Fixation of the central nervous system for electron microscopy by aldehyde perfusion

Schultz, Robert L.; Karlsson, Ulf

doi:10.1016/s0022-5320(65)80015-6

Cited by 168 publications

(40 citation statements)

References 16 publications

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“…The osmolarity of the fixative solutions and their effects on cell and tissue morphology have received relatively little attention recently -indeed often appear ignored -despite the fact that there are studies in the literature highlighting this issue. For example, Schultz and Karlsson (1965) observed that hypertonic fixative solutions perfused into the central nervous system caused cellular shrinkage and increased extracellular spaces, whereas isotonic (300-320mOsm) and hypotonic fixatives could result in cell swelling. Mathieu et al (1978) have reported the importance of both the % of GA and the molarity of the vehicle used for the fixative solution for the preservation of lung tissue.…”

Section: Introductionmentioning

confidence: 99%

A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology

Loqman

Bush²,

Farquharson³

et al. 2010

eCM

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The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage) artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a) paraformaldehyde (PFA; 4%), (b) glutaraldehyde (GA; 2%) with PFA (2%) with ruthenium hexamine trichloride (RHT; 0.7%), (c) GA (2%) with RHT (0.7%), or (d) GA (1.3%) with RHT (0.5%) and osmolarity adjusted to a 'physiological' level of 280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescentlylabelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids (~280mOsm). These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues.

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Section: Introductionmentioning

confidence: 99%

A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology

Loqman

Bush²,

Farquharson³

et al. 2010

eCM

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“…Later, the brains were postfixed with 4% formaldehyde (Serva, Heidelberg, Germany), 2.5% glutaraldehyde (Science Services, Munich, Germany), and 0.5% NaCl in phosphate buffer pH 7.4 17. Finally, the corpus callosum was postfixed with 2% OsO 4 (Science Services) in 0.1 M phosphate buffer pH 7.3 and embedded in EPON (Serva) after dehydration with ethanol and propylene oxide.…”

Section: Methodsmentioning

confidence: 99%

The Cannabinoid CB1/CB2 Agonist WIN55212.2 Promotes Oligodendrocyte Differentiation In Vitro and Neuroprotection During the Cuprizone‐Induced Central Nervous System Demyelination

et al. 2016

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SummaryAim and methodsDifferent types of insults to the CNS lead to axon demyelination. Remyelination occurs when the CNS attempts to recover from myelin loss and requires the activation of oligodendrocyte precursor cells. With the rationale that CB1 receptor is expressed in oligodendrocytes and marijuana consumption alters CNS myelination, we study the effects of the cannabinoid agonist WIN55212.2 in (1) an in vitro model of oligodendrocyte differentiation and (2) the cuprizone model for demyelination.ResultsThe synthetic cannabinoid agonist WIN55212.2 at 1 μM increased the myelin basic protein mRNA and protein expression in vitro. During cuprizone‐induced acute demyelination, the administration of 0.5 mg/kg WIN55212.2 confers more myelinated axons, increased the expression of retinoid X receptor alpha, and declined nogo receptor expression. Controversially, 1 mg/kg of the drug increased the number of demyelinated axons and reduced the expression of nerve growth factor inducible, calreticulin and myelin‐related genes coupling specifically with a decrease in 2′,3′‐cyclic nucleotide 3′ phosphodiesterase expression.ConclusionThe cannabinoid agonist WIN55212.2 promotes oligodendrocyte differentiation in vitro. Moreover, 0.5 mg/kg of the drug confers neuroprotection during cuprizone‐induced demyelination, while 1 mg/kg aggravates the demyelination process.

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“…Schultz and Karlsson (1965) have demonstrated that the usual fixatives do not immobilize membranous components sufficiently to prevent their subsequent movement, owing, in their study, to changes in osmolarity.…”

Section: Advantages Of the Methodsmentioning

confidence: 99%

A Simple Freeze-Fracture Replication Method for Electron Microscopy

Bullivant

Ames

1966

The Journal of Cell Biology

152

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A simple method to achieve results similar to the freeze-etching technique of Moor et al. (1961) is described. The frozen tissue is cut under liquid nitrogen with a razor blade outside the evaporator rather than inside with a cooled microtome. The conditions of the experiment do not favor sublimation, and it is proposed that the structure of the replica be explained by local faults in the cleavage plane which leaves structures, such as membranes, standing above the ice. Micrographs of replicas of glycerol-protected frozen small intestine of mouse prepared by the method are presented and the structural details they show are discussed. The problem of vapor-deposited contamination is discussed. It is concluded that this is a practical method for obtaining electron micrographs that are relatively free of artifact, and that further improvements may be expected from the use of rapidly frozen fresh tissue and a clean vacuum system, possibly of the ion-pumped type.

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Fixation of the central nervous system for electron microscopy by aldehyde perfusion

Cited by 168 publications

References 16 publications

A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology

A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology

The Cannabinoid CB1/CB2 Agonist WIN55212.2 Promotes Oligodendrocyte Differentiation In Vitro and Neuroprotection During the Cuprizone‐Induced Central Nervous System Demyelination

A Simple Freeze-Fracture Replication Method for Electron Microscopy

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