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Sensitive radiometric assays for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyl transferase (PNMT), and glutamic acid decarboxylase (GAD) have been combined with microdissection techniques for quantitative localization of these synthetic enzymes in rabbit spinal cord. TH was present uniformly in gray and white matter. DBH was higher in the lateral horns than in the other gray matter areas, and was not detectable in white matter. PNMT was detectable in gray but not white matter, and was considerably lower in activity than the other catecholamine synthetic enzymes. GAD was higher in the dorsal horns at the cervical and lumbar levels than in other gray matter areas and relatively low in white matter. GAD activity was considerably higher than the catecholamine synthetic enzyme activities. The quantitative localizations are consistent with the qualitative immunohistochemical enzymes maps and distributions of the related putative neurotransmitters.

We found substantial alterations in reactions catalyzed by calcium/phospholipid-dependent and calcium/calmodulin-dependent protein kinases during CNS ischemia which suggested that phenothiazines, drugs capable of inhibiting these reactions, might reduce neurologic damage. To test this hypothesis, we used chlorpromazine and trifluoperazine. Both drugs reduced neurologic function deficits relative to controls in a rabbit multiple cerebral embolism model and a rabbit spinal cord ischemia model. Chlorpromazine was effective despite reduction of blood pressure, and trifluoperazine did not alter blood pressure. These findings suggest that phenothiazines may be useful for preserving neurologic function when administered shortly after the onset of CNS ischemia.

Protein phosphorylation was evaluated in a rabbit spinal cord ischemia model under conditions where cyclic AMP-dependent protein kinase (PK-A) and calcium/phospholipid-dependent protein kinase (PK-C) were activated. One hour of ischemia did not affect PK-A activity significantly; however, PK-C activity was reduced by more than 60%. In vitro phosphorylation of endogenous proteins by endogenous PK-C revealed that eight particulate and five cytosolic proteins showed stimulated phosphorylation by PK-C activators in control tissue, although this stimulation was virtually absent in ischemic samples. When control and ischemic particulate fractions were combined, the endogenous protein phosphorylation pattern under PK-C-activating conditions was similar to the ischemic sample, which suggests that inhibitory molecules may be present in the ischemic particulate fraction. In vitro phosphorylation of endogenous proteins under PK-A-activating conditions in ischemic tissue was similar to that in control tissue. The results suggest that the PK-C phosphorylation system is selectively impaired in ischemic spinal cord. In addition to reduced PK-C-dependent phosphorylation, an Mr 64,000 protein was phosphorylated in ischemic cytosolic samples, but not in control samples. The phosphorylation of the Mr 64,000 protein was neither PK-C-dependent nor PK-A-dependent. These altered phosphorylation reactions may play critical roles in neuronal death during the course of ischemia.

In an ischemia-induced model of an acute motor neuron disorder, there is anterior horn cell damage with Wallerian degeneration in ventral roots; dorsal root ganglia and dorsal roots are unaffected. In a mixed nerve there is axonal degeneration reflecting loss of motor fibers. The sural nerve is normal showing that it does not contain motor fibers. This observation is relevant to the neuropathology of motor neuron disease where axonal degeneration found in the sural nerve suggests involvement of sensory fibers.

Catecholaminergic neurons have been implicated in the mechanism of general anesthesia, but previous attempts at measuring changes in adrenergic neuron function during anesthesia have been limited by techniques to whole brain. Microdissection techniques and sensitive radioisotopic-enzymatic assays were used to measure levels of catecholamines in 20 different nuclei, fiber tracts or nerve terminal regions in brains of rats anesthetized for 90-105 min with 1% halothane or 18% cyclopropane. These two anesthetics were chosen because of their diverse effects on the electroencephalogram and on the cardiovascular and respiratory systems. Of the areas examined, significant increases in norepinephrine content with both anesthetic agents were found only in the nucleus accumbens, locus coeruleus and central gray catecholamine areas. Only in the nucleus accumbens was the dopamine level increased by both anesthetics; cyclopropane, but not halothane anesthesia, also increased the dopamine content of the caudate nucleus, while halothane, but not cyclopropane anesthesia, significantly decreased the dopamine level of the ventral nucleus of the thalamus. Changes in levels of transmitters do not distinguish cause from effect of anesthesia, and further experiments are needed to delineate what role, if any, the specific areas play in muscle relaxation, analgesia, sleep or anesthesia. This study shows that a drug can affect one nucleus or region without significantly affecting other regions that contain the same transmitter substance, and that changes in transmitter levels can occur selectively in different regions of brain even if the nerve endings are derived from contiguous cell bodies.

Embolic stroke was induced in rabbits using autologous blood clot. One hour after stroke, animals received heparin anticoagulation (AC) for five hours (acute AC) or five days (chronic AC). Animals received excessive AC (partial thromboplastin time greater than 3.0 times control), adequate AC (partial thromboplastin time, 1.2 to 2.5 times control), or saline. After the animals were killed, the brains were examined for macroscopic evidence of intracerebral hemorrhage. There was no significant increase over control in the incidence or severity of hemorrhage in any of the four treatment groups. The data suggest that heparin AC does not promote intracerebral hemorrhage after experimental embolic stroke.

A highly predictive spinal "stroke" model in the unanesthetized rabbit was utilized to compare the effects of naloxone and the delta-selective opiate antagonist M154,129 on neurological recovery following ischemic injury to the central nervous system. Naloxone treatment protected against both moderate (20 min aortic occlusion) and severe (25 min aortic occlusion) degrees of ischemic spinal injury, whereas treatment with M154,129 failed to improve recovery in either model. These findings confirm that naloxone therapy can alter the pathophysiological sequelae caused by a critical reduction in blood flow to the central nervous system and suggests that its beneficial effects do not relate to actions at the delta-opiate receptor.

Tissue plasminogen activator (tPA) dissolves intravascular thrombus and restores blood flow after thromboembolic vascular occlusion. The utility of this agent for treatment of stroke in humans may be limited by post-reperfusion hemorrhagic complications. We studied tPA-mediated thrombolysis in an animal model of cerebrovascular occlusion in order to determine what factors, if any, predispose tPA-treated animals to suffer hemorrhage. Small blood clot emboli were injected into the internal carotid arteries of rabbits. Angiograms confirmed occlusion of the middle cerebral artery or internal carotid artery in 100% of subjects. tPA or saline was administered as a 30-minute infusion at various times after embolization. Hemorrhage rates were similar in all groups regardless of treatment. tPA increased the prothrombin time and the thrombin time but not the partial thromboplastin time. There was no correlation between these changes in blood coagulation and the finding of cerebral hemorrhage. We observed a significant association between stroke severity and cerebral hemorrhage. We conclude that tPA treatment successfully causes thrombolysis of cerebral emboli without causing an increase in the incidence of cerebral hemorrhage in rabbits.

A rabbit spinal cord ischemia model was used to study the effects of focal ischemia on the tissue concentrations of serotonin, 5-hydroxyindole acetic acid, and norepinephrine. Ischemia induced by abdominal aorta occlusion caused both serotonin and norepinephrine concentrations to decline in the most ischemic areas of the spinal cord by 55 minutes. In marginally perfused adjacent areas, serotonin concentrations transiently declined at 14 and 20 min. After the onset of ischemia and then returned to normal. The minimum was reached at the same time when previous studies showed damage had become irreversible in more ischemic regions. Concentrations of 5-hydroxyindole acetic acid did not change at any time and norepinephrine declined only in the most ischemic areas after damage was irreversible. Thus, permanent serotonin and norepinephrine decreases occur only in areas destined to be destroyed by infarction, but the serotonin returns to normal in marginal tissue that remains viable. These studies suggest that serotonin may be involved in the early stages of irreversible changes during central nervous system ischemia.

Newly developed colloid volume-expanding agents with mean molecular weights lower than currently available agents may improve outcome after stroke with fewer allergic and coagulation system side effects. The smaller molecule, however, may exacerbate ischemic cerebral edema if it accumulates in areas where ischemia has damaged the blood-brain barrier. We administered low-molecular-weight hydroxyethyl starch to rabbits after embolic infarction and measured specific gravity and total water content. We found evidence of ischemic edema in the hemisphere ipsilateral to the embolic arterial occlusion, but the measures of edema were not different in treated and control groups. Of those rabbits suffering severe neurologic deficit, mortality was 2 of 13 in the treated compared with 7 of 12 in the control groups (p less than 0.01). Thus, hemodilution with low-molecular-weight hydroxyethyl starch did not exacerbate cerebral edema and may have improved survival in this model.

The sequence of events in focal cerebral ischemia has been difficult to study quantitatively in humans. Experimental investigation of these phenomena has been impeded because reproducible animals models that simulate human stroke are lacking. We have developed a rabbit spinal cord ischemia model that resembles human stroke patterns in many respects. Using this model, we have found that (1) brief ischemia produces completely reversible neurological deficits; (2) intermediate ischemic periods may produce transiently reversible deficits that later progress without further manipulation; (3) prolonged ischemia produces irreversible lesions in all animals. The model should be useful for studies of the pathophysiological mechanisms of CNS ischemia.

Several serotonin inhibitors have been shown to reduce neurologic deficits in experimental CNS ischemia. Using biochemical and histological methods we tested the effects of the serotonin inhibitors cyproheptadine and brom-LSD in a highly reproducible rabbit spinal cord ischemia model. Detailed mapping of regional spinal cord blood flow was used to guide sampling for the biochemical studies. We found that it is possible to study biochemical and morphological aspects of spinal cord ischemia in great detail using a combination of quite precise techniques. However, at this level of resolution there were no substantial changes in biogenic amine concentrations in severely ischemic or marginally perfused tissue after the durations of ischemia that cause the onset of irreversible tissue damage. Treatment with doses of serotonin inhibitors that produce preservation of neurological function did not cause significant alterations of tissue concentrations of biogenic amines or tissue morphology in treated versus untreated animals.

Three patients had status epilepticus appearing de novo as the presenting manifestation of Epstein-Barr virus (EBV) encephalopathy. Clinical findings of infectious mononucleosis were absent and EBV-specific serologic tests made or confirmed the diagnosis in each case. Epstein-Barr virus should be considered as a potential cause when status epilepticus occurs in the absence of previous seizures or an identified cause of seizures. The diagnosis may be made in some cases only with the use of EBV-specific serologic testing.

The possible role of endogenous opioids in the pathophysiology of spinal cord injury was evaluated utilizing a variety of experimental models and species. In the cat, we have shown that beta-endorphin-like immunoreactivity was increased in plasma following traumatic spinal injury; such injury was associated with a decrease in spinal cord blood flow (SCBF) which was reversed by the opiate receptor antagonist naloxone. Naloxone treatment also significantly improved functional neurological recovery after severe injury. Thyrotropin-releasing hormone (TRH), possibly through its "anti-endorphin" actions, was even more effective than naloxone in improving functional recovery in the cat. In a rat model, utilizing a similar trauma method, TRH proved superior to naloxone in improving SCBF after injury. In addition, naloxone at high doses attenuated the hindlimb paralysis produced by temporary aortic occlusion in the rabbit. The high doses of naloxone required to improve neurological function after spinal injury suggest that naloxone's actions, if opiate receptor mediated, may be mediated by non-mu receptors. Dynorphin, an endogenous opioid with a high affinity for the kappa receptor, produced hindlimb paralysis following intrathecal administration in rats. Taken together, these findings suggest that endogenous opioids, possibly acting at kappa receptors in the spinal cord, may serve as pathophysiological factors in spinal cord injury.