Biomedical subjects
R L Isaacson
Publications and source records attributed to R L Isaacson.
Cellular alterations produced by the experimental increase in intracellular calcium and the nature of protective effects from pretreatment with nimodipine.
The immortalized septal cell line, SN56 B5 G4, generated by the fusion of mouse septal area cells and neuroblastoma cells, was used to determine if nimodipine, an antagonist of voltage sensitive calcium 'L' channels, might act in a neuroprotective fashion when intracellular calcium levels were raised by incubation in ouabain and monensin. Fluorescent indicator dyes and the automated spectrofluorometer, the CytoFluor 2300, were used to analyze specific cellular targets and functions affected by ouabain and monensin and possible protection by prior incubation with nimodipine. Ouabain and monensin were used together to create a time- and dose-dependent toxic episode. Increases in the emission intensity of Fluo3-AM demonstrated that the concentration of intracellular calcium was monotonically increased by increasing levels of ouabain-monensin. The calcein-AM fluorescent probe indicated that there were no changes in plasma membrane permeability during the toxic episode. Lysosomal integrity decreased as indicated by decreases in neutral red retention. The concentration of free radicals increased as shown by the increase in emission intensity of 2',7'-dichlorfluorescein. Nimodipine pretreatment of the cells incubated with ouabain and monensin resulted in apparent protection of lysosomes and a reduction in the level of free radicals. While nimodipine, by itself, produced a small decrease in intracellular calcium, it actually augmented the ouabain-monensin induced increase in intracellular calcium. The data suggest that in immortalized septal cells, (a) nimodipine offers protection to certain of the responses induced by ouabain-monensin, (b) the protection offered by nimodipine may be independent of antagonism of voltage sensitive calcium channels, and (c) that the protective changes can occur at the same time that intracellular calcium is increasing. These latter observations question the hypothesis that the protection against cell death and dysfunction offered by nimodipine is due solely to maintaining calcium homeostasis.
Behavioral and anatomical consequences of unilateral fornix lesions and the administration of nimodipine.
Male Wistar rats subjected to unilateral fimbria-fornix transection by mechanical knife cut or to sham operations were tested in a water maze and in an open field. Half the animals in each group were treated with either 0.06 mg/kg nimodipine or vehicle, administered i.p. for 7 days, beginning the day of surgery. Animals were sacrificed and brains were processed for acetylcholine esterase (AChE) histochemistry. In the water maze, lesioned rats showed a significant impairment relative to the sham-operated animals. Nimodipine treatment did not improve performance. There were no differences among the groups in the observed frequencies of the open field behaviors of locomotion, hole-poke, rearing and grooming. A significant reduction of AChE-positive cell bodies was found in the medial septal region on the side of the lesion. There were no differences in water maze performance among groups of rats treated with 0.0, 0.5, 1.0, or 5.0 mg/kg nimodipine for 7 days, beginning the day of fimbria-fornix transection, in an attempt to determine any dose-dependent effect of the drug.
Actions and interactions of calcium modulators and ethanol on rat atrial membrane potentials.
The effects of ethanol and dihydropyridines on atrial membrane potentials (MP) were studied. Rat atrial strips superfused with Tyrode's solution (36 degrees C) were driven at 5 Hz while recording MP with intracellular microelectrodes. Bay K 8644 7 X 10(-7) M (BAYK) increased the amplitude of the action potential (AAP) without affecting the resting membrane potential (RMP) or the Vmax of phase 0 (Vmax 0). The velocity of change in voltage decreased at the beginning of the repolarization, causing an increase in the action potential duration (APD), but it was not modified at negative voltages. Nimodipine 4.2 X 10(-6) M reduced the AAP without affecting RMP or Vmax 0. The velocity of change in voltage increased at the beginning of the repolarization, causing a decrease in the APD, but was not modified at negative voltages. Ethanol 5.3 X 10(-2) M exerted actions similar to those of nimodipine. Simultaneous exposure to ethanol and nimodipine resulted in changes not different from those obtained with each of the two compounds. The MP remained unchanged when the preparations were exposed to ethanol and BAYK simultaneously. In summary, ethanol and nimodipine exerted similar actions on the atrial MP while BAYK had opposite actions. The effects of ethanol and BAYK cancelled each other.
Inotropic effects of ethanol and dihydropyridines on the guinea pig heart atrial muscle.
The effects of ethanol and/or dihydropyridines (DHPs) on force of contraction of atrial muscle were studied. Guinea pig atrial strips superfused with Tyrode's solution (36 degrees C) were driven (1.5 Hz) while recording muscle tension. Bay K 8644 (BAYK) increased, while nimodipine or ethanol reduced, the peak tension developed and the maximum velocity of development of tension. The effects of ethanol were readily reversible, but those of the DHPs were not. The combined actions of ethanol and DHPs were the result of the synergism or antagonism of the drugs tested. The shorter duration of the action of ethanol resulted in the effect of DHPs being still evident well after the exposure to the drugs ended. In summary, ethanol and nimodipine exerted depressant actions on atrial contractile force, while BAYK had opposite effects. The different mechanisms of action may explain the different duration of the effects of ethanol (physical agent) and DHPs (receptor-binding chemicals).
Pretreatment effects on nitrite-induced methemoglobinemia: saline and calcium channel antagonists.
This study was undertaken to evaluate the effects of pretreatment with calcium channel antagonists (nimodipine or verapamil) on the formation of methemoglobin produced by sodium nitrite. Unexpectedly, the pretreatment of animals with control injections of physiological saline 2 hours before the nitrite administration reduced the amount of methemoglobin found in the blood 25 minutes later. When either of the calcium channel antagonists was given 2 hours before the administration of sodium nitrite, the saline effect was eliminated. When the injections of physiological saline or either of the calcium channel blockers were divided 24 hours before the nitrite administration, all reduced the amount of methemoglobin formed relative to rats that received no pretreatment. A tentative hypothesis is that the reduction of the nitrite-induced methemoglobin can be induced by the stress of handling and intraperitoneal injection and that this stress effect can last at least 24 hours. It is likely that whatever stress-related mechanism is involved in reducing methemoglobin levels, this effect can be reduced by the presence of "L channel" voltage-sensitive calcium antagonists or their active metabolites.
The residual spatial abilities of hippocampally lesioned rats can be enhanced by peripheral sympathetic-adrenal interventions.
In order to test the possible effectiveness of peripheral interventions with the adrenergic system for the alleviation of certain disorders that typically follow bilateral hippocampal lesions, rats with bilateral lesions of the hippocampus, the overlying neocortex, or sham operations were tested at two postoperative times in the Morris water maze, a frequently used "spatial task." Half of the animals in all groups were exposed to the adrenergic manipulations, i.e., a chronic, 7-day, systemic bretylium regime (5 mg/kg) and, in addition, a peripheral injection of norepinephrine (4 micrograms/kg) 30 min before the start of each training day. The other half received saline chronically and a single saline injection before each training day. Five days of training were given at each of the two training periods. The first began 7 days after surgery while the second began 33 days after surgery. As expected, the hippocampally lesioned animals were severely impaired in the task. The adrenergic treatment produced enhanced performances in the rats with hippocampal lesions at both training sessions, although the improvement was greatest at the later period. Although the animals receiving the pharmacologic treatment located the general area of the hidden platform better than the saline-treated animals with hippocampal lesions, the treated animals were still impaired, swimming directly to the hidden platform on fewer trials than did animals in the other groups.
Nimodipine accelerates recovery from the hyperemotionality produced by septal lesions.
Rats with large electrolytic lesions of the septal area were given the calcium channel antagonist nimodipine (70 micrograms/kg, ip) or its vehicle on the day of surgery and for 3 subsequent days. They were tested for emotionality and compared to control rats for 10 days beginning on the third day after surgery. Forty days after surgery all animals were trained in a two-way active avoidance task for 120 trials. Nimodipine reduced the hyperemotionality found after septal lesions as early as the first test day, and increased the rate of recovery toward control levels on subsequent days. Both septal-lesion groups exhibited more avoidance responses than controls, even though the intertrial crossings were enhanced only in rats with septal lesions that did not receive nimodipine.
Brain plasticity after damage.
This is a brief summary of our research on the prevention or reduction of the effects of brain damage. I believe that the results contain a number of exciting promises. At this time, the greatest hope for effective remedial interventions comes from the choline and metyrapone studies. However, I am also reminded of the components a psychiatrist who worked with children in the heart of Philadelphia made some 20 years ago at a conference on mental retardation. He said that the most significant contribution to the elimination of mental retardation in his city would be the elimination of lead-based paints from the walls of ghetto apartments, an intervention second only in effectiveness to the elimination of the ghettos themselves. The importance of early interventions with individuals suffering brain damage, either general or specific, is clearly recognized. In research now well underway, Yong Shen, William Smotherman, and I have found encouraging results indicating that both short- and long-term effects of hypoxia induced by clamping the rat's umbilical cord can be attenuated by the prior administration of a calcium L channel antagonist. If those results are upheld in further studies, the principles of prevention of damage described in this article for the adult may prove to be even more potent in the young.
Brief ganglioside treatment produces delayed enhancement of functional recovery after medial septal lesions.
The effects of a 5-day ganglioside (GM1) treatment (30 mg/kg) on body weight and water intake subsequent to medial septal lesions were evaluated for 44 consecutive days. In addition, activity, rearing, and repetitive motor acts were measured on postsurgery days 5, 10, 40, and 60. The rate of increase in the body weights of rats with medial septal lesions treated with GM1 was equivalent to that of controls, while untreated rats with such lesions had reduced body weights. Rats with medial septal lesions treated with GM1 also exhibited movement times and frequency of repetitious motor acts similar to those of control rats by postsurgery day 60. No differences were found in water intake between any of the groups. Rats with medial septal lesions, whether treated with GM1 or not, had equivalent frequencies of rearings that were lower than control rats. This study emphasizes that even brief regimes of GM1 administration can exert behavioral changes in brain-damaged rats well after the treatment was administered, i.e. 40-60 days after surgery.
Actions and interactions of dihydropyridines and ethanol on the rat sinus node.
The actions and interactions of dihydropyridines (DHPs) and ethanol on sinus node (SN) automaticity were studied using rat sinoatrial preparations superfused with Tyrode's solution at 37 degrees C. Intracellular microelectrodes were used to monitor SN rate (SNR). The automaticity of the SN was not affected by nimodipine 10(-10) M, but it was depressed by a higher concentration of this calcium antagonist (10(-8) M). The racemic compound (+/-)Bay K 8644 had a dose-dependent biphasic chronotropic action: At 10(-8) M it decreased the SNR, while at 10(-7) M it increased the SNR. The negative component was blocked by atropine. A small concentration of ethanol (1.8 x 10(-2) M) had a positive chronotropic effect on the SN, and this action was blocked by nimodipine 10(-10) M. This small concentration of ethanol did not modify the chronotropic effects of either of the DHPs. A higher concentration of ethanol (5.3 x 10(-2) M) did not by itself affect the SNR, but it antagonized the chronotropic actions of the two DHPs.
Posttraining or pretest administration of nimodipine fails to affect retention of a simple learned association.
In previous experiments, the antagonist of voltage-sensitive calcium channels, nimodipine, given before training, enhanced the retention of an acquired preference for white-left relative to black-right in a two compartment apparatus. In the present experiments we attempted to determine if the retention effects produced by the drug could be attributed to effects occurring in the posttraining period. Various groups of rats received posttraining injections of nimodipine or its vehicle and two hours prior to testing received a second injection of nimodipine or its vehicle or no injection at all. All rats were tested for retention of the association twice, both 24 and 48 hr after acquisition. No posttraining or pretesting drug effects were found. This would support the view that the enhanced retention found after treatment with nimodipine found by ourselves and by others is due to a change in the animals at the time of acquisition.
The effects of unilateral destruction of fimbria-fornix and supracallosal pathways in the rat.
Rats with unilateral lesions of either the supracallosal regions (including the dorsal cingulate cortex) and the fimbria-fornix either on the same (S) or the opposite (O) sides of the brain were studied in a 16-hole open field without pharmacologic intervention and, subsequently, after 0.1 and after 1.0 mg/kg scopolamine HBr. Their performances were compared with those of unoperated control animals subjected to the same testing regime. Certain of their behaviors were compared with those of a larger number of animals with bilateral hippocampal destruction (and their control groups) from prior studies. Unilateral lesions of fimbria-fornix and supracallosal afferents to the hippocampal formation produced a decrease in hole poking activity relative to control animals. A further decrease in hole-poking behavior, coupled with increased locomotion, was observed in rats with fimbria-fornix and cingulate cortex lesions on opposite sides of the brain (group O). The smaller dose of scopolamine accentuated these effects. Indeed, the behavior of group O after scopolamine treatment was similar to animals with large bilateral hippocampal lesions. The large dose of scopolamine induced stereotyped rearing or hole poking in the brain-damaged animals but not in the control group. These findings suggest that both the fimbria-fornix and the supracallosal pathway is necessary for normal hippocampal function and that the behavioral deficit is greater when these structures are damaged on the opposite sides of the brain.
Spontaneous bodily rotations and direction of locomotion at different times after radio frequency lesions at sites in and near the substantia nigra.
Rats were prepared with radio frequency lesions of the dorsolateral or ventromedial regions of the substantia nigra. Other rats were prepared as operated and unoperated controls for each type of lesion. Their behavior was evaluated in an open field at postoperative days 2, 7, 10, and 15. Three types of behavioral changes were observed over time: those noticeable for a brief period, i.e., a few days, after the lesion (rotational behavior), those lasting 7-10 days after the lesion (turning preferences) and those lasting through the end of the experiment that may be permanent (enhanced locomotion). The early effect of the medioventral lesions was pronounced contralateral rotation while the early effect of the dorsolateral lesion was ipsilateral rotation. This effect of the dorsal lateral lesions was reversed on test days 7 and 10. Lesion-induced turning changes associated with forward locomotion were observed on these two test days as well. By 15 days after surgery the only demonstrable effect of either lesion was enhanced locomotion. The results are discussed in terms of various theories of substantia nigra regulation of motor activities.
The effect of a calcium antagonist on the retention of simple associational learning.
Using a newly developed training paradigm, rats were trained to associate a spatial location and a black interior with mild footshock and another adjacent location with white interior with the absence of footshock in three independent experiments. Retention of these associations was measured 24 and 48 hr after training in situation in which the animals could move freely between the black and white locations over a 90 sec test. Other rats were subjected to a control procedure in which shock was received on both the black and the white sides of the apparatus. In each of the experiments, half of the animals in the experimental and the control groups were trained following the administration of the calcium slow channel blocking agent, nimodipine (5 mg/kg), and the other half after saline administration. In experiment 1, the injections were given 15 min before training. In experiment 2, the injections were given chronically, over a 6 day period before training. In experiment 3, the animals were given a single injection 7 days before training. In all cases, retention was examined both 24 and 48 hr after training. The results were that the experimental procedures produce a strong aversion to the black portion of the apparatus. The greatest amount of retention was found in animals that had received the chronic injections, whether they were of saline or of nimodipine. In every aspect of retention in which the saline-treated rats were less than perfect in retention, the nimodipine animals exhibited superior performance.
Changes in the distribution of the neuron-specific B-50, neurofilament protein and glial fibrillary acidic proteins following an unilateral mesencephalic lesion in the rat.
Following a unilateral electrolytic lesion in the ventral rat mesencephalon, changes in the immunocytochemical distribution of the neuron-specific B-50, neurofilament (NF) protein and glial fibrillary acidic (GFAP) proteins were studied around the lesion after 0, 3, 10 and 28 days. At all recovery times, the controls displayed on immunostaining with anti-B-50 and anti-neurofilament antibodies, a characteristic pattern of synaptic and neuritic localization of these antigens, whereas anti-GFAP staining revealed a distribution typical for astrocytes. The lesion was characterized by a center of coagulated material that exhibited immunoreactivity to B-50 (BIR) and NF (NFIR), but never GFAP-immunoreactivity. From 3 days on, the center became surrounded by disintegrating cells which were unreactive to the antibodies. The antigen distribution changed temporally, predominantly at the lesion rim. By 10 and 28 days postlesion, additional BIR was observed as punctuate dots in fibers and membranes of neurons. Enhanced NFIR was detected in fibers and cell bodies. Many astrocytes were detected around the lesion rim, forming by 28 days postsurgery a barrier between the lesion cavity and the uninjured tissue. Our study shows that distribution changes in B-50, NF and GFAP around the lesion may indicate local degenerative and adaptative processes as a temporal response to brain trauma.
Excessive grooming induced by the administration of codeine and morphine.
Previous studies have shown that peripheral injections of the codeinone RX 336-M can induce excessive grooming in rats in an age-dependent fashion. The present experiment demonstrates that codeine, itself, also induces excessive grooming but with apparent equal effectiveness at all ages tested. Because of the structural similarity between codeine and morphine, the effects of intraperitoneally administered morphine were examined as well. Morphine was found to produce a temporal course of excessive grooming quite different from that produced by codeine. Morphine did not affect grooming in the first half (30 min) of the observation period, but accentuated it, briefly, for the next 30-45 min. Intraventricular administration of codeine at doses of 0.3 or 1.0 microgram/3 microliter had no effect on grooming in animals previously shown to demonstrate excessive grooming in response to either dose.
Excessive grooming in response to opiate drugs. The ontogeny of responsiveness.
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