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Continuous intracerebroventricular or intrathecal infusions of neurotrophic factors have been reported to prevent neuronal degeneration, stimulate axonal sprouting and ameliorate behavioral deficits in various models of CNS injury and aging. In the present study, the ability of intrathecal infusions of recombinant human nerve growth factor (NGF) to reduce functional deficits following spinal cord ischemia was investigated. Adult rabbits underwent intrathecal cannulation and continuous infusions of either 300 microg/ml recombinant human NGF or artificial CSF (vehicle) at a rate of 143 microl/day for 7 days prior to induction of spinal cord ischemia. Continuous infusions were maintained after induction of ischemia. Four days later, both NGF-treated and vehicle-infused subjects showed a significant amelioration of functional motor deficits compared to lesioned, non-infused subjects (P<0.05). The average duration of tolerated ischemia increased from 23.4+/-1.8 min in lesioned, non-infused subjects to 35.5+/-3.1 min in lesioned, artificial CSF-infused subjects and 35.6+/-4.7 min in NGF-infused subjects (mean+/-S.E.M.). Significantly elevated NGF protein levels were attained within the spinal cords of both NGF-treated subjects and artificial CSF-infused subjects, although levels were substantially higher in NGF-treated subjects (9.8+/-3.8 ng/g in NGF-infused vs. 2.0+/-0.4 ng/g in vehicle-infused and only 0.4+/-0.2 ng/g in lesioned, non-infused animals). These findings indicate that the process of intrathecal cannulation and fluid infusion elicits alterations in the spinal cord environment that are neuroprotective, including spontaneous elevations in NGF levels.

Ischemia causes long-term effects on brain volume and neurologic function but the relationship between the two is poorly characterized. We studied the relationships between brain volume and three measures of rodent behavior after cerebral ischemia was induced by injecting several thousand microspheres into the internal carotid arteries of rats. Forty eight hours later, each subject was rated using a global neurologic rating scale. Several weeks later, the subjects were tested for open field activity and visual spatial learning. Post-mortem we measured the volume of the cerebral hemispheres and estimated the volume densities of cortex, white matter, hippocampus, basal ganglia, thalamus, ventricle, and visible infarction. Ischemia caused significant impairment, as measured by the global rating scale; the probability of an abnormal rating was correlated with the number of microspheres trapped in the brains. Visual spatial learning was significantly impaired by ischemia, but this deficit was independent of the count of microspheres, whether the subject was abnormal at 48 h, and whether the left or right hemisphere was embolized. Cerebral hemisphere volume was reduced from 430 mm3 to 376 mm3 (P < 0.05). The cortex was reduced from 22 to 19% of cerebrum (P < 0.05) and the white matter compartment was reduced to similar degree. The lesion volume was 6% of cerebrum, comparable to that seen with other ischemia methods. The global outcome rating was significantly related to total cerebral volume, but not to volume changes in any single compartment. On the other hand, visual spatial learning was significantly influenced by volume changes in the cortex and white matter, but not by the topography of the visible infarctions. Open field activity was not altered by infarction. Our data suggests that the total volume of brain tissue lost to infarction may partially determine global neurological rating independently of the topography of the volume loss. Integrative functions such as learning may depend more on the integrity of specific compartments and less on the total volume of intact brain. The volume of visible cystic infarction was not related to long term behavioral outcome. These results should be confirmed using another method of inducing ischemia.

Protein kinase C (PKC) is an important intracellular regulator, and its activity may play a central role in the modulation of neuronal ischemic damage. Staurosporine and the compound H-7 are potent in vitro inhibitors of PKC, and 1,2-oleoylacetylglycerol (OAG) is an effective activator. We administered these compounds through a spinal subarachnoid catheter and demonstrated in vivo alteration of spinal cord PKC activity. We then tested the effects of altering PKC activity in a well-established rabbit model of reversible spinal cord ischemia. Animals within each experimental group were subjected to a range of spinal cord ischemic durations by temporary occlusion of the infrarenal abdominal aorta. Compared to control, both staurosporine and H-7 significantly shortened the duration of ischemia that the animals could tolerate, without developing permanent paraplegia. OAG resulted in an insignificant lengthening of the ischemic duration that the animals could withstand. The worsening of ischemic outcome by PKC inhibitors suggests that the enzyme is important for maintaining neurologic function under ischemic conditions, possibly secondary to modulation of intracellular calcium levels.

Calcium/calmodulin (Ca2+/CaM)-dependent protein phosphorylation was evaluated in a rabbit spinal cord ischemia model. One hour of ischemia reduced particulate (5% of control) and cytosolic (35% of control) Ca2+/CaM-dependent protein kinase activity significantly (p less than 0.01). In vitro phosphorylation of endogenous proteins by endogenous Ca2+/CaM-dependent protein kinase showed that phosphorylation of 14 particulate and 7 cytosolic proteins was stimulated in the presence of Ca2+/CaM in control tissue. However, after 1 hour of ischemia, Ca2+/CaM-dependent protein phosphorylation was virtually absent in the particulate fraction and significantly reduced in the cytosol. When equal amounts of control and ischemic tissue samples were combined and assayed, Ca2+/CaM-dependent protein kinase activity was 43% of control in particulate and 70% of control in cytosolic fractions. This suggests that reduced Ca2+/CaM-dependent protein phosphorylation is probably not due to the presence of an inhibitory activity in ischemic tissue. These results show that the Ca2+/CaM-dependent protein phosphorylation system is impaired after ischemia durations which cause irreversible damage. These altered phosphorylation reactions may play critical roles in mediating irreversible neurologic injury.

Therapeutic efficacy of calcium channel blockers in stroke remains controversial, but previously used agents bind almost exclusively to L-type calcium channels. The newly-discovered N-type calcium channel is specific to neurons, and therapy involving blockade of this site has not been previously attempted. We assessed the neuroprotective effect of omega-conotoxin GVIA (CgTx), a blocker of N-type calcium channels, using both in vitro hypoxic injury to rat cortical neurons and an in vivo model of reversible spinal cord ischemia in the rabbit. In cell cultures, CgTx inhibited hypoxia-induced 45Ca accumulation and neuronal injury minimally, compared to the NMDA antagonist ketamine. In vivo, the duration of spinal cord ischemia which produced permanent paraplegia in 50% of control animals (ET50) was 24.0 +/- 2.6 min. Animals treated 2 h prior to ischemia with 0.5 nmol CgTx in the subarachnoid space had an ET50 of 26.9 +/- 1.8 min (P = 0.36). Animals treated 24 h prior to ischemia (all had persistent systemic tremor) had a ET50 of 28.9 +/- 1.8 min (P = 0.13). We conclude that pharmacologic modulation of the N-type calcium channel does not provide a significant protective effect against neuronal hypoxic-ischemic injury.

Tyrosine hydroxylase and dopamine-beta-hydroxylase have been measured in 34 discrete areas and nuclei of the limbic system of the rat. Both enzymes showed an uneven distribution in this system. The ratio between tyrosine hydroxylase and dopamine-beta-hydroxylase activities showed a significant correlation when compared with the ratio of dopamine and norepinephrine concentrations for the areas studied. The results strongly suggest that dopaminergic terminals are present in discrete areas of the limbic cortex, and several septal and amygdaloid nuclei, and allow the precise localization of dopaminergic and noradrenergic areas in the limbic system.

Spinal cord blood flow can be accurately and rapidly measured in rabbits by intravenous injection of [14C]iodoantipyrine, collection of sequential arterial samples, and abrupt termination of blood flow to the spinal cord by severing the aorta with a previously implanted ligature. The radioactive tracer concentrations in arterial and spinal cord samples can be rapidly measured in a scintillation counter. The spinal cord blood flow can then be calculated from these blood and tissue tracer concentrations. This method is suitable for use in anesthetized or unanesthetized small animals.

Recently developed highly sensitive radiometric assays for biogenic amines have been combined with microdissection techniques to localize and quantify these amines in different structures, and at various levels of rabbit and rat spinal cords. Assays were performed for serotonin, norepinephrine, dopamine and epinephrine. It was generally found that the highest concentrations of those biogenic amines were found in the lateral and ventral horns, slightly lesser concentrations were found around the central canal, and still lower concentrations were found in the dorsal horns. Relatively low amine concentrations were found in the white matter. These biochemical measurements correlate well with localizations indicated by histofluorescence methods but provide precise quantification. Comparisons with previously available, less sensitive biochemical measurements are discussed.

We developed a small animal embolic stroke model for pharmacological screening trials. Microspheres are injected into the carotid circulations and group embolus dose-response relationships are calculated. Emboli quantity is related to neurologic injury, and small changes in neurologic function are detectable. Rabbits tolerated twice as many microspheres when cyproheptadine-treated after embolization. This demonstrated both the sensitivity of the model and the value of serotonin antagonists in reducing neurological injury.

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.

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.