Ultra-low exposure to α-7 nicotinic acetylcholine receptor partial agonists elicits an improvement in cognition that corresponds with an increase in α-7 receptor expression in rodents: implications for low dose clinical efficacy.

Αlpha-7 neuronal nicotinic receptors (NNRs) are considered targets for cognitive enhancement in schizophrenia and Alzheimer's disease. AZD0328 is an alpha-7 NNR partial agonist that enhances cognition in rodents and nonhuman primates at sub-microgram to microgram doses. We hypothesized that increased expression of the alpha-7 receptor contributes to this beneficial activity at low doses and tested this by examining the effect of AZD0328 using in vivo and ex vivo binding, RT-PCR and cognitive function in rodents. AZD0328 (0.00178 mg/kg) was subcutaneously administered to mice 4, 24, 48 and 72 hours prior to testing in novel object recognition and produced a significant increase in cognition at 4, 24 and 48 h post-dosing. In vivo binding was examined in rat brain using [(3)H]AZ11637326 and there was a dose-dependent reduction in receptor binding at higher doses of AZD0328 (0.001-3 mg/kg), and a second alpha-7 partial agonist, SSR180711 (0.01-30 mg/kg). Lower doses of both compounds (0.0001 mg/kg) produced a significant increase in binding of [(3)H]AZ11637326. Ex vivo binding using [(125)I]-α-bungarotoxin, showed a significant increase in receptor number (B(max.)) in the frontal cortex or hippocampus with no significant effect on receptor affinity (K(d)) 2 h post administration of AZD0328. [(3)H]AZ11637326 administered 1.5 h following AZD0328 produced a significant increase in specific binding in rat brain regions. We found that the effect on receptor number was long-lasting, with [(125)I]-α-bungarotoxin binding increased in rats given AZD0328 for 2-48 h, but this was not accompanied by increased mRNA synthesis. SSR180711 produced a similar increase in B(max.) and specific binding with no effect on K(d). Therefore, trace dose of alpha-7 partial agonists has rapid onset and produces a profound, sustained effect on novel object recognition in mice that corresponds by dose to an increase in receptor number in rat brain. These findings provide an explanation for the acute and sustained benefit of alpha-7 receptor activation in working memory in nonhuman primates and guidance for drug development initiatives and treatment regimens for nicotinic partial agonists.

Differential neuroprotective effects for three GABA-potentiating compounds in a model of hypoxia-ischemia.

Clomethiazole (CMZ) is a GABA(A)-potentiating compound; however, it is unclear whether this mode of action is responsible for its neuroprotective effects in animal models of ischemia. This study compared the neuroprotective efficacies of muscimol and midazolam, two potent GABA(A)-potentiating compounds, to that of CMZ in a model of hypoxia-ischemia (H-I). To establish a neuroprotective profile for CMZ, CMZ (60, 95, or 125 mg kg-1, i.p.) was administered to post-natal day 25 male rats at numerous post-hypoxic time points and the rats were sacrificed 1 or 4 weeks later. Varying degrees of histological protection were evident when CMZ was administered 1, 2, or 3 h post-hypoxia with the 125 mg kg-1 dose producing complete histological protection if administered 3 h post-hypoxia. To determine whether midazolam or muscimol could match the protection provided by CMZ administered 3 h post-hypoxia, H-I rats received varying doses of these compounds 3 h post-hypoxia and were sacrificed 1 week later. Under identical conditions, no dose of muscimol or midazolam provided equivalent neuroprotection to that provided by CMZ. In fact, muscimol showed no neuroprotective ability whatsoever. Thus, CMZ, administered as late as 3 h post-hypoxia, was able to completely prevent H-I-induced cell death while a full dose range of other GABA-potentiating agents did not. Such direct comparison of these compounds in this model suggests the mechanism underlying the protective effects of CMZ may not rely solely on GABA(A)-potentiating properties. Elucidation of a novel mechanism of action for CMZ may expose new therapeutic targets in stroke treatment.

Effect of NXY-059 on infarct volume after transient or permanent middle cerebral artery occlusion in the rat; studies on dose, plasma concentration and therapeutic time window.

1. The efficacy of the free radical trapping agent NXY-059 in reducing the infarct volume following both transient and permanent focal ischaemia has been examined in rats. 2. In the transient ischaemia model, rats were subjected to a 2 h occlusion of the middle cerebral artery (MCA). Intravenous infusion of NXY-059 (1, 10 and 30 mg kg(-1) h) for 21.75 h starting 2.25 h after the occlusion, produced a dose-dependent decrease in both neurological impairment and the histologically measured infarct volume (a mean 59% decrease at 10 mg kg(-1) h). 3. In the permanent ischaemia model, animals were injected (s.c.) with a loading dose of NXY-059 of 32.5, 53.8 or 75.4 mg kg(-1) and osmotic minipumps were implanted which had been primed to deliver respectively 30, 50 or 70 mg kg(-1) h. When treatment was initiated 5 min after MCA occlusion there was a dose dependent protection of both cortical and sub-cortical tissue (cortex: 63% at the mid-range dose). Protection was related linearly to plasma concentration (plasma unbound NXY-059 concentration at 1 h: 37+/-16 micromol l(-1) at the mid-range dose). 4. When the mid range dose was administered between 5 min - 4 h after MCA occlusion, a marked and statistically significant protection was seen at all time points (44% protection in cortex at 4 h). 5. These data demonstrate the substantial neuroprotective efficacy of NXY-059 at plasma concentrations that can be achieved clinically and indicate that NXY-059 also has a therapeutic window of opportunity that is clinically relevant.

Long-term deficits following cerebral hypoxia-ischemia in four-week-old rats: correspondence between behavioral, histological, and magnetic resonance imaging assessments.

We examined whether following a hypoxic-ischemic insult in young animals there are long-lasting functional deficits that correlate either to histological tissue damage or to potential compensatory plasticity changes. Four-week-old rats were subjected to an episode of cerebral hypoxia-ischemia (right carotid artery occlusion + 30 min of hypoxia) or a sham operation. In hypoxic-ischemic animals there were gross neurological deficits 1, 24, and 48 h postinsult with recovery by 1 week. Behavioral deficits were observed in both the acquisition and the performance of a response duration differentiation test and a fine motor control test (staircase test) 3 months after the hypoxia-ischemia. Functional magnetic resonance imaging studies demonstrated less activation in the sensory-motor cortex of hypoxic-ischemic animals in response to left vs right forepaw stimulation 4 months postinsult. Histological assessment delineated striatal, cortical, and hippocampal damage in the hypoxic-ischemic hemisphere and a reduction in cortical thickness, bilaterally. GFAP immunoreactivity was increased in injured striatum and cortex. Neurofilament heavy chain (NF200) immunoreactivity was normally most intense in white matter and decreased in areas of ipsilateral cortical damage. Synaptophysin immunoreactivity was reduced around areas of infarction and somewhat increased in adjacent undamaged striatum and in layer IV of parietal cortex. The histological damage or chronic degenerative changes could account for much of the variance in functional outcome detected with sensitive behavioral tests so that overall the compensatory or plasticity changes evident within the juvenile brain are rather modest.

Clomethiazole is neuroprotective in models of global and focal cerebral ischemia when infused at doses producing clinically relevant plasma concentrations.

We investigated the neuroprotective effect of infusing various doses of clomethiazole in models of global and focal cerebral ischemia. In a model of global ischemia, gerbils were infused with clomethiazole (intravenous), attaining steady state plasma concentrations of between 1 and 13 microM for 24 h. In a transient middle cerebral artery occlusion model in rats, clomethiazole was administered subcutaneously over 22.75 h using osmotic minipumps producing steady state plasma concentrations of between 1 and 13 microM. Clomethiazole was protective in these models at plasma concentrations of respectively 6.1 microM and above and 3.5 microM and above. Clomethiazole is thus neuroprotective in both global and focal ischemia at plasma concentrations known to be well tolerated in stroke patients.

Long-term functional end points following middle cerebral artery occlusion in the rat.

The purpose of the present study was to assess the magnitude and stability of a number of functional deficits in rats subjected to occlusion of the middle cerebral artery (MCAO). Three groups of rats, treated with 90-min, 120-min, or sham occlusion were used in functional studies for 22 weeks following surgery. The following tests were used: methamphetamine-induced rotation, the staircase test, acquisition of operant responding, running-wheel behavior, and performance of operant differential reinforcement of a low-rate responding (DRL) schedule of reinforcement. Histology performed at 23 weeks following infarct showed on average modest damage of a 19% reduction in hemispheric volume. Of the behavioral tests conducted, rotation, the staircase test, and the operant DRL were sensitive to ischemic damage, and were under some circumstances related to lesion size. These data show that long-term functional deficits following MCAO are demonstrable, and hence, assessment of long-term neuroprotection is feasible.

The low-affinity, use-dependent NMDA receptor antagonist AR-R 15896AR. An update of progress in stroke.

Use-dependent N-methyl-D-aspartate (NMDA) receptor antagonists protect neurons from the lethal consequences of excessive stimulation by excitatory amino acids. Clinical development of high-affinity compounds such as MK801 have been limited due to untoward side effects. Toward this end, the lower-affinity use-dependent NMDA antagonists have greater margins of safety and have advanced to clinical trials for stroke, epilepsy, head trauma and chronic neurodegenerative disorders. AR-R 15896AR is currently in Phase II trials for stroke and has been repeatedly demonstrated to afford neuroprotection in a variety of in vivo and in vitro models associated with ischemia/excitotoxic conditions.

Efficacy of AR-R15896AR in the rat monofilament model of transient middle cerebral artery occlusion.

The monofilament technique of transient middle cerebral artery occlusion (MCAO) was used in 3 separate studies to evaluate the efficacy of the low-affinity, use-dependent N-methyl-d-aspartate receptor antagonist, AR-R15896AR. First, a dose-response curve was attempted. Wister Kyoto rats received 2 hours of MCAO. Five minutes later, a 30-minute intravenous infusion of AR-R15896AR was given, followed by subcutaneous implantation of Alzet minipumps that were calibrated to maintain specified plasma levels (approximately 682, 1885, or 2682 ng/mL) of AR-R15896 (free base) for 1 week. The highest plasma level attained significantly decreased the percentage of damage to the subcortex, cortex, and total brain. Second, the high-dose, 1-week treatment regimen was repeated to determine if neuroprotection would extend to 8 weeks after MCAO. Indeed, in separate groups of animals, significant reduction in the percentage of damage, which was generally confined to the cortex and subcortex, was observed at 1, 2, 4, and 8 weeks. Third, verification was achieved in another laboratory. Lister Hooded rats received 60 minutes of transient MCAO. At 70 minutes, an acute dose of AR-R15896AR (20.3 mg/kg) was injected intraperitoneally and the rats were killed 23 hours later. This treatment group also exhibited significant reduction in the volume of infarction in the subcortex, cortex, and total brain. The outcome of these investigations supports the ongoing Phase II clinical trials in patients with acute stroke.

The effect of oedema and tissue swelling on the measurement of neuroprotection; a study using chlormethiazole and permanent middle cerebral artery occlusion in rats.

The effect of chlormethiazole on hemispheric swelling and cortical tissue water content has been investigated in a model of permanent middle cerebral artery (MCA) occlusion. Chlormethiazole (1 mmol/kg i.p.) or saline was administered 60 min after the induction of ischaemia and the animals sacrificed after 24 hours. The cross sectional area of the left hemisphere was increased by 21.8 + 1.9% in saline treated rats, but only by 8.4 + 2.4% in chlormethiazole treated rats. However, the reduction in the absolute area of neurodegenerative damage (mm2) following chlormethiazole administration was considerably greater than the reduction in hemispheric swelling. Cortical tissue water content of ischaemic brain increased from 76.4% to 84.2% and this was attenuated to 78.8% by chlormethiazole administration. These data demonstrate that, providing damage is measured by fitting tissue slices onto prematched stereotactic maps, the decrease in oedema which accompanies a decrease in neurodegeneration does not result in erroneous estimates of neuroprotection.

The neuroprotective effect of chlormethiazole on ischaemic neuronal damage following permanent middle cerebral artery ischaemia in the rat.

The ability of chlormethiazole to protect against ischaemic cell damage in a rat model of permanent focal ischaemia has been examined. Chlormethiazole (1 mmol/kg) was administered intraperitoneally either 1 or 3 h after occlusion of the middle cerebral artery with an intraluminal filament. Twenty four hours after the start of occlusion there was histological evidence for ischaemic damage in both cortex and striatum. The volume of ischaemic damage in control (saline injected) animals was 310 +/- 25 mm3 (mean +/- SEM; n = 6). Chlormethiazole administered 1 h after occlusion reduced this damage by 58% (128 +/- 40 mm3; n = 6; P < 0.01), protection being observed in both brain regions. The drug was ineffective when given 3 h after occlusion (304 +/- 25 mm3; n = 5). Chlormethiazole had no effect on body temperature, mean arterial blood pressure, blood pH, pO2 or pCO2, but did induce mild bradycardia. Chlormethiazole therefore appears to be an effective neuroprotective agent in this model of permanent ischaemia, complementing previous data on the efficacy of this drug in other focal and global models of cerebral ischaemia.

The effect of chlormethiazole on neuronal damage in a model of transient focal ischaemia.

1. The effect of chlormethiazole has been studied in a transient middle cerebral artery (MCA) occlusion model of cerebral ischaemia in the rat. The MCA was occluded for 1 h by use of an intraluminal suture technique, with reperfusion for 24 h following removal of the occluding filament. Neuronal damage was determined by measurement of the area of necrosis following Cresyl Violet staining of sections taken through the ischaemic region. 2. In the initial experiment, occlusion of the MCA produced a large volume of ischaemic damage in both cortex and striatum, characterized by necrosis and pyknosis (total volume of damage, 287 +/- 13 mm3, n = 9). Rats injected with chlormethiazole (1000 mumol kg-1, i.p.) 60 min before occlusion had a reduced volume of damage in both regions (104 +/- 11 mm3; n = 9; P < 0.001). 3. In a subsequent study systemic physiological parameters (heart rate, blood pressure, blood pH, blood gases and rectal temperature) were measured throughout the ischaemic period. 4. Chlormethiazole (1000 mumol kg-1) pretreatment produced little change in systemic physiology and the neuroprotective effect of the drug when given 60 min prior to the MCA occlusion was confirmed. Chlormethiazole was also neuroprotective when given 10 min following the start of reperfusion (control group: 244 +/- 52 mm3, n = 10; chlormethiazole pretreatment group: 102 +/- 23 mm3, n = 10; P < 0.001; chlormethiazole post-ischaemia group: 122 +/- 16 mm3; P < 0.001, n = 10). 5. It is concluded that chlormethiazole is an effective neuroprotective agent in this model of transient focal ischaemia. The observation that chlormethiazole is protective when given after reperfusion indicates that the effect of the drug is unlikely to be due to an alteration of intra-ischaemic cerebral blood flow, but is more probably a direct effect on the development of ischaemic damage.

Calcitonin gene-related peptide reduces brain injury in a rat model of focal cerebral ischemia.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide is an endogenous vasodilating neuropeptide with a dense concentration in the trigeminocerebrovascular system. It is hypothesized that depletion of this peptide contributes to delayed cerebral ischemia after subarachnoid hemorrhage and that an exogenous supply of calcitonin gene-related peptide will augment ischemic cerebral blood flow and reduce neuronal injury. METHODS: In this study we have investigated the effect of an intravenous infusion of calcitonin gene-related peptide (100 ng/kg per minute), started 1 hour before and continued throughout 4 hours of focal cerebral ischemia, on cerebral blood flow and the volume of brain injury in a rat model of middle cerebral artery occlusion. RESULTS: Calcitonin gene-related peptide produces a significant improvement in ischemic cerebral blood flow (32 +/- 2 compared with 13 +/- 2 mL/100 g per minute in the controls; t = 6.92, P < .0001) with a concomitant reduction in the volume of ischemic brain injury (102 +/- 22 compared with 234 +/- 19 mm3; t = 4.47, P < .001). CONCLUSIONS: These findings lend support for the potential use of this peptide in the prophylactic treatment of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.