Modification of AgNOR staining to reveal the nucleolus in thick sections specified for stereological and pathological assessments of brain tissue.

BACKGROUND: Various stains have been devised to reveal degenerative or reactive cell phenotypes, or the disintegrative and/or neuropathic lesions associated with Alzheimer's, Parkinson's, and Pick's diseases, Down's syndrome, or chemical toxicity. Utilization of silver staining has allowed researchers to elucidate neural pathways promoting a greater understanding of the functional connections between brain regions. All of these methods employing silver can be characterized as 'directed staining technologies'. NEW METHODS: The argyrophilic proteins (AgNOR) staining protocol was modified to stain nucleoli in thick sections prepared for stereological evaluation of brain tissue. Nucleoli appeared as black dots against a pale amber background. Tissue sections were counterstained with Toluidine Blue, or reduced-strength Tyrosine Hydroxylase immunohistochemistry to facilitate visualization of basic cellular morphology and regional nucleus identification. Here, we present a modified method for nucleolar staining in free-floating thick sections of brain embedded in a gelatin matrix. The modifications in our procedure include incubation in HCl to denature ('unravel') the DNA, a bleaching step to reduce non-specific background silver staining, and counterstaining with Toluidine Blue or reduced-strength tyrosine hydroxylase immunohistochemistry. COMPARISON WITH OLD METHODS: Prior to the development of immunohistochemistry, silver staining was used primarily to identify pathological profiles and trace axon pathways; however, in many cases, a combination of silver staining and immunohistochemistry are required to fully visualize pathomorphology. The mechanism of these stains requires the binding of silver ions to cellular components and the subsequent reduction of the ions to metallic silver. Dilutions of TH primary antibody were evaluated to maximize identification of neurons and the nucleolus amongst the soma and processes present in the thick section. The use of stereology as a tool to estimate cell number has become increasingly prevalent in neuroscience experiments. As requirements for the preparation of experimental tissue have been refined, researchers have begun to use thicker sections, between 40 to 80 microns, to increase the number of optical planes available for analysis. These thick sections require modified staining protocols to assure complete penetration of stains throughout the tissue section. CONCLUSIONS: This method is particularly useful in nucleolar identification for Stereology, and automated counting methods. Use of the nucleolus avoids some of the problems associated with use of the nucleus. The nucleolus is smaller than the nucleus and is less susceptible to transection during sectioning. It has a higher density than the nucleus and is easier to visualize. It is generally darker staining than the immunohistochemical reaction product that provides the identification marker for the cells to be counted. Examples of the method in several brain sections of the rat are shown, though the method has been also proven in other mammalian models.

Life without a brain: Neuroradiological and behavioral evidence of neuroplasticity necessary to sustain brain function in the face of severe hydrocephalus.

A two-year old rat, R222, survived a life-time of extreme hydrocephaly affecting the size and organization of its brain. Much of the cortex was severely thinned and replaced by cerebrospinal fluid, yet R222 had normal motor function, could hear, see, smell, and respond to tactile stimulation. The hippocampus was malformed and compressed into the lower hindbrain together with the hypothalamus midbrain and pons, yet R222 showed normal spatial memory as compared to age-matched controls. BOLD MRI was used to study the reorganization of R222's brain function showing global activation to visual, olfactory and tactile stimulation, particularly in the brainstem/cerebellum. The results are discussed in the context of neuroadaptation in the face of severe hydrocephaly and subsequent tissue loss, with an emphasis on what is the "bare minimum" for survival.

Inhibition of growth of ehrlich ascites tumor in strong A mice: a preliminary report

Experiments performed with the end in view of totally inhibiting the growth of Ehrlich tumor in Strong A mice were performed. Preliminary results indicate that such a tumor inhibition is possible and is caused by local reactions the nature of which deserve further investigationThese studies were done at the Department of Pathology, Northwestern University Medical School, Chicago 11, Illinois, U.S.A. Work along this and allied lines will be started at the Institute of Medicine, Far Eastern University. (Summary)

Respuesta cardiovascular al ejercicio a gran altura en trabajadores crónicamente expuestos a hipoxia hipobárica intermitente / Cardiovascular response to exercise at high altitude in chronically exposured workers to intermittent hypobaric hypoxia

The isottonic work perfomance was assessed in 34 workers aged 35 ñ 5.8 years old that had working of four days at 4500 m over the sea level and resting periods of other four days at the sea level during at least two years. Subjects were assessed in one occasion at the sea level, and at the first and fourth day of the working shift at 4500 m over the sea level. resting arterial oxygen saturation in theses three periods was 97 ñ 1.1, 88 ñ 18 and 91 ñ 1.1 percent respectively (p<0.01) and markedly decreased during maximal and submaximal exercise at 4500 m over sea level. Evercise duration in the three periods was 931 ñ 210, 775 ñ 105 and 778 ñ 105 seg respectively (p<0.001). Heart rate in the resting period was at least 10 percent higher and maximal and submaxilmal rates were lower at the high altitude. No differences in blood pressure or packed red cell volume were observed. Exercise duration correlated inversely with age (r=-0.49 p=0.03) and directly with maximal heart rate (r=0.44 p=0.009) at the sea level. No correlation between aerobic capacity and other measured parameters was observed. These results show no differences in the cardiovascular response to exercise between the first and fourth day of stay at high altitude in workers chronically exposed to intermittent hypobaric hypoxia

Octopamine-sensitive adenylyl cyclase and G proteins in Ceratitis capitata brain during aging.

The loss of the ability in signalling transduction constitutes an attractive hypothesis to explain the age-related loss of functions in the nervous system. In this paper we have examined adenylyl cyclase and G proteins in Ceratitis capitata brain during aging. The intermediate level of complexity of the Mediterranean fruit fly and its short lifespan make it a particularly interesting system for aging studies. Adenylyl cyclase basal activity decreased in the course of aging. By contrast, neither guanine nucleotide-induced activation of adenylyl cyclase nor Gs protein levels were modified. However, adenylyl cyclase activation by octopamine, which is a major neurotransmitter, neuromodulator and neurohormone in insects, was lost during aging. This observation correlated with a decrease in octopamine binding to brain plasma membranes that was due to a decrease in both receptor affinity and binding sites. On the other hand, we observed an increase in the expression of C. capitata Go protein with age, as revealed by pertussis toxin-catalysed ADP-ribosylation and immunoblotting experiments, that was not correlated with an increase in beta subunit levels. This report constitutes the first direct evidence for the participation of a Go protein in aging in the nervous system.

Differential effects of fluoride and a non-hydrolysable GTP analogue on adenylate cyclase and G-proteins in Ceratitis capitata neural tissue.

We have examined the effects of fluoride on guanine nucleotide-binding regulatory proteins (G-proteins) in neural membranes from the dipterous Ceratitis capitata. Fluoride effects on the Gs-protein were monitored by determining adenylate cyclase activity and cholera toxin-catalysed ADP-ribosylation whereas those on the G(o)-protein were studied by measuring ADP-ribosylation with pertussis toxin. Data are discussed in relation to the effects of a non-hydrolysable GTP analogue. G-protein activation carried out by fluoride seems not to mimic, at least in insects, activation by non-hydrolysable GTP analogues, in opposition to that proposed for transducin, the G-protein of the mammalian visual system, and other G-proteins.