Roland N. Auer

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.

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.

Immediate and long-lasting effects of MK-801 on motor activity, spatial navigation in a swimming pool and EEG in the rat

In a series of experiments, rats received the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK-801 and measures were made of motor behavior, spatial navigation in a swimming pool, and electroencephalographic (EEG) activity. High doses (0.25–10 mg/kg IV) produced somnolence and akinesia, impaired food consumption, locomotion and swimming, and also impaired navigation to a hidden platform but complete recovery on all measures was obtained between 3 and 5 days postinjection. Lower doses (0.05–0.10 mg/kg, IV) impaired acquisition of a new place response in a swimming pool and produced hyperactivity but did not impair performance on a new cue response or on a well-learned place response. Two forms of hippocampal EEG activity, atropine-sensitive and atropine-resistant EEG were present with the low doses. The results demonstrate that a single dose of MK-801 causes changes in motor behavior and learning lasting a few days, but complete recovery occurs within 5 days of administration of even very high doses of MK-801. They further demonstrate that low doses of the drug selectively impair acquisition of new place responses. Although the general changes in behavior produced by MK-801 suggest that NMDA receptors are involved in many aspects of the control of behavior, the results additionally suggest that NMDA receptors are improtant for place learning.

Effect of Age in Rodent Models of Focal and Forebrain Ischemia

BACKGROUND AND PURPOSE The majority of animal experiments examining the nature and treatment of stroke have used relatively young animals ranging in age from 2 to 6 months. However, significant morphological, neurochemical, and behavioral changes occur with aging in rodents particularly during the first 24 months of age. This study examines the effect of age in two models of transient ischemia a forebrain and a focal model in male Wistar rats. METHODS We induced forebrain ischemia of 12 minutes duration by bilateral carotid artery occlusion with controlled hypotension at a mean blood pressure of 45 mm Hg and using an intraluminal filament technique, induced focal middle cerebral artery occlusion of 100 minutes duration at a mean blood pressure of 60 mm Hg. Physiological parameters were monitored and maintained within normal limits. On day 7 after ischemia, the rats were perfusion-fixed and the brains removed for quantitative histopathology. RESULTS After forebrain ischemia, older rats showed significantly less CA1 neuronal necrosis than the younger group (P < .003), whereas both striatal and neocortical injury were significantly greater in the older group (P < .05). Among animals subjected to focal ischemia, the volume of infarcted tissue and the number of necrotic neurons in the area adjacent to the infarction were both greater in older rats (P < .05). CONCLUSIONS This study emphasizes the importance of age in models of forebrain and focal ischemia. The interaction between age-related changes in morphology, neurochemistry, and behavior on the ischemic cascade complicates the interpretation of mechanistic data, and pharmacological effects observed in younger animals may not necessarily translate to an older population.

Primary intracerebral hemorrhage.

This article reviews the epidemiology, pathophysiology and management of primary intracerebral hemorrhage. In North American and European populations, 15% of strokes are due to intracerebral hemorrhage. Pathologically in hypertension, early arteriolar proliferation of smooth muscle is followed later by smooth muscle cell death and collagen deposition. This eventually leads to occlusion or ectasia of arterioles. The latter leads to Charcôt-Bouchard aneurysm formation and possible intracerebral hemorrhage. Amyloid deposition in the tunica media causes similar brittle arterioles. Fibrin globes in concentric spheres attempt to seal off the site of bleeding. But vasculopathy (either amyloid or hypertensive) inhibits the contractile capability of arterioles. The size of the final sphere of blood at cessation of bleeding determines the clinical spectrum, from asymptomatic to fatal. Since arteriolar bleeding is slower than arterial bleeding, several hours exist where intervention may be useful. While medical intervention is controversial, guidelines for blood pressure, intracranial pressure, glucose and seizure management exist. Surgical trials have tended to show no benefit. Recombinant factor VIIa is undergoing investigation as hemostatic therapy for intracerebral hemorrhage, to limit clot expansion and possibly also as a hemostatic adjunct to surgery.

The nature and timing of excitotoxic neuronal necrosis in the cerebral cortex, hippocampus and thalamus due to flurothyl-induced status epilepticus

SummaryFlurothyl-induced status epilepticus was studied by light and electron microscopy (LM, EM) to determine the time course and structural features of neuronal necrosis in the vulnerable brain regions in epilepsy. The cerebral cortex, hippocampus and thalamus were examined after closely spaced recovery periods of up to 1 week. The results showed that acidophilic neurons appeared simultaneously in neurons of the neocortex, hippocampus and thalamus, and that this occurred within 1 h following the end of the epilepsy. The corresponding features of acidophilic neurons by EM were mitochondrial flocculent densities and large discontinuities in cell and nuclear membranes. Dark neurons were ubiquitous during the epilepsy, but recovered almost universally. A few dark neuronal forms persisted and underwent cytorrhexis after 12-h recovery or longer. Axon-sparing dendritic lesions characteristic of excitotoxic neuronal death were found in the neuropil of the neocortex, and in both vulnerable CA1 and resistant CA3 neurons of the hippocampus. Other than acute edema, glial changes were absent. The findings support an excitotoxic mechanism in epilepsy-induced selective neuronal necrosis also in brain regions outside the hippocampus, and contrast with previous reports in ischemia and hypoglycemia in that neuronal necrosis occurs virtually immediately after an epileptic insult. No “maturation” of cell damage, as described in ischemia, was seen. Furthermore, even exceedingly dark neuronal forms and massive dendritic swelling must be considered sub-lethal or prelethal cellular changes. Lethal cellular changes include acidophilia by LM, cell membrane breaks, and mitochondrial flocculent densities by EM.

Postischemic insulin reduces spatial learning deficit following transient forebrain ischemia in rats.

We investigated the ability of postischemic insulin administration to modify the structural and neurobehavioral consequences of cerebral ischemia in rats. Forebrain ischemia was induced in fed rats by combining controlled systemic hypotension with bilateral carotid artery clamping for 10 1/2 minutes. Following clamp release, one group of six rats [corrected] was given insulin (2 IU/kg s.c. b.i.d.) for 1 week. An ischemic-control group of five rats [corrected] received no postischemic treatment. A sham-ischemia group of rats was used as a behavioral control. Throughout the recovery period until sacrifice, the drinking water of all rats was supplemented with 25% glucose. Rats were trained on two water maze place navigation tasks 1-2 months after ischemia. Escape latencies and swim patterns were recorded. Performance in the insulin-treated group was better than that in the ischemic-control group (p less than 0.05) on both tasks and did not differ significantly from that of the sham-ischemia group. Improvement in behavior correlated with a significant reduction in CA1 hippocampal necrosis in the insulin-treated group (p less than 0.05). Our findings demonstrate that postischemic treatment with insulin improves neurobehavioral performance in addition to lessening ischemic neuronal necrosis.

Non‐Pharmacologic (Physiologic) Neuroprotection in the Treatment of Brain Ischemia

Abstract: Clinical trials for ischemic stroke have been characterized by a disappointing series of negative results, using a panoply of pharmacologic agents. This paper emphasizes five physiologic measures that can be taken to mitigate ischemic brain damage. These are (1) hypothermia, (2) insulin, (3) arterial hyperoxemia, (4) blood pressure control and (5) magnesium. Hypothermia is protective in both focal and global ischemia, even postischemically protecting against selective neuronal necrosis and infarction. The total equation for protection includes the (i) postischemic delay, (ii) depth, and (iii) duration of hypothermia. Insulin operates by lowering glucose levels to the normal range in focal ischemia. It is possible that very low glucose levels are detrimental in focal ischemia with paradoxical augmentation of the infarct size, and that spreading depression plays a role in this. Controlled arterial hyperoxemia seems effective experimentally in reducing infarct size, operating mechanistically by either a direct effect of oxygen, or vasoconstriction causing shunting of blood into the infarct, or both. Blood pressure is a critical determinant of infarct size, and raising blood pressure improves collateral blood flow and reduces stroke size. To be used clinically, however, hemorrhage must be ruled out. The most dramatic clinical effects of blood pressure are seen in aneurysm patients with vasospasm, where minor increases in blood pressure reverse temporary hemiparesis by reducing ischemia. Magnesium is likely the safest NMDA antagonist, with a long history of safe administration to pregnant women with eclampsia. There is potential interaction with insulin, in that magnesium causes hyperglycemia, which requires insulin to counteract it. Magnesium and insulin together have been shown effective in experimental brain ischemia. In the absence of safe and effective pharmacologic neuroprotection agents, clinical trials should be designed and launched to test these physiologic measures, singly and in combination, to reduce brain damage after ischemia.

Graded Hypotension and MCA Occlusion Duration: Effect in Transient Focal Ischemia

The first 2 h of middle cerebral artery occlusion (MCAO) are likely critical in determining the final outcome in ischemic stroke. To study this early postischemic period, male Wistar rats (n = 161) were subjected to right MCAO with closely spaced step variations in both duration of MCAO and blood pressure (BP), using the intraluminal suture technique. Quantitative neuropathology was performed at 25 coronal planes of the brain after 1-week survival. Atrophy was measured as the difference between the two hemispheres and was added to cortical and striatal necrosis to obtain total tissue loss. Damage consistently increased monotonically with increasing duration of occlusion only when infarct size was expressed as percentage of the contralateral hemisphere, but not when expressed as mm3, because of variable tissue size. The results showed that already at 1 week, the quantity of tissue loss due to resorption and transsynaptic effects approached the quantity of geographically traceable necrosis in cortex and striatum. Minimum brain damage (5%) occurred after 60 min at a BP of 80 mm Hg, with almost no cortical necrosis. Damage was extremely sensitive to hypotension and MCAO duration. At a BP of 40 mm Hg, 60 min of MCAO produced 25% damage, accelerating every 20 min during the 2-h period studied. At BP 80 mm Hg, 120 min of MCAO produced the same damage as only 80 min of MCAO at BP 60 mm Hg. At 60-, 80-, 100-, and 120-min duration of MCAO, infarct size was significantly reduced with increasing BP. Analysis of the independent contribution of BP and MCAO duration revealed that BP played a greater role in determining infarct size than did MCAO duration. Ipsilateral hippocampal damage was seen in CA1 and, in some animals, CA3. Necrotic neurons in hippocampus were found in 21 animals, including four with bilateral hippocampal damage, largely but not exclusively distributed in the hypotensive groups. Contralateral necrotizing damage was seen in cortex and hippocampus as selective neuronal necrosis and as cortical infarction in two animals. Ipsilateral and contralateral hippocampal damage reproduced the pattern of selective vulnerability seen in global ischemia. The histologic penumbra (rim of selective neuronal necrosis surrounding the infarct) increased over time at BP 80 mm Hg but remained constant at a larger, presumably maximal level at BP 40 mm Hg in spite of increasing infarct size. We conclude that the first 2 h after MCAO is a very dynamic period, with increasing infarct size every 20 min, a process extremely sensitive to hypotension during occlusion. Transsynaptic effects can produce necrotizing damage in the hemisphere opposite to the ischemia.

Neuropathy with onion bulb formations and pure motor manifestations

A thirty-eight-year-old man presented with a six year history of symptoms resembling an anterior horn cell disorder. There was progressive upper extremity wasting and weakness in the absence of sensory complaints. Electrophysiologic abnormalities were confined to motor nerve conduction and indicated a demyelinating process involving the brachial plexus and major proximal upper extremity nerve trucks bilaterally. Biopsy of the proximal right ulnar nerve revealed changes suggesting a chronic demyelinating process, and onion-bulb formations were present. Immunohistochemical staining for S-100 protein was positive in the cells comprising the onion-bulbs, indicating a Schwann cell, not a perineurial origin of these cells. After 8 years, symptoms have failed to appear in the lower limbs. Recent reports in the literature have begun to delineate the syndrome, which appears to represent an unusual, localized or multifocal, sometimes inflammatory, clinically benign neuropathy that can mimic motor neuron disease in its earlier stages. We report the first such case with underlying pathology.