Histopathological and immunohistochemical analysis of the cerebral white matter after transient hypoglycemia in rat.

Patients with hypoglycemic coma show abnormal signals in the white matter on magnetic resonance imaging. However, the precise pathological changes in the white matter caused by hypoglycemic coma remain unclear in humans and experimental animals. This study aimed to reveal the distribution and time course of histopathological and immunohistochemical changes occurring in the white matter during the early stages of hypoglycemic coma in rats. Insulin-induced hypoglycemic coma of 15-30-min duration was induced in rats, followed by recovery using a glucose solution. Rat brains were collected after 6 and 24 hr and after 3, 5, 7, and 14 days. The brains were submitted for histological and immunohistochemical analysis for neurofilament 200 kDa (NF), myelin basic protein, olig-2, Iba-1, and glial fibrillary acidic protein (GFAP). Vacuolation was observed in the fiber bundles of the globus pallidus on days 1-14. Most of the vacuoles were located in GFAP-positive astrocytic processes or the extracellular space and appeared to be edematous. Additionally, myelin pallor and a decrease in NF-positive signals were observed on day 14. Microgliosis and astrogliosis were also detected. Observations similar to the globus pallidus, except for edema, were noted in the internal capsule. In the corpus callosum, a mild decrease in NF-positive signals, microgliosis, and astrogliosis were observed. These results suggest that after transient hypoglycemic coma, edema and/or degeneration occurred in the white matter, especially in the globus pallidus, internal capsule, and corpus callosum in the early stages.

Magnetic resonance imaging and diffusion-weighted imaging changes after hypoglycemic coma.

The authors report a case of severe hypoglycemic encephalopathy in an elderly patient. The magnetic resonance images showed bilateral cortical signal changes and basal ganglia lesions, which spared the thalami. The lesions were bright on fluid-attenuated inversion recovery and diffusion-weighted images and dark on the apparent diffusion coefficient map, being more conspicuous on the diffusion-weighted images than on the fluid-attenuated inversion recovery images. A literature review of the imaging features and pathophysiological mechanism in comparison with those of hypoxic ischemic injury is discussed.

Hypoglycemic brain damage.

Hypoglycemia was long considered to kill neurons by depriving them of glucose. We now know that hypoglycemia kills neurons actively rather than by starvation from within. Hypoglycemia only causes neuronal death when the EEG becomes flat. This usually occurs after glucose levels have fallen below 1 mM (18 mg/dL) for some period. At that time abrupt energy failure occurs, the excitatory amino acid aspartate is massively released into the limited brain extracellular space and floods the excitatory amino acid receptors located on neuronal dendrites. Calcium fluxes occur and membrane breaks in the cell lead rapidly to neuronal necrosis. Significant neuronal necrosis occurs after 30 min of electrocerebral silence. Other neurochemical changes include energy depletion to roughly 25% of control, phospholipase and other enzyme activation, tissue alkalosis, and a tendency for all cellular redox systems to shift towards oxidation. Hypoglycemia often differs from ischemia in its neuropathologic distribution, in that necrosis of the dentate gyrus of the hippocampus can occur and a predilection for the superficial layers of the cortex is sometimes seen. Cerebellum and brainstem are universally spared in hypoglycaemic brain damage. Hypoglycemia constitutes a unique metabolic brain insult.

Is neuronal injury caused by hypoglycemic coma of the necrotic or apoptotic type?

In this study, we explored if a 30 minute period of hypoglycemic coma yields damage which shows some features associated with apoptosis. To that end, we induced insulin-hypoglycemic coma of 30 min duration, and studied brain tissues after the coma period, and after recovery period of 30 min, 3 h, and 6 h. Histopathological data confirmed neuronal damage in all of the vulnerable neuronal populations. Release of cytochrome c (cyt c), assessed by Western Blot, was observed in the neocortex and caudoputamen after 3 and 6 h of recovery. In these regions, the caspase-like activity increased above control after 6 h of recovery. By laser-scanning confocal microscopy, a clear expression of Bax was observed after 30 min of coma in the superficial layers of the neocortex, reaching a peak after 30 min of recovery. Punctuate immunolabeling surrounding nuclei in soma and dendrites in cortical pyramidal neurons likely represents mitochondria, which suggests that Bax protein assembled at the surface of mitochondria in vulnerable neocortical neurons. It is concluded that although previous morphological data have suggested that cells die by necrosis, neuronal damage after hypoglycemic coma shows some features of apoptosis.

Cerebral protection by AMPA- and NMDA-receptor antagonists administered after severe insulin-induced hypoglycemia.

Excitatory amino acids are implicated in the development of neuronal cell damage following periods of reversible cerebral ischemia or insulin-induced hypoglycemic coma. To explore the importance of glutamate receptor activation in the posthypoglycemic phase, we exposed rats to 20 min of insulin-induced severe hypoglycemia. The rats were treated immediately after the hypoglycemic insult with four regimes of glutamate receptor antagonists: (1) the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propriate)-receptor antagonist NBQX [2.3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline] given as a bolus dose of 30 mg.kg-1 i.p., followed by an i.v. infusion of 225 micrograms.kg-1.min-1 for 6 h; (2) the non-competitive NMDA-receptor antagonist, dizocilpine (MK-801) 1 mg.kg-1 given i.v.; (3) a combined NBQX treatment, (a bolus dose of 10 mg.kg-1 i.p., followed by an i.v. infusion of 225 micrograms.kg-1.min-1 for 6 h), with dizocilpine 0.33 mg.kg-1 given twice i.p. at 0 and 15 min after recovery and (4) the competitive NMDA-receptor blocker CGP 40,116 [D-(E)-2-amino-4-methyl-5-phosphono-3- pentenoic acid] 10 mg.kg-1 given i.p. In the striatum, all glutamate receptor blockers significantly decreased neuronal damage by approximately 30%. An approximately 50% decrease in neuronal damage was demonstrated in neocortex and hippocampus following the combined treatment with NBQX and dizocilpine, while protection was variable following the treatment with a single glutamate-receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective lesions of mesostriatal dopamine neurons ameliorate hypoglycemic damage in the caudate-putamen.

Unilateral 6-hydroxydopamine lesion of the mesostriatal dopaminergic system was found to ameliorate neuronal necrosis in the caudate-putamen following 30 min of insulin-induced hypoglycemic coma. We propose that increased release of dopamine in the striatum during hypoglycemia or in the recovery period potentiates a deleterious neuronal hyperexcitation, probably induced by excessive release of glutamate or related compounds, thereby aggravating neuronal necrosis.

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. In the first case, an extensive necrotizing injury with gliosis was present in the cerebral cortex with temporal preponderance, as well as in the amygdalae and hippocampus. Lesions were also present in the putamen and caudate nucleus whereas the globus pallidus and thalamus were less severely destroyed. The distribution of the lesions was therefore somewhat different from that commonly seen in hypoxic-ischemic brain injury, which, together with some previously published data, suggests some difference in the pathogenesis of hypoglycemic vs. hypoxic-ischemic brain injury. In the second case only a slight loss of cortical neurons with secondary gliosis could be attributed to the hypoglycemic insult. This case demonstrates the danger of accidental intake of sulfonylurea preparations, which can cause an irreversible brain injury due to their prolonged hypoglycemic effect.

Effects of insulin-induced hypoglycemia on cerebrovascular permeability to horseradish peroxidase.

The effects of hypoglycemia on cerebrovascular permeability to a protein, horseradish peroxidase (HRP), were studied in mice given 3 or 8 units of crytalline zinc insulin intraperitoneally. HRP (10 mg in 0.1 ml saline) was injected intravenously 15 to 20 minutes prior to sacrifice. Both mildly and severely hypoglycemic groups of mice showed a drastic reduction in the normal transit of HRP across cerebral arterioles. The number of normally-stained vessel segments and of HRP-filled endothelial vesicles decreased in insulin-treated mice. In the brains of severely hypoglycemic mice, however, increased parenchymal HRP accumulation occurred. A ruptured blood vessel was found in the center of one-fourth of the focal exudates examined. Electron microscopic examination revealed thrombin, sometimes extending through the vessel wall, and hemorrhage, yet inter-endothelial tight junctions remained intact. Seizures were associated with severe hypoglycemia in 6 out of 10 mice with serum glucose levels below 40 mg/100 ml following 8 units of insulin, but the number of focal exudates per brain was similar in all 10 mice. We conclude that insulin-induced hypoglycemia is associated with decreased HRP transit across cerebral arterioles, and that severe insulin shock is also accompanied by actual rupture of vessel walls and extravasation of blood and HRP into the parenchyma of the brain.