Y. Olsson

The distribution of hypoglycemic brain damage

SummaryRats were exposed to insulin-induced hypoglycemia resulting in periods of cerebral isoelectricity ranging from 10 to 60 min. After recovery with glucose, they were allowed to wake up and survive for 1 week. Control rats were recovered at the stage of EEG slowing. After sub-serial sectioning, the number and distribution of dying neurons was assessed in each brain region. Acid fuchsin was found to stain moribund neurons a brilliant red.Brains from control rats showed no dying neurons. From 10 to 60 min of cerebral isoelectricity, the number of dying neurons per brain correlated positively with the number of minutes of cerebral isoelectricity up to the maximum examined period of 60 min.Neuronal necrosis was found in the major brain regions vulnerable to several different insults. However, within each region the damage was not distributed as observed in ischemia.A superficial to deep gradient in the density of neuronal necrosis was seen in the cerebral cortex. More severe damage revealed a gradient in relation to the subjacent white matter as well. The caudatoputamen was involved more heavily near the white matter, and in more severely affected animals near the angle of the lateral ventricle. The hippocampus showed dense neuronal necrosis at the crest of the dentate gyrus and a gradient of increasing selective neuronal necrosis medially in CA1. The CA3 zone, while relatively resistant, showed neuronal necrosis in relation to the lateral ventricle in animals with hydrocephalus. Sharp demarcations between normal and damaged neuropil were found in the hippocampus. The periventricular amygdaloid nuclei showed damage closest to the lateral ventricles. The cerebellum was affected first near the foramina of Luschka, with damage occurring over the hemispheres in more severely affected animals. Purkinje cells were affected first, but basket cells were damaged as well. Rare necrotic neurons were seen in brain stem nuclei. The spinal cord showed necrosis of neurons in all areas of the gray matter. Infarction was not seen in this study.The possibility is discussed that a neurotoxic substance borne in the tissue fluid and cerebrospinal fluid (CSF) contributes to the pathogenesis of neuronal necrosis in hypoglycemic brain damage.

Hypoglycemic Brain Injury in the Rat: Correlation of Density of Brain Damage with the EEG Isoelectric Time: A Quantitative Study

Thirty-eight male Wistar rats were exposed to insulin-induced hypoglycemia resulting in periods of cerebral isoelectricity ranging from 10 to 60 min. Plasma glucose levels during cerebral isoelectricity ranged from 0.12 mM to 1.36 mM. Control rats were injected with insulin, but hypoglycemia was terminated with glucose at the stage of large δ-wave EEG slowing. After recovery, the rats were allowed to wake up and survive for 1 wk. The number of dying neurons was assessed with acid-fuchsin/cresyl-violet-stained, whole-brain, sub-serial sections using direct visual counting of acido-philic, cytoclastic neurons. Brains from control rats that were not allowed to become isoelectric showed no dying neurons. Ten minutes of cerebral isoelectricity produced very minimal brain damage. The density of neuronal necrosis was positively related to the number of minutes of cerebral isoelectricity up to the maximum examined period of 60 min, but showed no correlation with the blood sugar levels. The cerebral cortex, hippocampus, caudate nucleus, spinal cord, and, to a lesser extent, cerebellar Purkinje cells were affected. The distribution of neuronal necrosis was not identical with that seen in ischemia, but, rather, suggested a CSF-borne neurotoxin operant in contributing to the pathogenesis of neuronal necrosis in hypoglycemie brain damage. Neuronal death does not occur in hypoglycemia unless the EEG becomes isoelectric, whatever the blood sugar level. Serious brain damage does not occur until electrocerebral silence has been established for at least several minutes. Neuronal death accelerates after 30 min of EEG isoelectricity in the rat. Most animals recovered well after up to 40 min of cerebral isoelectricity.

The temporal evolution of hypoglycemic brain damage

SummaryIn the course of a study on the pathogenesis of neuronal necrosis in severe hypoglycemia, the morphological characteristics reflecting reversible and irreversible neuronal lesions were examined as a function of time following normalization of blood glucose. To that end, closely spaced time intervals were studied in the rat cerebral cortex before, during, and up to 1 year after standardized pure hypoglycemic insults of 30 and 60 min of cerebral isoelectricity.Both the superficial and deep layers of the cerebral cortex showed dark and light neurons during and several hours after the insult. By electron microscopy (EM) the dark neurons were characterized by marked condensation of both karyoplasma and cytoplasm, with discernible, tightly packed cytoplasmic organelles. The light neurons displayed clustering of normal organelles around the nucleus with clearing of the peripheral cytoplasm. Some cells, both dark neurons and neurons of normal electron density, contained swollen mitochondrial with fractured cristae.Light neurons disappeared from the cerebral cortex by 4 h of recovery. Some dark neurons in the superficial cortex and almost all in the deep cortex evolved through transitional forms into normal neurons by 6 h recovery. Another portion of the dark neurons in the superficial cortex became acidophilic between 4 and 12 h, and by EM they demonstrated karyorrhexis with stippled electron-dense chromatin. The plasma membrane was disrupted, the cytoplasm was composed of amorphous granular debris, and the mitochondria contained flocculent densities. These definitive indices of irreversible neuronal damage were seen as early as 4–8 h recovery. Subsequently, the acidophilic neurons were removed from the tissue, and gliosis ensued.Thus, even markedly hyperchromatic “dark” neurons are compatible with survival of the cell, as are neurons with conspicuous mitochondrial swelling. Definite nerve cell death is verified as the appearance of acidophilic neurons at which stage extensive damage to mitochondria is already seen in the form of flocculent densities, and cell membranes are ruptured.Our previous results have shown that hypoglycemic neocortical damage affects the superficial laminae, chiefly layer 2. The present results demonstrate that, following the primary insult, this damage evolves relatively rapidly within the first 4–12 h. We have obtained no evidence that additional necrotic neurons are recruited after longer recovery periods.

Age-related reduction of human growth hormone-binding sites in the human brain

Previous studies have shown that alterations in various neuroendocrine functions occur with increasing age. We here report a study of growth hormone (GH)-binding sites in different areas of post-mortem human brains collected from individual males and females of different age. The results indicate that there exists a significant negative correlation between the density of GH-binding sites and increasing age. This phenomenon was observed in both sexes in brain areas such as choroid plexus, hippocampus, hypothalamus, pituitary and putamen but not in e.g. thalamus. In all tissues (except for choroid plexus), the GH binding was significantly higher in those originating from females than those from males. This discrepancy was found likely to be associated with the affinity of GH to lactogenic rather than to somatogenic sites as no pronounced sex difference in binding was observed in the presence of excessive amounts of human prolactin. Data also indicate that the putative GH receptors in the various brain regions differ with regard to binding constants and to the estimated molecular size of the hormone-binding units. The loss of GH receptors in brain of elderly people may have consequences in several physiological courses. The decrease in GH binding at hypothalamic and pituitary levels may be of importance for the mechanisms behind the release or secretion of the hormone.

Hypoglycemic brain injury. I. Metabolic and light microscopic findings in rat cerebral cortex during profound insulin-induced hypoglycemia and in the recovery period following glucose administration

SummaryProfound hypoglycemia causing the disappearance of spontaneous EEG activity was induced by insulin in rats. For analysis of cerebral cortical concentrations of labile phosphates, glycolytic metabolites and amino acids, the brain was frozen in situ. For microscopic analysis of the corresponding cerebral cortical areas the brain was fixed by perfusion. Hypoglycemia with an isoelectric EEG for 30 and 60 min caused severe perturbation of the cerebral energy metabolites. After both 30 and 60 min of isoelectric EEG, two microscopically different types of nerve cell injury were seen. Type I injury was characterized by angulated, darkly stained neurons with perineuronal vacuolation, mainly affecting small neurons in cortical layer 3. Type II injured neurons, mainly larger ones in layers 5–6, were slightly swollen with vacuolation or clearing (depending on the histotechnique used) of the peripheral cytoplasm, but had no nuclear changes.Recovery was induced by glucose injection. Improvement in the cerebral energy state occurred during the 30 min recovery period even after 60 min of hypoglycemia. However, the persisting reduction in the size of adenine nucleotide and amino acid pools after 30 or 180 min recovery suggested that some cells remained damaged. In confirmation many type I injured neurons persisted during the recovery suggesting an irreversible injury. The disappearance of virtually all type II injuries indicated reversibility of these histopathological changes.The microscopic changes in hypoglycemia were different from those in anoxia-ischemia suggesting a dissimilar pathogenesis in these states despite the common final pathway of energy failure.

Brain lactic acidosis and ischemic cell damage: Quantitative ultrastructural changes in capillaries of rat cerebral cortex

SummaryExcessive tissue lactic acidosis has earlier been shown to aggravate structural damage of both neurons and glial cells in the rat cerebral cortex. To study the reactions of cortical capillaries, light- and electronmicroscopic morphometry was used. Rats were subjected to severe incomplete ischemia (cerebral blood flow below 5% of normal) for 30 min by clamping their carotid arteries and by lowering the blood pressure. Lactate production during ischemia was modified by preischemic administration of either saline (low lactic acidosis group) or glucose (high lactic acidosis group). In the animals with low lactic acidosis, only minimal vascular changes were seen after both 5 min and 90 min recirculation. In the high lactic acidosis group, the endothelial cells were swollen after 5 min of recirculation, and the changes grew markedly worse during 90 min of recirculation. Nuclear chromatin coarsened and mitochondria swelled up. Morphometry showed that the lumen narrowed as a result of endothelial swelling. In spite of variable degree of perivascular astrocytic edema, the outer capillary diameter was little changed in the experimental groups. It seems likely that endothelial swelling hampers postischemic circulation in incomplete ischemia accompanied by high lactic acidosis.

The dentate gyrus in hypoglycemia: Pathology implicating excititoxin-mediated neuronal necrosis

SummaryA detailed light- and electron-microscopic study of the damage to the rat dentate gyrus in hypoglycemia was undertaken, in view of the previously advanced hypothesis that hypoglycemic nerve cell injury is mediated by a released neurotoxin. The distribution of neuronal necrosis showed a relationship to the subarachnoid cisterns.Electron microscopy of the dentate granule cells and their apical dendrites revealed dendrosomal, axon-sparing neuronal pathology. Dentate granule cells were affected first in the dendrites in the outer layer of the stratum moleculare, sparing axons of passage and terminal boutons. Subsequently, the neuronal perikarya were affected, and Wallerian degeneration of axons followed. Cell membrane abnormalities preceded the appearance of mitochondrial flocculent densities and degradation of the cytoskeleton, and are suggested to be early lethal changes.The observed early dendrotoxic changes, and the dendrosomal, axon-sparing nature of the lesion implicate an excitotoxin-mediated neuronal necrosis in hypoglycemia.

Effects of severe hypoglycemia on the human brain neuropathological case reports

The neuropathological findings in two cases of irreversible hypoglycemic brain injury are described. A 26‐year‐old diabetic man injected insulin without adequate food intake and died after 2 months in coma. An 84‐year‐old nondiabetic man accidentally received 10 mg of glibenclamide and died after 3 months in relatively superficial coma.

A transient hypertensive opening of the blood-brain barrier can lead to brain damage

SummaryA transient increase in blood pressure was induced in 15 male Sprague Dawley rats by clamping the upper abdominal aorta for 8–10 min. Three rats served as controls. The brains were fixed by perfusion 2 h or 7 days later. Evans blue-albumin (EBA) was used for macroscopic evaluation of the blood-brain barrier (BBB) integrity. Extravasated plasma albumin, fibrinogen and fibronectin were demonstrated by immunohistochemistry on paraffin sections. Glial fibrillary acidic protein (GFAP) was visualized in the same way. Parallel sections were analyzed for possible parenchymal changes associated with the BBB breakdown. Multiple focal areas of BBB opening were seen in the brains of the three rats killed 2 h after the hypertensive episode. The plasma proteins were present in the vascular wall, extracellular space and within certain neurons. Shrunken acid fuchsin positive neurons were seen in some areas of extravasation. After 7 days, in 5 out of 12 rats a few local lesions with EBA leakage and positive immunostaining for plasma proteins were seen. Structurally these lesions were characterized by shrinkage, fuchsinophilia and disintegration of neurons and proliferation of astrocytes. Thus, a transient opening of the BBB by acute hypertension may lead to permanent tissue damage.

Cerebral microangiopathy in stroke-prone spontaneously hypertensive rats. An immunohistochemical and ultrastructural study.

SummaryThe morphology of cerebral microvessels was studied immunohistochemically and ultrastructurally in 6- to 9-month-old normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone SHR (SHRSP) with a systolic blood pressure of 138±15 mm Hg, 189±9 mm Hg, and 258±30 mm Hg respectively. Regions with major opening of the blood-brain barrier (BBB) were revealed by an i. v. injection of Evans Blue. Multifocal BBB opening with massive leakage of plasma constituents rich in fibrinogen-fibrin-related antigen occurred in SHRSP with a blood pressure above 210–220 mm Hg. BBB-leakage sites were found in the cerebral cortex and the basal ganglia, most frequently in the arterial border zones. The perivascular tissue spaces were dilated within the BBB-leakage sites, in particular around arterioles. Damaged endothelial and smooth muscle cells were replaced by fibrin-like material, multiple layers of basement membranes and bundles of collagen fibrils surrounded by proliferated fibroblasts. The degenerative-infiltrative-proliferative disease process transformed short segments of single arterioles into severely thickened, tortuous and stenotic vessels. Fibrinoid degeneration, formation of microaneurysms and fibrin-rich vascular occlusions were observed. In contrast, only minor or no vascular alterations were seen in regions with preserved BBB in SHRSP and SHR. A severely increased intraluminal pressure load appears to be of major pathogenetic importance for breakdown of the BBB and initiation of the vascular disease process in SHRSP. However, since only short segments of a limited number of widely separated vessels are severely affected, and the number of affected vessels increase towards arterial end and border zones, additional predisposing and aggravating factors may play significant roles in the development of fibrinoid vascular lesions in arterial hypertension.