Magnetic resonance imaging of propagating waves of spreading depression in the anaesthetised rat.

Gradient echo magnetic resonance (MR) imaging was used to demonstrate propagating waves of cortical spreading depression (SD) in the anaesthetised rat. SD was initiated by remote perfusion with 150 mM KCl applied for 0.5-2 min to the left parietal cortex. Gradient echo MR images were obtained every 12-30 s in either a vertical coronal section or a horizontal section including the superficial cortex in plan view. Within 2 min of application of KCl, we observed a zone of increased signal intensity (3-15%) on the MR image, up to 2 mm across, lasting approximately 1 min and propagating away from the site of initiation. The mean velocity for 27 of such waves seen in seven animals was calculated to be 2.79 mm/min, with means (+/- SD) in individual animals averaging 2.90 +/- 0.46 mm/min (n = 7). Increased signal intensity in gradient echo images has been attributed to an increased level of oxygenation within the venous blood. Our results are consistent with this interpretation although other physiological changes during SD may also contribute to the signal changes.

Compartmental analysis of diprenorphine binding to opiate receptors in the rat in vivo and its comparison with equilibrium data in vitro.

The regional binding of the opiate receptor ligand diprenorphine has been examined in rat brain both in vivo and in vitro. The time course of total label in specific brain regions was followed up to 2 h after intravenous bolus injection of [3H]diprenorphine, with or without a pulse chase of unlabelled diprenorphine at 30 min. In addition, total label was measured 30 min after injection of labelled diprenorphine at nontracer concentrations over a range of specific activities. Total data sets for each region were fitted simultaneously to a compartmental model to give estimates of maximal binding capacity (Bmax), the second-order apparent association rate constant, and the first-order dissociation rate constant of the receptor-ligand complex. The model incorporated the use of a reference region with low specific binding (cerebellum). The binding of diprenorphine to rat brain homogenates was measured in vitro under equilibrium conditions at 37 degrees C, pH 7.4, in the presence and absence of naloxone, to give corresponding regional estimates of Bmax and the half-saturation constant Kd. The results showed a close correlation between in vitro and in vivo regional estimates of Bmax over a wide range. There were no significant interregional differences either in Kd in vitro or in the Kd derived from the in vivo analysis, although in vitro and in vivo estimates differed by an order of magnitude. This work was carried out as part of a validation study with a view to the application of the compartmental model to data obtained in vivo in humans using positron emission tomography, when successive studies over a range of specific activities are not feasible.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered turnover and synthesis rates of lung surfactant following thoracic irradiation.

Between 2-6 weeks after thoracic irradiation with 10 Gy X rays, when levels of surfactant in the alveoli show the greatest increase, there is a reduction in the rate of radioactivity loss from 3H-choline labeled disaturated phosphatidylcholine from the lung. This indicates a reduced turnover of surfactant. Discrepancies between the halving times for specific activity and for total radioactivity of the disaturated phospholipids suggest that at between 2 and 3 weeks post-irradiation, removal and degradation of surfactant almost ceases, but that synthesis continues normally. However, by 3 weeks post-irradiation, choline-3H incorporation into disaturated phosphatidylcholine suggests that surfactant synthesis is increased about two-fold. The reduced number of macrophages recovered from alveolar lavage between about 2 and 6 weeks post-irradiation may indicate a reason for the lengthened turnover times of surfactant over this period. Nevertheless the stimulated surfactant production that takes place from about 3 weeks onward suggests an additional active response to radiation or to radiation damage by the type II pneumonocytes. These studies confirm that the maximum levels of alveolar surfactant seen at 3 weeks post-irradiation result from a different lung response than that responsible for the increase in surfactant, which occurs within hours of irradiation.

The induction of thermotolerance in the ear of the mouse by fractionated hyperthermia.

The development of thermotolerance in ears of mice was investigated after fractionated hyperthermia. Ears were heated at 43.5 degrees C by immersion in a water bath and the response was measured in terms of the heating time required to cause thermal necrosis in 50% of the ears (NT50). Three types of treatment were given: (1) single treatments, for which the NT50 was 42 minutes; (2) priming treatments, which caused little visible effect but induced thermotolerance. These treatments were given as 1-10 daily fractions, the total heating time ranging from 20-630 minutes; (3) test treatments which were given at various times after priming and were varied to estimate the NT50. Thermotolerance was defined as an increase in the test NT50 for preheated ears relative to the single treatment NT50. It has been suggested that thermotolerance induced by a single priming treatment may be increased by giving additional heat treatments which would not be tolerated by normal cells. In the mouse ear, the maximum thermotolerance induced by a single priming treatment of 20 min at 43.5 degrees C was seen after 24 hr when the test NT50 was about 2.5 times the single NT50. The effect of giving up to nine additional daily treatments of 70 min, each of which would cause necrosis in ears that had not received prior hyperthermia, was measured. The maximum thermotolerance observed was equal to that after a single 20 minute priming treatment but thermotolerance decreased as the number of 70 min treatments was increased from four to nine. The effects of repeating a treatment (20 min or 5 min) which was tolerated by normal ears and induced maximal or less than maximal resistance were compared. The interval between each fraction (24 hr or 12 hr respectively) was equal to the time at which maximal thermotolerance was observed after one treatment. For each regimen, the degree of resistance seen after 2 to 10 exposures was similar to that after the appropriate single treatment. This resistance was maintained throughout the course of priming treatment and decayed after the last fraction. Thus for this regimen, thermotolerance depended on the duration of each treatment rather than on the number of treatments given.

Increased extracellular dopamine in the nucleus accumbens of the rat during associative learning of neutral stimuli.

Brain microdialysis was used to study changes in dopamine in the nucleus accumbens and the dorsal striatum during associative learning between two neutral stimuli, flashing light and tone, presented on a paired schedule during stage 1 of a sensory preconditioning paradigm. The tone was subsequently paired with mild footshock using standard aversive conditioning procedures and the formation of a conditioned association between the flashing light and the tone in stage 1 was assessed by measuring the ability of the flashing light to elicit the same conditioned response as the tone when presented at test. The first experiment used behavioural monitoring only, to establish stimulus parameters for subsequent microdialysis experiments. Animals receiving paired presentation of the light and tone in stage 1 showed a conditioned suppression of licking to the light as well as to the tone, indicating that associative learning between the flashing light and the tone had occurred during stage 1, whilst in a separate group of animals given the same stimuli over the same time period but on an explicitly non-paired schedule, the conditioned emotional response was seen to the tone, but not to the light, showing that no association had been formed between the two stimuli during stage 1. In dialysis experiments using the same procedure, we measured a two-fold rise in dopamine in the nucleus accumbens during paired presentation of flashing light and tone, but not during non-paired presentation of the two stimuli. On subsequent test presentation of the two stimuli, we saw increases in accumbal dopamine on presentation of the tone in both groups, reflecting the formation of an association with the footshock in both. However the flashing light elicited an increase in dopamine only in the group which had received paired presentation at stage 1. Thus accumbal dopamine release at test is correlated to the ability of the stimulus to evoke a conditioned response measured behaviourally. Hypotheses of the behavioural function of the mesolimbic dopamine system centre on its role in mediating the effects of biological reinforcers, both rewarding and aversive, conditioned and unconditioned. The present results, showing increases in extracellular dopamine in the nucleus accumbens when an association is formed between two stimuli of which neither is a biological reinforcer nor, prior to formation of the association, affects dopamine levels, suggest a role for accumbal dopamine in the modulation of associative learning in general, not only that involving reinforcement.

Responses of mouse lung to irradiation. 2. Levels of alveolar protein in lung lavage fluid following neutrons or X-rays.

Alveolar protein exudate is shown to exhibit a biphasic increase after irradiation. During the first 6 weeks after doses of between 5 and 28 Gy X-rays or 3.5 and 14 Gy neutrons, mean alveolar protein levels increase to about 4 times control levels. Over this period there was no suggestion of any dose-response nor of an RBE greater than 1. It seems probable that this response is unrelated to cell killing by radiation. After sublethal doses there was recovery from this first phase of leakage. However, although after 5 Gy X-rays this recovery was complete, after higher doses a second and greater increase in alveolar protein took place. After 10 Gy X-rays and 8 Gy neutrons this peaked at about 15 weeks post-irradiation and 20-30 times control levels. This second dose-dependent response corresponded with the times of animal death after doses close to the LD50. A comparison of these changes in alveolar protein exudate with previously published data on surfactant level suggests that the surfactant and protein responses are largely unrelated.

Responses of mouse lung to irradiation. 1. Alterations in alveolar surfactant after neutrons and X-rays.

Surfactant levels in the alveolus after X-irradiation are shown to be increased over a period of about 6-8 weeks. Although the increase in phospholipid in alveolar lavage commences shortly after irradiation, peak levels occur about 3 weeks post-irradiation. The time course of the response was independent of radiation dose and quality but its magnitude was dose related with X-rays and neutrons being comparable in effectiveness (i.e. RBE approximately 1). Amounts of alveolar surfactant subsequently return to about control level and, during the period of radiation pneumonitis some 3-5 months post-irradiation, there is no indication of a further response by the surfactant system. Analysis of the amount of disaturated lipids in both lung tissue and alveolar lavage fluid suggests that increased surfactant levels are at least partly due to reduced turnover. Post-radiation changes in alveolar lavage phospholipid composition were not detected.

Vascular and epithelial damage in the lung of the mouse after X rays or neutrons.

The response of the lung was studied in CFLP mice after exposure of the whole thorax to X rays (250 kVp) or cyclotron neutrons (16 MeV deuterons on Be, mean energy 7.5 MeV). To measure blood volume and leakage of plasma proteins, 51Cr-labeled red blood cells and 125I-albumin were injected intravenously and 24 h later lungs were lavaged via the trachea. Radioactivities in lung tissue and lavage fluid were determined to estimate the accumulation of albumin in the interstitial and alveolar spaces indicating damage to blood vessels and alveolar epithelium respectively. Function of type II pneumonocytes was assessed by the amounts of surfactant (assayed as lipid phosphorous) released into the lavage fluid. During the first 6 weeks, lavage protein and surfactant were increased, the neutron relative biological effectiveness (RBE) being unity. During pneumonitis at 12-24 weeks, surfactant levels were normal, blood volume was decreased, and both interstitial and alveolar albumin were increased. Albumin levels then decreased. At late times after exposure (42-64 weeks) alveolar albumin returned to normal but interstitial albumin was still slightly elevated. Values of RBE for changes in blood volume and interstitial and alveolar albumin at 15 weeks and for changes in blood volume and interstitial albumin at 46 weeks were 1.4, comparable with that for animal survival at 180 days. The results indicate that surfactant production is not critical for animal survival. They suggest that changes in blood vessels and alveolar epithelium occur during acute pneumonitis; epithelial repair follows but some vascular damage may persist. The time course of the changes in albumin levels did not correlate with increases in collagen biosynthesis which have been observed as early as 1 month after exposure and persist for up to 1 year. Furthermore, a dose which had no effect on leakage caused a marked increase in collagen biosynthesis. Thus the present results do not support a causal relationship between exudation of vascular protein during pneumonitis and the later development of fibrosis.

Observations on the cellular effects of ethanol and hyperthermia in vivo.

The presence of ethanol in mice not previously exposed to the drug potentiates the damage caused by hyperthermia to the skin of the ear. Similarly, when thermotolerance is induced in the ear by a prior heat treatment, ethanol given before the second heat treatment reduces the amount of thermotolerance that is expressed. Conversely, mice that have developed ethanol tolerance after exposure to ethanol vapor for 7 days display resistance to hyperthermia-induced necrosis. It is possible that there may be similarities in the direct cellular effects of heat and ethanol, and that the development of tolerance to ethanol and hyperthermia may be by a similar mechanism.

In vivo labelling of the NMDA receptor channel complex by [3H]MK-801.

An in vivo radioligand binding assay for the N-methyl-D-aspartate (NMDA) receptor channel complex in the mouse brain has been developed using the non-competitive NMDA receptor antagonist [3H]MK-801. In vivo binding of [3H]MK-801 was displaced by MK-801 (ED50 = 0.17 mg/kg i.p.), (-)-MK-801 (1.0 mg/kg), thienylcyclohexylpiperidine (1.8 mg/kg), etoxadrol (5.1 mg/kg) and (+)-SKF 10,047 (34.5 mg/kg). The potency of these drugs in this in vivo binding assay was highly correlated (r = 0.97) with their functional effects as antagonists of N-methyl-DL-aspartate-induced tonic convulsions.

Evaluation of [O-methyl-3H]WAY-100635 as an in vivo radioligand for 5-HT1A receptors in rat brain.

N-(2-(4-(2-Methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridyl)cyclohexanecarboxamide trihydrochloride (WAY-100635) is a new, potent and selective 5-HT1A receptor antagonist. We have evaluated radiolabelled WAY-100635 as a prospective radioligand for positron emission tomography (PET) by studying biodistribution in rat ex vivo. After intravenous injection, [O-methyl-3H]WAY-100635 cleared rapidly from plasma but was retained in brain. Specific binding was quantified from kinetic studies, using a reference-tissue compartment model, fitting for binding potential (k3/k4). The regional variation in binding potential correlated with the known distribution of 5-HT1A receptors. Saturation studies gave Bmax values in vivo that were consistent with those reported in vitro. At 60 min after injection, the ratio of radioactivity in 5-HT1A receptor-rich regions (e.g. septum, entorhinal cortex and hippocampus) to that in cerebellum reached approximately 16. Pre-dosing the rats with WAY-100635 (2 mg/kg) reduced this ratio to one, whereas similar pre-dosing with citalopram (5-HT uptake site inhibitor), prazosin (alpha 1A-adrenoceptor antagonist) or idazoxan (alpha 2-adrenoceptor antagonist) caused little or no reduction. Substantial (77%) blockade of [3H]WAY-100635 binding was achieved with the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and the partial agonists, ipsapirone and buspirone. Thus, the properties of WAY-100635 are such that, when labelled with carbon-11, it could provide a radioligand suitable for clinical and pharmacological investigations of central 5-HT1A receptors in man using PET.

Long-term thermal sensitivity of previously irradiated skin.

The response of the mouse ear to hyperthermia was investigated at 7-64 weeks after irradiation with X rays. Thermal sensitivity was increased by 12 weeks after single doses of 18 to 20 Gy but showed no further changes up to 64 weeks after exposure. It is suggested that the increase in sensitivity to retreatment by hyperthermia several months after the initial course of radiation may be related to the turnover time of the tissue. Although there are reports which suggest that prior irradiation, given more than two months earlier, does not affect the response of human skin or superficial tumours to the mild hyperthermic treatment in current clinical use, more aggressive heat therapy may produce unexpectedly severe responses in previously irradiated sites.

A long-term effect of X rays on thermal sensitivity of the mouse ear.

The response of the mouse ear to hyperthermia was investigated after single doses of X rays. A dose of 15 Gy, which alone caused no visible effect, increased thermal sensitivity at 1-2 weeks after irradiation. There was a slight recovery at 4-6 weeks but thermal sensitivity was still increased at 6 months. When heat treatment was given at 1 week or 24 weeks after X-ray doses ranging from 9-18 Gy, it was found that thermal sensitivity increased as the dose was increased above 10 Gy. Although there are reports which indicate that prior radiotherapy given more than two months earlier does not affect normal tissue response to the mild hyperthermia currently being used to treat patients, more aggressive heat therapy may produce unexpected responses at previously irradiated sites.

Thermal enhancement of radiation damage in previously irradiated skin.

The responses of the mouse ear to hyperthermia alone, X rays alone or X rays combined with heat were investigated at various times up to one year after prior irradiation. The initial X-ray treatments, either a single dose of 17 Gy or 34 Gy given as 10 fractions in 11 days, caused the same skin reactions (a small area of moist desquamation in a few ears) and had the same effects on the responses to subsequent treatments. The response to heat alone was increased at 3-12 months after prior irradiation, so that the heating time at 43.5 degrees C required to cause necrosis was about 70% of that in age-matched controls. The responses to X rays alone were also increased. Prior irradiation, however, had different effects on the susceptibilities to develop acute radiodermatitis and late deformity. For acute radiodermatitis, the X-ray dose required to obtain a given response in previously irradiated ears was about 90% of the dose required in age-matched controls. For deformity at 6 months, the dose was about 70%. Prior irradiation had the same effects on the responses to X rays given 6 min before mild hyperthermia (43.5 degrees C, 15 min) as on those to X rays alone. As a consequence, the thermal enhancement ratio (TER), i.e., the dose of X rays required alone to cause an effect divided by that required with heat, did not depend on previous irradiation. The TER was 1.4 for both acute radiodermatitis and late deformity.

The effect of prior heat treatment on the thermal enhancement of radiation damage in the mouse ear.

The effects of prior heat treatment on the skin reaction produced by a subsequent treatment with combined heat and X-rays were investigated in the mouse ear. Ears were heated by immersion in hot water. The priming heat treatment was always 43.5 degrees C for 40 minutes. Its effect was transient, beginning between 24 and 48 hours after the priming treatment and reaching a maximum at 48 to 96 hours when there was a reduction in the skin response to combined heat and X rays, i.e. it caused a reduction in the thermal enhancement ratio (TER). The effect was lost by 192 hours. At 96 hours after the priming treatment the TER for 30 minutes at 42.5 degrees C or at 43.5 degrees C was reduced by a value equivalent to decreasing the temperature by about 0.4 degrees C. This was equivalent to increasing the heating at 43.5 degrees C required to produce a given enhancement of radiation damage by a factor of 1.4 relative to that required without prior heating. The effect was smaller than induced resistance to damage caused by severe heat treatment alone (i.e. necrosis) and it occurred later. These differences support the concept that two separate mechanisms underlie direct heat necrosis and thermal enhancement of radiation damage.

A long-term effect of prior irradiation on the thermal enhancement of radiation damage in the mouse ear.

The responses of the mouse ear to heat alone, X-rays alone or X-rays combined with heat were measured at 10 months after initial X-ray treatments (19 Gy or 10 X 3.8 Gy), which caused similar acute reactions. Fractionating the initial dose had little effect on the response to retreatment. Prior irradiation increased thermal sensitivity so that the heating time at 43.5 degrees C required to cause necrosis was about 65 per cent that in age-matched controls. Prior irradiation also increased the response to X-rays alone, but had different effects on the susceptibilities to develop acute radiodermatitis and late deformity. For acute radiodermatitis, the second X-ray dose required to cause a given response in previously irradiated ears was 80-90 per cent that in age-matched controls and for late deformity it was 60-65 per cent. Prior irradiation had the same effects on the responses to X-rays given 6 min before mild hyperthermia (43.5 degrees C, 12 min) as on those to X-rays alone but had little effect on the responses to X-rays given 6 min after hyperthermia. Consequently, the thermal enhancement ratios for heat given after X-rays did not depend on prior irradiation whereas those for heat given before X-rays were reduced. This reduction may be due to a reduced ability of irradiated blood vessels to elicit an hyperaemic response to heat.

Thermotolerance induced by fractionated hyperthermia: dependence of the interval between fractions.

The induction of thermotolerance by fractionated hyperthermia was investigated in the mouse ear. Ears were heated at 43.5 degrees C by immersion in water. One to ten treatments of 20 min were followed by test treatments. Thermotolerance was assessed as the increase in the duration of the test treatment required for a thermal response in 50 per cent of the ears (NT50). A single treatment induced thermotolerance which reached a maximum at 24 h when the NT50 was increased by a factor of 2.4. The same maximum was observed after each fractionated treatment used in the present study. The time course of development, however, depended on the interval between fractions. (1) When the interval was too short to allow development of thermotolerance after a single fraction (4 h), thermotolerance was not induced during fractionated treatment but it developed during the first 24 h after treatment. (2) When the interval between fractions allowed the maximal development of thermotolerance (24 h), this maximum was maintained during fractionated treatment and persisted for 24 h after treatment. (3) When the interval allowed some decay of thermotolerance (72 or 168 h) there was a further increase to maximal thermotolerance after each fraction. The decay of thermotolerance from the maximum did not depend on the interval between fractions. These results indicate that the degree of thermotolerance may fluctuate during fractionated hyperthermia.