Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil.

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.

A simple, inexpensive method of monitoring brain temperature in conscious rodents.

A method was developed to monitor brain temperature in conscious, unrestrained rodents. A commercially available thermocouple microprobe was modified so that it could be screwed firmly into a guide cannula that had been stereotactically implanted in the brain. Because the microprobe remains firmly in place, it is possible to record continuously for several hours from a single animal. Because the microprobe is easily removed, one can also record intermittently from several animals at once. The necessary equipment is relatively inexpensive and the modified microprobe can be sterilized and reused indefinitely. This method has proved especially useful for monitoring brain temperature during and after transient cerebral ischemia, an insult that destroys CNS neurons in a temperature-sensitive fashion.

Absence of electrographic seizures after transient forebrain ischemia in the Mongolian gerbil.

EEG was continuously recorded from Mongolian gerbils for 4 days after transient bilateral forebrain ischemia, to determine whether ischemic brain damage in this species is necessarily associated with seizures. Gerbils were chronically implanted with EEG recording electrodes in hippocampal area CA1, striatum and frontal neocortex and were subjected to a 5-10 min occlusion of both common carotid arteries. During the first few hours after the occlusion, the EEG was dominated by slow waves similar to those recorded from human brain after a damaging episode of cerebral ischemia. Amplitudes of the hippocampal and striatal EEG declined markedly with time, presumably as a result of neuronal degeneration. Ictal activity was never recorded, even from animals that suffered extreme damage to the hippocampal formation and striatum. Therefore ischemic brain damage in the gerbil does not result from seizure activity.

Effects of transient forebrain ischemia in area CA1 of the gerbil hippocampus: an in vitro study.

Selective delayed post-ischemic degeneration of CA1b neurons takes place in tissue slices in vitro as it does in brain in situ. Therefore neither selectivity nor the delay of the process can be explained by vascular factors. Changes of orthodromic evoked potentials precede morphologic signs of degeneration, but antidromic activation of neurons fails pari passu with histopathologic degeneration. The marked, transient, enhancement of excitatory synaptic potentials is compatible with the idea that increased release of excitatory amino acids contributes to neuron damage. The fact that degeneration proceeds in the absence of spontaneous activity or overt electrographic seizures indicates, however, that increased excitation cannot be the sole cause of the damage. Postsynaptic excitability of neurons decreases even while synaptic potentials are enhanced. The mechanism of decreased excitability is not clear, but its development could be interpreted as a compensatory change, counteracting enhanced excitatory transmission. We confirmed that it is possible to save neurons by drug treatment administered after the ischemic insult, and demonstrated that such protection is not due to an effect on blood vessels. These findings are relevant to the proposed clinical use of NMDA receptor antagonists to prevent ischemic brain damage (Meldrum, 1985; Rothman and Olney, 1986; Choi, 1988).

Potent stimulation of glycoprotein secretion in canine trachea by substance P.

The effects have been investigated of the regulatory peptides, substance P (SP) and bombesin, on the secretion of [14C]glucosamine-labeled trichloroacetic acid-phosphotungstic acid precipitable glycoproteins by canine tracheal explants. SP (10(10) to 10(-7) M) induced a dose-dependent increase in secretion of high-molecular-weight (greater than 2 X 10(6) radiolabeled glycoproteins predominantly from the submucosal glands. On a molar basis, SP [median effective concentration (EC50) = 8.2 X 10(-10) M] was about 1,000-fold more potent than methacholine (EC50 = 6.3 X 10(-7) M). Bombesin (10(-10) to 10(-4) M) had no effect on glycoprotein secretion. The time course of SP effect was characterized by an initial stimulation of glycoprotein secretion followed by a period of inhibition, suggesting that it rapidly exhausts a pool of glycoprotein, possibly that present within the duct lumen of the submucosal gland. Consistent with this are the findings that SP-induced secretion of glycoprotein was augmented by preincubation with methacholine while methacholine-induced secretion was diminished by preincubation with SP. Our findings show that SP is a potent stimulant of airway glycoprotein secretion in vitro and suggest that it acts by increasing the rate of clearance of mucus from the ducts of the submucosal gland, possibly by induced constriction of the secretory tubules and collecting duct. A role is discussed for SP in mucus hypersecretion induced by local axonal reflexes in the airway mucosa.

Postischemic synaptic excitation and N-methyl-D-aspartate receptor activation in gerbils.

Transient forebrain ischemia leads to the delayed degeneration of CA1b hippocampal pyramidal cells. In previous studies using the gerbil carotid occlusion model, we demonstrated that CA1b pyramidal cell degeneration is preceded by a period of enhanced excitatory transmission. Experiments with hippocampal slices prepared after 5 minutes of bilateral carotid artery occlusion show that ischemia enhances excitatory synaptic transmission and reduces pyramidal cell excitability before it abolishes synaptic function. In the present study, we tested the hypothesis that these effects require the activation of N-methyl-D-aspartate receptors during the postischemic period. Hippocampal slices were prepared 20-30 minutes after carotid occlusion, and Schaffer collateral-commissural input-output curves were constructed from recordings made every 30-60 minutes for 11-14 hours. Inclusion of the selective, reversible N-methyl-D-aspartate receptor antagonist 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid in the superfusion medium completely prevented the progressive loss of pyramidal cell excitability that normally follows this ischemic insult. This antagonist also prevented the postischemic increases in the duration and initial slope of the focally-recorded excitatory postsynaptic potential. The antagonist could still partially block the effects of transient forebrain ischemia when treatment was delayed for 4 hours. Our results confirm that the deleterious electrophysiologic changes in area CA1b depend on the continuing activation of N-methyl-D-aspartate receptors. Delayed ischemic neuronal death may result, in part, from excessive synaptic excitation during the postischemic period. However, other factors that are eliminated by preparing hippocampal slices appear to play an important role as well.

Postischemic synaptic physiology in area CA1 of the gerbil hippocampus studied in vitro.

After transient forebrain ischemia in the Mongolian gerbil, CA1b hippocampal pyramidal cells degenerate during a period of 2-4 d. We tested the hypothesis that this delayed neuronal death is preceded by excessive synaptic excitation. Hippocampal slices were prepared from gerbils that had been subjected to a 5 min occlusion of both common carotid arteries. Input/output curves demonstrated enhancement of the initial slope of the Schaffer collateral-commissural focally recorded EPSP at all stimulus currents between 5 and 10 hr after the ischemic insult. The duration of the focally recorded EPSP also increased. At the same time, the excitability of the CA1b pyramidal cells decreased. Thus, the EPSP brought fewer pyramidal cells to threshold than the same size EPSP in control slices. During the first 14 hr after ischemia, the antidromic population spike remained unaffected. By 24 hr after ischemia, however, the focally recorded EPSP and both orthodromic and antidromic population spikes were markedly depressed, and they declined further over the next 2 d. No recovery was detected. In the same slices, transient ischemia only mildly and reversibly affected the response of dentate granule cells to perforant path stimulation and did not affect their response to antidromic stimulation. Hippocampal slices adjacent to those used for electrophysiological recording were analyzed histologically. Examination of somatic argyrophilia confirmed that CA1b pyramidal cells suffered delayed neuronal death, whereas dentate granule cells remained intact. Pyramidal cell argyrophilia was, however, not detected until 2 d after these neurons had become virtually inexcitable. We conclude that CA1b pyramidal cells begin to lose electrophysiological function well before definite morphological signs of degeneration become visible. The observation of enhanced excitatory transmission 5-10 hr after reperfusion is consistent with the idea that delayed ischemic neuronal death results, at least in part, from excessive excitation.

Airway mucus: composition and regulation of its secretion by neuropeptides in vitro.

Human and canine airway mucosa in vitro synthesizes and secretes mucus glycoprotein, proteoglycans and lipids which can be separated by density gradient ultracentrifugation in caesium bromide. In secretions from unstimulated explants, the small amount of mucus glycoprotein present is found in association with proteoglycans. 'Free' mucus glycoprotein of typical buoyant density is present only after stimulation of submucosal gland secretion by methacholine. Lipids are synthesized, at least in part, by the airway mucosa and occur in explant secretions as a viscoelastic gel, suggesting that they significantly influence the rheological properties of airway mucus. In addition to cholinergic and adrenergic secretomotor neurons, the airway mucosa is innervated by peptidergic fibres containing immunoreactivity to vasoactive intestinal peptide (VIP) and substance P (SP). In explants of non-bronchitic human airway, VIP inhibits baseline glycoprotein and lysozyme secretion; in canine airway mucosa, by contrast, VIP is a weak partial secretory agonist. SP is the most potent agonist of canine airway glycoprotein release described to date and appears to evoke secretion by a direct action on a stereospecific SP receptor rather than by inducing release of other endogenous secretagogues. VIP and SP have little effect on glycoprotein discharge by mucous and serous cells of the submucosal gland; SP appears to induce secretion by causing contraction of submucosal gland ducts. This may represent the most rapid way for delivering mucus into the airway in response to injury or irritation of airway epithelium.

Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa.

The effects of leukotrienes C4 (LTC4) and D4 (LTD4) on the secretion by human bronchial mucosa of [14C]glucosamine-labeled, trichloro-acetic acid/phosphotungstic acid-precipitable glycoprotein and lysozyme were evaluated in vitro. LTC4 and LTD4, in the concentration range of 0.16 to 1600 nM, induced a dose-related increase in the release of radiolabeled glycoprotein, but not of lysozyme. This secretagogue effect was selective for high molecular weight glycoproteins of about 2-5 x 10(6) daltons, and the median effective concentrations (EC50) of LTC4 of 9.4 x 10(-9) M and of LTD4 of 2.44 x 10(-8) M, indicate that these leukotrienes are approximately 100-fold more potent than the cholinergic agonist methacholine. Incubation of [14C]glucosamine-labeled bronchial mucosal explants with LTC4 or LTD4 for six sequential 15-min periods revealed a rapid, progressive decrement in glycoprotein release, compatible with stimulatory action on secretion rather than augmentation of the rate of glycoprotein synthesis. This interpretation is also consistent with the finding that the specific activity (ratio of bound radiolabel: protein content) of the macromolecular glycoprotein secreted by the explants is not changed with stimulation of release by the leukotrienes. Based upon the activity of synthetic leukotriene analogs, the specific C-6 chirality of the sulfidopeptide of LTD4, the presence of a hydroxyl at C-5 and the presence of eicosanoid carbons 9-20 were of no importance for secretagogue activity. These findings contrast with the stereochemical requirements for the spasmogenic response to sulfidopeptide leukotrienes and suggest that leukotriene-induced secretion is not likely to be mediated via a specific receptor.