Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

Effects of high-fat chow on heart tissue in acute and chronic experimental murine schistosomiasis mansoni

SUMMARYThis study aimed to investigate myocardial injuries in mice infected with Schistosoma mansoni and fed a high-fat chow. Sections of myocardial tissue from S. mansoni-infected mice, and controls that had been killed 9 and 17 weeks post-infection, were stained with H&E and Picrosirius red. Histopathological examination, stereological design-based method (optical disector) and morphometry (vessels, cardiomyocytes and an amount of collagen) were used. Data were analysed using two-way ANOVA. Regardless of time of infection, myocardial tissue from the infected mice fed high-fat chow showed myocarditis characterized by a higher number of inflammatory foci, several areas displaying coagulation of cardiac fibres, a greater loss of cardiomyocytes and fibroblast proliferation than in the standard chow control. Comparing infected mice from acute and chronic infections, a higher cardiomyocyte hyperplasia (P < 0·0001) and higher amounts of collagen (P < 0·05) were observed than in standard chow control. In addition, all animals fed high-fat chow showed lower numerical density and total number of cardiomyocytes (P < 0·05), thicker vessel walls and narrowed luminal intramyocardial vessels (P > 0·05) than in the standard chow control. Altogether the data supported the view that a double burden has a synergistic deleterious effect on the myocardial tissue.

IMPROVING METHODOLOGICAL STRATEGIES FOR SATELLITE CELLS COUNTING IN HUMAN MUSCLE DURING AGEING

Stereological methods, based on the optical disector principle and fluorescent staining, were developed for estimating frequency of satellite cells in skeletal muscles. The parameter NL(sc, fib) (number of satellite cells per fibre length) was compared with the parameter NN(sc, nucl) (the percentage of satellite cell nuclei in all muscle nuclei), most often published in the literature, by applying unbiased sampling and counting procedures and using a confocal microscope. The methods were tested in autopsy samples of four young vs. four old human vastus lateralis muscles. Both parameters NL(sc, fib) and NN(sc, nucl) declined during ageing. However, it appears that the two parameters cannot be substituted one by the other because the number of nuclei per fibre length tends to be increased during aging. Using the introduced methods, it is more straightforward to estimate NL(sc, fib) than NN(sc, nucl).

ESTIMATION OF THE NUMBER OF NEURONS IN THE HIPPOCAMPUS OF RATS WITH PENICILLIN INDUCED EPILEPSY

Epilepsy is a neurological disease arising from strong and uncontrollable electrical firings of a group of neurons in the central nervous system. Experimental epileptic models have been developed to assess the physiopathology of epileptic seizures. This study was undertaken to estimate the number of neurons in the rat hippocampus with penicillin induced epilepsy, using a stereological method, "the optical fractionator". In the experimental group, 500 IU penicillin-G was injected intra-cortically, and in the control group, the same volume of saline was administered. A week later, the animals were decapitated and their brains were removed by craniatomy. Frozen brains were cut with a thickness of 150 ěm in a cryostat. Sections were collected by systematic random sampling and stained with hematoxylen-eosin. Microscopic images of pyramidal cell layers from hippocampus CA1, CA2 and CA3 subfields were then transferred to a monitor, using a 100x objective (N.A. = 1.25). Using the optical disector method, the neurons were counted in the frames and determined with a fractionator sampling scheme. The total pyramidal neuron number was then estimated using the optical fractionator method. The total pyramidal neuron number was found to be statistically lower in the experimental group (mean = 142,888 ± 11,745) than in the control group (mean = 177,953 ± 10,907) (p < 0.05). The results suggest that a decrease in the hippocampal neuronal number in a penicillin model of epilepsy can be determined objectively and efficiently using the optical fractionator method.

METRIC CHARACTERISTICS OF VARIOUS METHODS FOR NUMERICAL DENSITY ESTIMATION IN TRANSMISSION LIGHT MICROSCOPY – A COMPUTER SIMULATION

In the introduction the evolution of methods for numerical density estimation of particles is presented shortly. Three pairs of methods have been analysed and compared: (1) classical methods for particles counting in thin and thick sections, (2) original and modified differential counting methods and (3) physical and optical disector methods. Metric characteristics such as accuracy, efficiency, robustness, and feasibility of methods have been estimated and compared. Logical, geometrical and mathematical analysis as well as computer simulations have been applied. In computer simulations a model of randomly distributed equal spheres with maximal contrast against surroundings has been used. According to our computer simulation all methods give accurate results provided that the sample is representative and sufficiently large. However, there are differences in their efficiency, robustness and feasibility. Efficiency and robustness increase with increasing slice thickness in all three pairs of methods. Robustness is superior in both differential and both disector methods compared to both classical methods. Feasibility can be judged according to the additional equipment as well as to the histotechnical and counting procedures necessary for performing individual counting methods. However, it is evident that not all practical problems can efficiently be solved with models.

THE APPLICATION OF STEREOLOGICAL METHODS FOR THE QUANTITATIVE ANALYSIS OF THE ATHEROSCLEROTIC LESIONS IN RABBITS

Atherosclerosis is a chronic metabolic and inflammatory disease of vascular arteries. Insome cases, it comes the accumulation of the inflammatory cells and extracellular matrix in vessel intima, which lead to the narrowing of the vessel lumen. The aim of this study was introduce stereological methods for the quantification of atherosclerotic changes in the aorta of New Zealand White rabbits in dependence on the period of feeding with atherogenic diet containing 0.4% of cholesterol. The Cavalieri estimator was used for the estimation of the volume of atherosclerotic lesions. The area of the atherosclerotic lesions and the area fraction of free vessel lumen were determined with the image analysis software Lucia. The principle of the point counting method was used for the estimation of the volume fraction of collagen in atherosclerotic lesions and the principle of the optical disector was used for the estimation of the number of cells per unit volume in atherosclerotic lesions. The results obtained by the stereological methods confirmed the induction and progression of atherogenic changes in rabbit vessel wall in cholesterol fed animals. The methods applied in this study will be used in our prospective studies where the effect of hypolipidemic drug treatment on atherogenic process will be monitored

A morphometric examination of neuronal and glial cell pathology in the orbitofrontal cortex in late-life depression

ABSTRACTBackground: The orbitofrontal cortex has been implicated as a key component in depression by several imaging studies. This study aims to examine morphometrically glial cell and neuronal density and neuronal volume in the orbitofrontal cortex of late-life major depression patients.Methods: Post mortem tissue from 13 patients with major depression and 11 matched controls was obtained and analyzed using the optical disector and nucleator methods.Results: No changes were found in glial cell, pyramidal or non-pyramidal neuron density, or in non-pyramidal and pyramidal neuron volume in the orbitofrontal cortex.Conclusions: Based on previous findings, this study suggests variability in morphological changes within the orbitofrontal cortex, as well as the prefrontal cortex as a whole.