation of the chromophil neuron induced by postmortem trauma and of the ocellate neuron elicited by perfusion with hypertonic saline.

Is the solitary dark neuron a manifestation of postmortem trauma to the brain inadequately fixed by perfusion?

Dark neurons, classified as solitary because of their sparse occurrence, were discerned in the transitional zones between gray and white matter in various species of laboratory animals fixed by perfusion. These neurons, histologically indistinguishable from dark neurons in immersion fixed material, tended to develop when the saline perfusion was delayed or slow, the amount of the Bouin fixative was excessive, or the autopsy was performed shortly after the perfusion. Under these conditions, the white matter manifested a softer consistency and a paler color than the gray matter. These observations suggest that, as the consequence of regional differences in intensity and speed of fixation, distortion during extraction of the brain may activate a stress force in the transitional zones where incompletely fixed neurons become affected and acquire an abnormal affinity for aniline dyes and silver.

Cerebral and retinal fat emboli in normal animals fixed by perfusion.

The brains and retinas of laboratory animals fixed by perfusion occasionally contain isolated round fat emboli, which increase in number if the two organs are covered with oil during the autopsy. These emboli, in contrast to emboli induced by intravenous injection of oil, are present in smaller numbers, occur without adjacent aggregation of erythrocytes and do not cause widening of the occluded vascular channel. The fat emboli in the normal brain are attributed to connective tissue fat aggregating on the exposed cerebral surface and flowing through openings cut in the leptomeninges and the vascular walls during removal of the brain. Their formation could not be entirely prevented by covering the brain with running water or by submerging the forepart of the animal's body in water during the autopsy. Nevertheless, such a procedure is recommended to avoid introduction of extraneous fat when in a given experiment the question of fat embolism arises. Fat emboli demonstrable in the flattened retina of the cat and the mulatta monkey are ascribed to aspiration of retrobulbar connective tissue fat; they can be prevented by placing a ligature around the optic nerve prior to removal of the eye.

Factors contributing to denucleation of cerebral mast cells.

When mast cells, identified by metachromatic color in cresyl-echt-violet-stained paraffin sections, aggregate in large numbers, the intensity of cytoplasmic granulation varies. The intensely stained cytoplasmic granules of heavily granulated cells may be pushed over the microscopic section by the microntome blade either in the direction of cutting or against it. In the pale mast cells, in which a progressive pyknosis and atrophy of the nuclei by compaction of DNA particles take place, dislodgment and displacement of the pyknotic nuclei may occur in 1.1-2.3% of these cells. The pyknotic nuclei are expelled, usually in the direction of cutting, and deposited on top of the mi-roscopic section at various distances from their original site. While this expulsion is regarded as artifactitious, caused by the mechanical actioh of the microtome blade, the nuclear pyknosis, which is a prerequisite, is associated with intravital alterations.

The effect of cortisone treatment and reoperation on reactive changes in the facial nucleus after axotomy.

The material, with few exceptions, consists of PAS-gallocyanin stained paraffin sections from 4- to 6-month-old male rabbits fixed by perfusion first with saline and then with Bouin's solution. (1) In animals treated with cortisone prior to and subsequent to axotomy, the neurons exhibit an accelerated dispersal and delayed reconstitution of Nissl substance (ribosomes). While mitotic activity is depressed at various sites, formation of new microglial cells is evident. Neuronal degeneration with karyorrhexis is occasionally noted in single neurons in the lateral parts of the facial nucleus. An increase in intraneuronal glycogen deposition is manifested by a greater number of glycogen-rich neurons; such neurons are depleted of their glycogen after axotomy. (2) In other animals, reoperation of the facial nerve on the 6th, 22nd, 60th and 120th day, followed by survival of 3 days, results in dispersal of restored Nissl substance and in increased extravascular mitotic activity which is of less intensity than in single-operated animals.