This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell dea...

Ischemic Cell Death in Brain Neurons

Swelling of primary astrocyte cultures by exposing them to hypotonic media caused release of label after the cells had been allowed to accumulate 3H-L-glutamate, 3H-D-aspartate, or 3H-taurine. Comparable release of endogenous L-glutamate or taurine, as measured by high- pressure liquid chromatography (HPLC), was also found. Release of label was not affected by treating the cells with cytochalasin B, indicating that microfilament polymerization was not significantly involved. Hypotonic-induced release did not appear to principally involve reversal of the Na(+)-dependent uptake system since increasing external K+ to depolarize the cells by replacement of external Na+, thus maintaining isotonic conditions, increased release to a lesser extent. Threo beta-hydroxyaspartate, a potent 3H-L-glutamate uptake blocker, added externally stimulated efflux of 3H-L-glutamate independently of the swelling-induced efflux. Upon restoration of swollen cells to isotonic medium they showed an unimpaired ability to take up 3H-L- glutamate. The swelling-induced release of label was inhibited by a number of anion transport inhibitors, one of which has been shown to significantly improve outcome in an experimental brain trauma/hypoxia model in which astrocyte swelling is an early event.

https://www.jneurosci.org/content/jneuro/10/5/1583.full.pdf

Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures

Immunocytochemistry is one of the most powerful techniques employed in defining cellular location and analyzing molecular and cellular organizations. It includes the preparation and fixation of cells without adversely affecting the visualization of subcellular structures by usage of highly specific reagents - predominantly labelled antibodies - and electron microscopy analysis. The book describes all practical steps involved in the transition of living cells to a labelled thin section in the electron microscope. It includes theoretical background to allow lab workers to modify and apply the procedures to their particular nature of work and to interpret microscopical results. Stereological methods for quantitative analysis are also covered. The work helps to obtain a realistic picture of the organization of a living cell.

Fine Structure Immunocytochemistry

This study explores the influence of severe lactic acidosis in the ischemic rat brain on postischemic recovery of the tissue energy state and neurophysiological parameters. Severe incomplete brain ischemia (cerebral blood flow below 5% of normal) was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. We varied the production of lactate in the tissue by manipulating the blood glucose concentrations. A 30-min period of incomplete ischemia induced in food-deprived animals caused lactate to accumulate to 15-16 mumol g-1 in cortical tissue. Upon recirculation these animals showed: (1) a considerable recovery of the cortical energy state as evaluated from the tissue concentrations of phosphocreatine, ATP, ADP, and AMP; and (2) return of spontaneous electrocortical activity as well as of somatosensory evoked response (SER). In contrast, administration of glucose to food-deprived animals prior to ischemia caused an increase in tissue lactate concentration to about 35 mumol g-1. These animals did not recover energy balance in the tissue and neurophysiological functions did not return. In other experiments the production of lactate during 30 min of complete compression ischemia was increased from about 12 mumol g-1 (normoglycemic animals) to 20-30 mumol g-1 by preischemic hyperglycemia and, in separate animals, combined hypercapnia. The recovery of the cortical energy state upon recirculation was significantly poorer in hyperglycemic animals. It is concluded that a high degree of tissue lactic acidosis during brain ischemia impairs postischemic recovery and that different degrees of tissue lactic acidosis may explain why severe incomplete ischemia, in certain experimental models, is more deleterious than complete brain ischemia.

https://journals.sagepub.com/doi/pdf/10.1038/jcbfm.1981.34

Brain lactic acidosis and ischemic cell damage: 1. Biochemistry and neurophysiology.

Recent developments in the field of cerebral circulation and metabolism are reviewed, with emphasis on circulatory and metabolic events that have a bearing on brain damage incurred in ischemia. The first part of the treatise reviews aspects of cerebral metabolism that provide a link to the coupling of metabolism and blood flow, notably those that lead to a perturbation of cellular energy state, ionic homeostasis, and phospholipid metabolism. In the second part, attention is focused on the derangement of energy metabolism and its effects on ion fluxes, acid-base homeostasis, and lipid metabolism. It is emphasized that gross brain damage, involving edema formation and infarction, is enhanced by tissue acidosis, and that neuronal damage, often showing a pronounced selectivity in localization, appears related to a disturbed Ca2+ homeostasis, and to Ca2+-triggered events such as lipolysis and proteolysis.

Cerebral circulation and metabolism

The feline right cerebral hemisphere was subjected to regional (incomplete) ischemia after clipping the middle cerebral artery for 5, 10, 15, 30 or 60 min, respectively. After each ischemic episode, a 10-min recirculation period was allowed, following which the brain was fixed and processed for electron microscopy. The earliest alterations, detected in the cerebral cortex after 15 min, increased in severity with longer ischemic episodes and were distributed multifocally. There was: (a) marked neuronal mitochondrial matrical swelling and progressive condensation of cytoplasm and nucleoplasm; (b) cytoplasmic swelling of astrocytes with preservation of glial mitochondrial volume; (c) capillaries, oligodendrocytes, myelin sheaths and axis cylinders did not change significantly, even after the longest interval studied: 60 min. This type of tissue reaction appears to be common for those forms of cerebral ischemia, in which circulation is either sustained partially (via collateral arteries) or restored after a period of absolute ischemia. Under these conditions, as yet undefined permeability changes in cell membranes lead to pronounced volumetric alterations of cellular compartments. Although no softening is detectable by digital examination, we suggest that such a set of structural abnormalities constitutes encephalomalacia, or the earliest stage of a lesion which is designated infarction, once it reaches irreversibility.

Cellular events during partial cerebral ischemia. I. Electron microscopy of feline cerebral cortex after middle-cerebral-artery occlusion.

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO4), and osmium tetroxide (OsO4) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO4 or OsO4 but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO4 but little or no effect with GA or GA-OsO4. Osmolality of the buffer had a marked effect on cell volume with OsO4, whereas with GA or GA-OsO4 it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO4 alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO4. After fixation with GA and/or OsO4 cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.

/pdf/influence-of-glutaraldehyde-and-or-osmium-tetroxide-on-cell-2y2eisp4b3.pdf

Influence of glutaraldehyde and/or osmium tetroxide on cell volume, ion content, mechanical stability, and membrane permeability of ehrlich ascites tumor cells

Permanent, complete global cerebral ischemia was induced in cats by filling the cardiovascular system with a plasma substitute (37° C). At variable intervals and up to 120 min thereafter, these feline brains were perfused with aldehydes and processed for electron microscopy. The resulting cellular alterations were homogeneous and uniform throughout the entire brain; they included early chromatin clumping, gradually increasing electron lucency of the cell sap, distention of endoplasmic reticulum and Golgi cisternae, transient mitochondrial condensation followed by swelling and appearance of flocculent densities, and dispersion of ribosomal rosettes. The marked contrast between the structural alterations in permanent, complete ischemia and incomplete cerebral ischemia, suggest differences in their pathogenesis. A basic determinant factor of the structural changes appears to be the volume of flow (serum, plasma, other) which is available at the time of the injury. This analysis of global cerebral ischemia provides some insight on the nature of cellular changes occurring shortly after somatic death.

The ultrastructure of "brain death". II. Electron microscopy of feline cortex after complete ischemia

Rat brains were fixed either with glutaraldehyde (GA) or formaldehyde (FA). After 20 min or 24 h fixation the osmotic sensitivity of the tissue was tested by immersion in (a) distilled water (b) 0.15 M or (c) 0.3 M cacodylate buffer. GA-fixed material retained some sensitivity to osmotic stress after 20 min fixation but was entirely resistant after 24 h fixation. Ultrastructural preservation was good after only 20 min GA-fixation, provided that the subsequent treatment was with isotonic solutions. The fixation with FA was less efficient and slower. Dark neurons and other artifacts were commonly seen after the 20 min fixation with FA. Prolongation of the FA-fixation overnight gave markedly better preservation, but however, never equivalent to that with GA.

Hannu Kalimo

Papers

Cellular events during partial cerebral ischemia. I. Electron microscopy of feline cerebral cortex after middle-cerebral-artery occlusion.

Influence of glutaraldehyde and/or osmium tetroxide on cell volume, ion content, mechanical stability, and membrane permeability of ehrlich ascites tumor cells

The ultrastructure of "brain death". II. Electron microscopy of feline cortex after complete ischemia

The efficiency of aldehyde fixation for electron microscopy: stabilization of rat brain tissue to withstand osmotic stress.

Comparison Between Regional Cerebral Ischemia and Total Cerebral Ischemia. an Ultrastructural Study in the Cat