Use of frozen sections to determine neuronal number in the murine hippocampus and neocortex using the optical disector and optical fractionator

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.

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MODERN STEREOLOGICAL EVALUATION IN THE AGING HUMAN SUBSTANTIA NIGRA

Image Analysis & Stereology ◽

10.5566/ias.v22.p73-80 ◽

2011 ◽

Vol 22 (2) ◽

pp. 73 ◽

Cited By ~ 2

Author(s):

Shuang Y Ma ◽

Frank M Longo ◽

Matias Röyttä ◽

Yrjö Collan

Keyword(s):

Substantia Nigra ◽

Quantitative Estimation ◽

Degenerative Change ◽

Reduction Rate ◽

Cell Number ◽

Aging Brain ◽

Tissue Sections ◽

Neuronal Number ◽

Optical Fractionator ◽

Human Substantia Nigra

Quantitative estimation of neuronal numbers in the human substantia nigra (SN) can be achieved by a conventional single section (SS) count or by the more modern stereological disector (DS) count. However, counting results from SS counts are potentially biased and might not accurately reflect the total neuronal number in the SN or the changes in the total number of neurons occurring during aging or with neurodegenerative disease. Potential sources of bias include the lack of linearity between cell number per area of section and cell number per volume; the variation in the counting level and orientation of tissue sections; and shrinkage of tissue. Modern stereological DS counting overcomes these problems and has played a crucial role in many recent studies in neuropathology, neuroanatomy, neuropharmacology and neurogenetics. Over the past decades, four stereology based counting methods including physical DS, physical fractionator, optical DS and optical fractionator, have been established for quantitative measurement. Recently, stereological estimates have revealed a linear reduction rate of total nigral neuronal numbers with age of about 10% per decade. These findings suggest that the surviving nigral neurons undergo a degenerative change leading to neuronal dysfunction with aging. Furthermore, as an advanced quantitative tool, modern stereological evaluation may provide new insights into the aging of the human SN thereby enabling us to better understand the pathophysiological processes in aging brain.

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Stereological Estimation of Neuronal Number: The Optical Disector/Cavalieri Combination

Neurotrophin Protocols ◽

10.1385/1-59259-060-8:251 ◽

2003 ◽

pp. 251-268

Author(s):

Victor Nurcombe ◽

Nigel G. Wreford ◽

John F. Bertram

Keyword(s):

Optical Disector ◽

Neuronal Number

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Immunoferritin localization of intracellular antigens in positively stained frozen sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008170x ◽

1978 ◽

Vol 36 (2) ◽

pp. 168-169

Author(s):

K. T. Tokuyasu

Keyword(s):

Surface Tension ◽

Water Surface ◽

Thin Layers ◽

The Other ◽

Positive Staining ◽

Frozen Sections ◽

The Past ◽

Ultrathin Frozen Sections ◽

Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

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Localization of pancreatic enzymes in ultrathin frozen sections stained with metal labeled antibody

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081310 ◽

1978 ◽

Vol 36 (2) ◽

pp. 90-91

Author(s):

Kenjiro Yasuda

Keyword(s):

Fluorescent Antibody ◽

Pancreatic Enzymes ◽

Tissue Preparation ◽

Zymogen Granules ◽

Frozen Sections ◽

En Bloc ◽

Antibody Method ◽

Ultrathin Frozen Sections ◽

Fluorescent Antibody Method

Localization of amylase,chymotrypsinogen and trypsinogen in pancreas was demonstrated by Yasuda and Coons (1966), by using fluorescent antibody method. These enzymes were naturally found in the zymogen granules. Among them, amylase showed a diffuse localization around the nucleus, in addition to the zymogen granules. Using ferritin antibody method, scattered ferritin granules were also found around the Golgi area (Yasuda et al.,1967). The recent advance in the tissue preparation enables the antigen to be localized in the ultrathin frozen sections, by applying the labeled antibodies onto the sections instead of staining the tissue en bloc.The present study deals with the comparison of the localization of amylase and lipase demonstrated by applying the bismuth-labeled, peroxidase-labeled and ferritin-labeled antibody methods on the ultrathin frozen sections of pancreas, and on the blocks of the same tissue.

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Ultrastructural Localization of Acid Mucosubstance in Skeletal Muscle

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060271 ◽

1967 ◽

Vol 25 ◽

pp. 52-53 ◽

Cited By ~ 2

Author(s):

William J. Dougherty ◽

Samuel S. Spicer

Keyword(s):

Striated Muscle ◽

Divalent Cations ◽

Ultrastructural Localization ◽

Major Elements ◽

Frozen Sections ◽

Thorium Dioxide ◽

Iron Solution ◽

Coupling System ◽

Psoas Muscles ◽

Formalin Fixed

In recent years, considerable attention has focused on the morphological nature of the excitation-contraction coupling system of striated muscle. Since the study of Porter and Palade, it has become evident that the sarcoplastic reticulum (SR) and transverse tubules constitute the major elements of this system. The problem still exists, however, of determining the mechamisms by which the signal to interdigitate is presented to the thick and thin myofilaments. This problem appears to center on the movement of Ca++ions between myofilaments and SR. Recently, Philpott and Goldstein reported acid mucosubstance associated with the SR of fish branchial muscle using the colloidal thorium dioxide technique, and suggested that this material may serve to bind or release divalent cations such as Ca++. In the present study, Hale's iron solution adapted to electron microscopy was applied to formalin-fixed myofibrils isolated from glycerol-extracted rabbit psoas muscles and to frozen sections of formalin-fixed rat psoas muscles.

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The Application of Ultracryotomy to Immunocytochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073969 ◽

1973 ◽

Vol 31 ◽

pp. 704-709

Author(s):

R. G. Painter ◽

K. T. Tokuyasu ◽

S. J. Singer

Keyword(s):

Frozen Sections ◽

Protein Antigens ◽

Carbon Coated ◽

Antibody Conjugates ◽

Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

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Ultrathin frozen sections of yeast without aldehyde prefixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081152 ◽

1978 ◽

Vol 36 (2) ◽

pp. 58-59

Author(s):

K. J. Böhm ◽

a. E. Unger

Keyword(s):

Aqueous Solutions ◽

Biological Material ◽

Yeast Cells ◽

Frozen Sections ◽

Good Preservation ◽

Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

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X-Ray Analysis of Frozen Hydrated Sections:The Unconventional Approach

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100056065 ◽

1982 ◽

Vol 40 ◽

pp. 754-757

Author(s):

R. Beeuwkes ◽

A. Saubermann ◽

P. Echlin ◽

S. Churchill

Keyword(s):

Ratio Method ◽

Frozen Sections ◽

Technical Problems ◽

Sample Handling ◽

X Ray ◽

Common Group ◽

Ideal Method ◽

Classic Paper ◽

Physical Principles ◽

The Ideal

Fifteen years ago, Hall described clearly the advantages of the thin section approach to biological x-ray microanalysis, and described clearly the ratio method for quantitive analysis in such preparations. In this now classic paper, he also made it clear that the ideal method of sample preparation would involve only freezing and sectioning at low temperature. Subsequently, Hall and his coworkers, as well as others, have applied themselves to the task of direct x-ray microanalysis of frozen sections. To achieve this goal, different methodological approachs have been developed as different groups sought solutions to a common group of technical problems. This report describes some of these problems and indicates the specific approaches and procedures developed by our group in order to overcome them. We acknowledge that the techniques evolved by our group are quite different from earlier approaches to cryomicrotomy and sample handling, hence the title of our paper. However, such departures from tradition have been based upon our attempt to apply basic physical principles to the processes involved. We feel we have demonstrated that such a break with tradition has valuable consequences.

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