The cholinergic hypothesis of geriatric memory dysfunction.

Biochemical, electrophysiological, and pharmacological evidence supporting a role for cholinergic dysfunction in age-related memory disturbances is critically reviewed. An attempt has been made to identify pseudoissues, resolve certain controversies, and clarify misconceptions that have occurred in the literature. Significant cholinergic dysfunctions occur in the aged and demented central nervous system, relationships between these changes and loss of memory exist, similar memory deficits can be artificially induced by blocking cholinergic mechanisms in young subjects, and under certain tightly controlled conditions reliable memory improvements in aged subjects can be achieved after cholinergic stimulation. Conventional attempts to reduce memory impairments in clinical trials hav not been therapeutically successful, however. Possible explanations for these disappointments are given and directions for future laboratory and clinical studies are suggested.

Intravenous RMP-7 selectively increases uptake of carboplatin into rat brain tumors.

Rats implanted with RG-2 gliomas were administered i.v. RMP-7 and [14C]carboplatin. Changes in the permeability of the blood-brain barrier to carboplatin were determined using quantitative autoradiography. i.v. infusions of RMP-7 induced an increase in the permeability of the vascular barrier within the tumor to carboplatin. Additionally, permeability of brain tissue proximal to, but clearly outside the tumor mass, was also increased. Progressively less uptake of [14C]carboplatin was observed as distance from the tumor border increased. The increases in permeability induced by RMP-7 occurred in a dose-related fashion. No increase in carboplatin level was observed in several nonbrain tissues, including sciatic nerve, retina, heart, lung, liver, kidney, and spleen. Finally, the permeabilizing effects of RMP-7 were shown to occur independent of histaminergic or hypotensive mechanisms. These data provide additional insight into the permeabilizing effects and mechanism of RMP-7 and offer additional support for the therapeutic utility of this novel compound as an adjunctive treatment for human gliomas.

Time-related neuronal changes following middle cerebral artery occlusion: implications for therapeutic intervention and the role of calpain.

Changes in neocortex and striatum were characterized over time following focal ischemia to the brain. Rats were subjected to permanent middle cerebral artery occlusion (MCA-O) and sacrificed 1, 3, 6, 12, or 24 h later. The affected tissue was processed for tetrazolium chloride (TTC) and cresyl violet staining, as well as for Western blots to detect calpain-induced spectrin proteolysis. Significant changes in cell size and spectrin breakdown occurred within the first hour of occlusion, with further, dramatic changes in these two early markers continuing over time. Initial evidence of cell loss was noted at 1 h postocclusion in the striatum and at 3 h in the neocortex. However, even in the center of the most affected portion of the neocortex, the majority of cells appeared to be intact through 6 h. By this time, a significant TTC-defined infarct also emerged. These quantitative data indicate that calpain-induced proteolysis occurs very soon after the ischemic insult, is correlated with earliest changes in cell hypotrophy, and precedes or occurs in tandem with evidence of significant cell loss. They also demonstrate that, while some cell loss occurs earlier than previously believed, the majority of cells remains morphologically intact well beyond what is typically thought to be the window of opportunity for intervention. The results thus raise the question of how long after the ischemic event pharmaceutic intervention might be employed to salvage substantial numbers of neurons.

Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage.

Experiments were conducted to determine whether a potent, reversible calpain inhibitor could reduce the cortical ischemic brain damage associated with focal ischemia in the rat. AK275 (Z-Leu-Abu-CONH-CH2CH3), the active isomer of the diastereomeric mixture, CX275, was employed in conjunction with a novel method of perfusing drug directly onto the infarcted cortical surface. This protocol reduced or eliminated numerous, nonspecific pharmacokinetic, hemodynamic, and other potentially confounding variables that might complicate interpretation of any drug effect. Focal ischemia was induced using a variation of the middle cerebral artery occlusion method. These studies demonstrated a reliable and robust neuroprotective effect of AK275 over the concentration range of 10 to 200 microM (perfused supracortically at 4 microliters/h for 21 h). Moreover, a 75% reduction in infarct volume was observed when initiation of drug treatment was delayed for 3 h postocclusion. Our data further support an important role of calpain in ischemia-induced neuropathology and suggest that calpain inhibitors may provide a unique and potentially powerful means of treating stroke and other ischemic brain incidents.

Tetrahydroaminoacridine, 3,4 diaminopyridine and physostigmine: direct comparison of effects on memory in aged primates.

The effects of tetrahydroaminoacridine (THA), 3,4 diaminopyridine (3,4 DAP) and physostigmine were evaluated for their ability to reduce memory impairments in aged, test-sophisticated cebus monkeys (18 to 26 years old). Several doses of each drug were tested (PO) in each of ten different monkeys, allowing for direct and extensive comparison of each drug's efficacy in this model. The results of this comparative test revealed several potentially interesting findings: (1) all drugs produced improvement in a portion of the monkeys tested; (2) as in many past tests with aged monkeys and humans, wide variations in most effective dose, per subject, were observed; (3) different monkeys responded more effectively to one drug than another; and (4) under these tightly controlled conditions, physostigmine produced the most reliable and robust effects (p less than 0.005), in more monkeys, than did either THA (p less than 0.05) or 3,4 DAP (p less than 0.10).

Lack of efficacy of clonidine on memory in aged cebus monkeys.

Using a dosing paradigm and an automated behavioral testing procedure designed to maximize potential drug efficacy, we evaluated the ability of clonidine to improve performance of aged monkeys on a trial-specific memory task. These results indicated that neither acutely administered (0.0025-0.04 mg/kg) or subchronically administered (0.01-0.06 mg/kg for 3 consecutive days) clonidine improved the performance of aged monkeys. These data are discussed in view of previously reported, conflicting data, as well as their implication regarding probable neurochemical factors involved in age-associated memory impairment and approaches to future palliative treatments.

Memory deficits in aged cebus monkeys and facilitation with central cholinomimetics.

Cebus monkeys of 3 different age groups were trained to perform an automated behavioral task (delayed response), intended to measure recent memory ability. In in initial study, the aged monkeys (18 years and older) exhibit prprogressively greater performance impairments (relative to young monkeys) as they were required to remember the location of a visual stimulus for increasingly longer durations (0 to 20 sec). This deficits replicated previously published results from aged Rhesus monkeys and appeared similar to the primary memory deficits reported in elderly humans and demented patients. In subsequent studies, the effects of three different cholinomimetics were evaluated for their ability to improve the aged monkey's performance on this task. Each monkey was tested under several acute doses of the cholinergic precursor, choline, the anticholinesterase, physostigmine, and the cholinergic muscarinic receptor agonist, arecoline. The results revealed clear differences in the ability of these drugs to improve performance on this task. Choline exerted no apparent effects in the aged monkeys at any dose tested. Physostigmine clearly enhanced performance in certain aged monkeys, but the optimal dose varied dramatically between subjects, replicating previously published results with aged Rhesus monkeys and humans. Arecoline produced clear improvement within a restricted dose range, with little variation in optimal dose between subjects. In addition to demonstrating differences in the effects of different cholinomimetics on memory performance in aged primates, these data also suggest a possible rationale for future investigations. Assuming that each of these drugs primarily affected cholinergic function in the manner conventionally attributed, these data suggest that, within the cholinergic system, the more directly one stimulates the receptor, the more one might expect robust and consistent effects on memory performance in aged subjects.

Neuropeptide effects on memory in aged monkeys.

The effects of several neuropeptides were evaluated using a non-human primate model of age-related memory impairments. Several doses of ACTH4-10, lysine vasopressin, arginine vasopressin, oxytocin and somatostatin were each tested in several aged monkeys. Because data from a large number of non-drug control sessions was collected before, during and after this study, it was possible to define the normal range of control performance for each monkey and statistically determine whether a change in performance under any single dose of drug reflected a significant change from the particular monkey's normal baseline performance. Although none of the neuropeptides produced consistent group effects, evaluations of individual subjects against their own baseline performance revealed reliable changes at certain doses. Arginine vasopressin appeared to produce the best overall effects with three of the five monkeys exhibiting reliable changes in performance from baseline. These same three monkeys also responded positively to the lysine form. Oxytocin impaired memory in three of the six aged monkeys tested over a wide range of doses. Three of six aged monkeys performed better under ACTH4-10 compared to baseline; however, in two of these cases only a single dose was effective. The performance of only one subject was improved under somatostatin, and this was at a single dose only. The data reported here provide evidence for neuropeptides producing behavioral improvement in non-human primates using an appetitive task, eliminating a popular criticism that the data in this literature has depended too heavily on the testing of rodents in shock-motivated tasks. Additionally, the improvements observed in this study involve a behavior that it naturally impaired by age and one which has many operational similarities and some empirical relevance to measures of recent memory in humans. However, these positive findings must be tempered by the lack of robust effects and high individual variation observed.

Profound effects of combining choline and piracetam on memory enhancement and cholinergic function in aged rats.

In an attempt to gain some insight into possible approaches to reducing age-related memory disturbances, aged Fischer 344 rats were administered either vehicle, choline, piracetam or a combination of choline or piracetam. Animals in each group were tested behaviorally for retention of a one trial passive avoidance task, and biochemically to determine changes in choline and acetylcholine levels in hippocampus, cortex and striatum. Previous research has shown that rats of this strain suffer severe age-related deficits on this passive avoidance task and that memory disturbances are at least partially responsible. Those subjects given only choline (100 mg/kg) did not differ on the behavioral task from control animals administered vehicle. Rats given piracetam (100 mg/kg) performed slightly better than control rats (p less than 0.05), but rats given the piracetam/choline combination (100 mg/kg of each) exhibited retention scores several times better than those given piracetam alone. In a second study, it was shown that twice the dose of piracetam (200 mg/kg) or choline (200 mg/kg) alone, still did not enhance retention nearly as well as when piracetam and choline (100 mg/kg of each) were administered together. Further, repeated administration (1 week) of the piracetam/choline combination was superior to acute injections. Regional determinations of choline and acetylcholine revealed interesting differences between treatments and brain area. Although choline administration raised choline content about 50% in striatum and cortex, changes in acetylcholine levels were much more subtle (only 6-10%). No significant changes following choline administration were observed in the hippocampus. However, piracetam alone markedly increased choline content in hippocampus (88%) and tended to decrease acetylcholine levels (19%). No measurable changes in striatum or cortex were observed following piracetam administration. The combination of choline and piracetam did not potentiate the effects seen with either drug alone, and in certain cases the effects were much less pronounced under the drug combination. These data are discussed as they relate to possible effects of choline and piracetam on cholinergic transmission and other neuronal function, and how these effects may reduce specific memory disturbances in aged subjects. The results of these studies demonstrate that the effects of combining choline and piracetam are quite different than those obtained with either drug alone and support the notion that in order to achieve substantial efficacy in aged subjects it may be necessary to reduce multiple, interactive neurochemical dysfunctions in the brain, or affect activity in more than one parameter of a deficient metabolic pathway.

Presynaptic cholinergic mechanisms in brain of aged rats with memory impairments.

Presynaptic cholinergic mechanisms were investigated in various brain regions of aged Fisher 344 rats with documented 24 hr retention deficits measured in a single-trial passive avoidance tasks. Sodium-dependent high affinity choline uptake was found to be decreased by 22% in hippocampus of 23-26 month old animals as compared to 6 month old controls. Prior depolarization of hippocampal or cortical synaptosomes with K+ resulted in stimulation of choline uptake which was similar in aged rats and young controls. No age-related differences were observed either in hippocampal, cortical, striatal acetylcholine or choline concentrations, or in the activity of choline acetyltransferase in hippocampus. Synthesis of acetylcholine in hippocampal and cortical slices under basal conditions, as well as under K+-stimulated concentrations, did not differ in the two age groups examined. These neurochemical findings are consistent with an age-related decrease in hippocampal cholinergic neuronal activity without an actual loss in cholinergic neuron number. It is further suggested that this reduction in cholinergic neuronal activity may be related to the deficit in cognitive performance observed in aged Fisher rats.

Behavioral effects of phosphatidylserine in the aged Fischer 344 rat: amelioration of passive avoidance deficits without changes in psychomotor task performance.

A series of studies was conducted to evaluate the effects of phosphatidylserine (PS) in aged Fischer 344 rats. No effects were observed in any of four psychomotor tasks in which aged rats normally show deficits, nor on measures of shock sensitivity. However, significant dose-related effects on retention of passive avoidance were observed when PS was given both 30 min prior to training and retention. Further, in a second experiment similar positive effects were observed when PS was given only 30 min prior to training, as well as only 5 min following training. These results suggest that one effect of PS may include an ability to enhance neural events involved in the encoding or consolidation of new information into memory.

Brain cholinergic dysfunction and memory in aged rats.

Age related alterations in mnemonic ability and in the functional status of muscarinic receptors were evaluated and compared to biochemical measures of pre and post-synaptic cholinergic functioning. Retention of a single trial passive avoidance task was considerably disturbed as a function of aging. The functional status of muscarinic receptors, as measured by the ability of microiontophoretically applied acetylcholine to stimulate the firing of hippocampal pyramidal cells, was similarly disturbed in aged rats. A small, but significant decrease in muscarinic receptors was detected in the dorsal hippocampi of these same aged rats, while choline acetyltransferase activity did not change. When considered with prior psychopharmacological studies, these data suggest that specific muscarinic receptor impairments may play a critical role in the memory disturbances associated with old age.

Cereport (RMP-7) increases carboplatin levels in brain tumors after pretreatment with dexamethasone.

Accumulating evidence suggests that dexamethasone might decrease permeability of the blood-brain tumor barrier, further limiting the delivery of agents into brain tumors. The bradykinin B2 receptor agonist, Cereport (RMP-7), selectively increases permeability of the vasculature supplying brain tumors in both animal models and humans. The present study was conducted to characterize the effects of dexamethasone on the blood-brain tumor barrier and its potential interaction with Cereport's ability to enhance penetration of radiolabeled carboplatin. Dexamethasone (1.5 mg/kg/day, twice a day) was given to RG2 glioma-bearing rats via oral gavage for 3 consecutive days. After treatment, animals received a 15-min intracarotid infusion of Cereport (4.5 micrograms/kg) and a bolus of [14C]carboplatin. The levels of [14C]carboplatin (nCi/g) in the tumor and nontumor regions were determined at 1, 14, or 24 h after the last dose of dexamethasone. Dexamethasone, alone, significantly decreased the levels of radiolabeled carboplatin permeating the tumor (19%), although there were no significant differences between any of the time points examined. Cereport administration significantly increased levels of carboplatin in the tumor, independent of whether or not dexamethasone was given (46% with and 49% without). Although the relative effects of Cereport on tumor carboplatin levels were not affected by dexamethasone, the absolute levels achieved with Cereport were modestly reduced (44 nCi/g versus 55.5 nCi/g of [14C]carboplatin, with and without dexamethasone, respectively). Thus, while the data support the use of Cereport as adjunctive therapy in the treatment of glioma patients, they also warn that the use of dexamethasone may reduce delivery of chemotherapeutic agents to brain tumors, even when special pharmacologic measures are employed to enhance delivery.

Synthesis and biological activity of N6-(p-sulfophenyl)alkyl and N6-sulfoalkyl derivatives of adenosine: water-soluble and peripherally selective adenosine agonists.

A series of N6-(p-sulfophenyl)alkyl and N6-sulfoalkyl derivatives of adenosine was synthesized, revealing that N6-(p-sulfophenyl)adenosine (10b) is a moderately potent (Ki vs [3H]PIA in rat cortical membranes was 74nM) and A1-selective (120-fold) adenosine agonist, of exceptional aqueous solubility of > 1.5 g/mL (approximately 3 M). Compound 10b was very potent in inhibiting synaptic potentials in gerbil hippocampal slices with an IC50 of 63 nM. At a dose of 0.1 mg/kg ip in rats, 10b inhibited lipolysis (a peripheral A1 effect) by 85% after 1 h. This in vivo effect was reversed using the peripherally selective A1-antagonist 1,3-dipropyl-8-[p-(carboxyethynyl)phenyl]xanthine (BW1433). The same dose of 10b in NIH Swiss mice (ip) was nearly inactive in locomotor depression, an effect that has been shown to be centrally mediated when elicited by lower doses of other potent adenosine agonists, such as N6-cyclohexyladenosine (CHA) (Nikodijevic et al. FEBS Lett. 1990, 261, 67). HPLC studies of biodistribution of a closely related and less potent homologue, N6-[4-(p-sulfophenyl)butyl]adenosine indicated that a 25 mg/kg ip dose in mice resulted in a plasma concentration after 30 min of 0.46 micrograms/mL and no detectable drug in the brain (detection limit < 0.1% of plasma level). Although 10b at doses > 0.1 mg/kg in mice depressed locomotor activity, this depression was unlike the effects of CHA and was reversible by BW1433. These data suggest that 10b is a potent adenosine agonist in vivo and shows poor CNS penetration.

The development of the bradykinin agonist labradimil as a means to increase the permeability of the blood-brain barrier: from concept to clinical evaluation.

Labradimil (Cereport; also formerly referred to as RMP-7) is a 9-amino-acid peptide designed for selectivity for the bradykinin B2 receptor and a longer plasma half-life than bradykinin. It has been developed to increase the permeability of the blood-brain barrier (BBB) and is the first compound with selective bradykinin B2 receptor agonist properties to progress from concept design through to tests of efficacy in patients. In vitro studies demonstrate that labradimil has a longer half-life than bradykinin and selectively binds to bradykinin B2 receptors, initiating typical bradykinin-like second messenger systems, including increases in intracellular calcium and phosphatidylinositol turnover. Initial proof of principle studies using electron microscopy demonstrated that intravenous labradimil increases the permeability of the BBB by disengaging the tight junctions of the endothelial cells that comprise the BBB. Autoradiographic studies in rat models further demonstrated that labradimil increases the permeability of the BBB in gliomas. Intravenous or intra-arterial labradimil increases the uptake of many different radiolabelled tracers and chemotherapeutic agents into the tumour in a dose-related fashion. These effects are selective for the tumour and for the brain surrounding the tumour, and are particularly robust in tumour areas that are normally relatively impermeable. The increased chemotherapeutic concentrations are maintained for at least 90 minutes, well beyond the transient effects on the BBB. The increase in permeability with labradimil occurs rapidly but is transient, in that restoration of the BBB occurs very rapidly (2 to 5 minutes) following cessation of infusion. Even with continuous infusion of labradimil, spontaneous restoration of the barrier begins to occur within 10 to 20 minutes. Collectively, these data demonstrate that the B2 receptor system that modulates permeability of the BBB is highly sensitive and autoregulated and that careful attention to the timing of labradimil and the chemotherapeutic agent is important to achieve maximal effects. Survival studies in rodent models of both gliomas and metastatic tumours in the brain demonstrate that the enhanced uptake observed with the combination of labradimil and water-soluble chemotherapeutics enhances survival to a greater extent than achieved with chemotherapy alone. Finally, preliminary clinical trials in patients with gliomas provide confirmatory evidence that labradimil permeabilises the blood-brain tumour barrier and might, therefore, be used to increase delivery of agents such as carboplatin to tumours without the toxicity typically associated with dose escalation.

Histopathologic consequences of moderate concussion in an animal model: correlations with duration of unconsciousness.

Although duration of unconsciousness is commonly used as a prognostic index following traumatic brain injury (TBI), few controlled studies have statistically evaluated the relationship between unconsciousness and histologic pathology, particularly after moderate head injury. Using a pendulum-striker concussive device, a reproducible model of TBI in rats was developed. This model is uncomplicated by skull fractures, contusions, or experimenter-induced craniotomies. In the present study, the severity of the histopathology observed in this model of moderate closed-head injury at 48 h posttrauma is linearly related to the duration of unconsciousness (p < 0.0001). The pathology, assessed with a silver stain for neurodegeneration, is particularly striking if unconsciousness persists for 4 minutes or more. These data suggest that the initial period of unconsciousness may be a useful predictor of clinical brain histopathology associated with moderate closed-head injury, predicting either the degree of pathology and/or the rate it progresses if left untreated.

Dissociation of p75 receptors and nerve growth factor neurotrophic effects: lack of p75 immunoreactivity in striatum following physical trauma, excitotoxicity and NGF administration.

While the function and regulation of the low affinity (p75) nerve growth factor (NGF) receptor in the central nervous system (CNS) remains a mystery, one of the more intriguing observations involves its response to injury in the adult rat striatum. Following mechanical injury to the striatum, a re-expression of striatal p75 receptors and mRNA purportedly occurs (apparently mediated by elevations in NGF), thus reversing the natural loss of these phenotypic markers that is known to occur during development. This observation has important implications for understanding both the regulation of NGF neurotrophic activity and the role of the p75 receptor, for it implies that the presence of this receptor may be required for NGF trophic activity in the CNS. In an effort to gain a greater understanding of the function and regulation of the low affinity p75 NGF receptor, we performed a series of experiments to study the injury-induced, re-expression phenomenon in the striatum. In the first experiment, we duplicated the mechanical, cannula-induced injury used in the original study. In a follow-up study, we exacerbated that injury by infusing quinolinic acid directly into the striatum. In a third study, the mechanical injury was complemented with chronic striatal infusions of NGF. In a final study, we examined striatal tissue from rats who had been protected from striatal quinolinic acid neurotoxicity by administration of NGF. In no instance was the re-expression of p75 striatal receptors observed, despite positive controls for (a) effective neural trauma, confirmed by histologic and immunocytochemical methods, (b) effective antibody staining, confirmed by appropriate basal forebrain p75 immunoreactivity, and (c) effective biological activity of exogenous NGF, confirmed by hypertrophy of choline acetyltransferase (ChAT)-positive striatal neurons and protection of ChAT-positive striatal neurons against excitotoxicity. At least two important conclusions can be drawn from these studies: (1) the presence or induction of low affinity p75 receptors is not necessary, while the presence of constitutive high affinity tropomyosin related kinase (trk) NGF receptors seem sufficient for NGF trophic activity in the CNS, and (2) the variables necessary to induce re-expression of p75 striatal receptors in adult rats have not yet been elucidated and are apparently complex.

Temporal ordering of pathogenic events following transient global ischemia.

Rats were subjected to transient global ischemia (four vessel occlusion) and time-related changes in the selectively vulnerable hippocampal field CA1 were characterized. The assessment included ex vivo field responses to afferent stimulation, silver staining, calpain-induced spectrin breakdown, chromatolysis, and cell death, beginning at 6 h post-ischemia and continuing until total disintegration of the pyramidal cells occurred several days later. The earliest change observed was a modest increase in the slope and amplitude of field CA1 potentials (at 6 h). The hyperresponsiveness was most apparent at higher stimulation currents and persisted unchanged at 16 h post-ischemia. Three effects became detectable within 24 h, post-ischemia: (a) an increase in concentrations of calpain-mediated, spectrin breakdown products; (b) enhanced silver staining in the deep pyramidal neurons of the field CA1 with lesser, though still apparent, staining of stratum radiatum, and (c) a decrease in amplitude and slope of field CA1 responses to afferent stimulation. Both the concentration of spectrin breakdown products and the intensity of silver staining progressively increased to a maximum at four days post ischemia, while the amplitude and slope of the field responses dropped to a very low level between 24 and 48 h. Disturbances of Nissl staining were finally evident at 48 h, with nearly complete disappearance of staining at five days post-ischemia. This study provides the first demonstration of a close and early temporal relationship between calpain proteolysis, subcellular damage to the pyramidal cells and their loss of function following global ischemia, prior to their eventual death.