The human brain is very complex and is based on specialised cells designed to transmit information, called neurones. Neurones are an integral part and basic functional unit of the brain, which contains almost one billion of these cells. The neurones consists of a cell body containing a nucleus and an electricity-conducting fibre called an axon, which also gives rise to many branches before ending at nerve terminals. Neurones send signals by transmitting electrical impulses along their axons. When the signals reach the end of the axons, they trigger the release neurotransmitters, which then bind to receptors in adjacent neurones. This point of vital contact is called the synapse. The synaptic response involves the closing and opening of ion channels, which pass through the cell membranes and enable the ions to flow through them. This phenomenon creates an electrical current that provides tiny voltage changes across the membrane which leads to altered synaptic connectivity.

This network is capable of controlling a vast array of activities, including heart rate, body movement, perception, sexual function, emotions, learning and memory. The organisation and neurotransmitter content of intrinsic cerebral cortical and hippocampal neurones, and those of extrinsic inputs to these regions, are described below with respect to neuronal systems known to be affected in Alzheimer-type dementia. For the intrinsic neurones, particular attention is focused on the neuropeptides such as cholecystokinin, vasoactive intestinal polypeptide, somatostatin and neuropeptide Y, which are apparently stable post mortem and provide biochemical markers that can be used to judge the integrity of neuropeptide-containing cells in dementia.

Neurochemistry of cognition and cognitive dysfunction: cholinergic hypothesis

The first neurochemical was identified 70 years ago as acetylcholine. The neurones that release acetyl-choline are called cholinergic neurones and control the heartbeat and voluntary muscles, causing them to contract. Acetylcholine also serves as a neurotrans-mitter in many regions of the brain and plays an important role in learning and memory function. Mammalian brain contains several groups of cholinergic projection neurones located within the basal forebrain and brainstem. Cholinergic axons exert their neurotransmitter effect through the mediation of nicotinic and muscarinic receptors. Cholinergic neurones and cholinergic neurotransmitter pathways are highly implicated in cognition and cognitive dysfunction. The cholinergic neurones are centred on the medial septum, around the vertical limb of the diagonal band of Broca, around the horizontal limb of the diagonal band of Broca, and are also found around the nucleus basalis of Meynert.

All cholinergic neurones of the human basal fore-brain and brain stem contain the cholinergic enzymes choline acetyltransferase and acetylcholinesterase. Much of the research on the participation of neurotransmitter systems in cognitive decline associated with ageing and Alzheimer’s disease has concentrated on the role of acetylcholine, because of its correlation with the degree of cognitive dysfunction, and learning and memory deficits produced in humans, even though the role of interaction between acetylcholine and other neurotransmitters such as noradrenaline, dopamine, serotonin, γ-aminobutyric acid (GABA) and several neuropeptides affecting cognition are also important. To improve cholinergic transmission, different strategies have been suggested including increased acetylcholine synthesis, the augmentation of pre-synaptic acetylcholine release, and stimulation of postsynaptic acetylcholine muscarinic and nicotinic receptors and reduction of acetylcholine synaptic degradation with cholinesterase inhibitors. Several aspects of the functional features of the cholinergic system are shown in Figure: Functional features of the cholinergic system.

Figure: Functional features of the cholinergic system

Assessment of neurodegeneration

Animal models used for the assessment

Drugs effective in neurodegenerative disease should have several aims: to improve the cognitive impairment, control the behavioural and neurological symptoms, delay the progression of the disease and to prevent the onset. To attain these targets, cell and animal models are needed in which pathogenetic hypothesis and potential effectiveness of new drugs are to be tested, exploiting links between the molecular and biochemical studies on the disease and the reality of human pathology. Animal models of Alzheimer’s disease can provide insight into the neurological and pathological mechanisms of cognitive and behavioural changes in patients. Monitoring of behavioural changes in animal models could both provide insight into the neurobiology of these behavioural changes and help validate the felicity of the model to Alzheimer’s disease. Animal models will play a critical role in further defining the events and processes underlying the final phenotypic expression.

Aged animals models

Aged animals (rats, mice and monkeys) have been investigated on a variety of learning and memory tasks. Aged rodents have been shown to have memory impairment on tasks such as the Morris water maze and passive avoidance tests. This behavioural impairment has provided a model that resembles the neuropsychiatric symptoms commonly observed in Alzheimer’s disease. The senescence-accelerated mouse (SAM) exhibits age-related deficits in learning and memory in the Morris water maze and radial arm maze and decreased acetylcholine synthesis in hippocampus pyramidal neurones. The effect of nerve growth factor (NGF) on cognition of aged rats has been assessed by intraven-tricular infusion of NGF, delayed alternation, Morris water maze, and sensory motor tasks. NGF has been most thoroughly assessed in this setting and has shown effects in rats, which have provided a model for the assessment of cholin-ergic neurones and the cholinoprotective effects of compounds potentially useful in the treatment of Alzheimer’s disease.

Brain lesion models

In concert with the recognition of the importance of the cholinergic deficit in Alzheimer’s disease, early models of the disease concentrated on surgical or chemical lesions of the basal forebrain. Transection of the fornix results in degeneration of cholinergic cells in the basal forebrain. These experiments involved primarily rats, monkeys and baboons, which demonstrated deficits in attention and memory, tested in various maze paradigms such as passive active avoidance, Morris water maze and the eightarm radial arm maze. Lesion studies have focused primarily on the behavioural changes of the animals, with only limited attention to the pathological to the neuropsychiatric symptoms commonly observed in Alzheimer’s disease. Ageing studies are all likely to contribute information important to our understanding of the disease, and none are likely to represent a completely isomorphic model that is fully predictive of the pathogenesis, course, and treatment of human Alzheimer’s disease.

Amyloid beta protein infusion induced model of Alzheimer’s disease

Artificially created amyloid (AS) deposits in normal rats, and transgenic mice overexpressing amyloid precursor protein (APP) are the models in which possible treatments are tested. They are aimed at preventing formation of AS deposits or its transformation in neuritic plaques. Synthetic amyloid beta protein (Aβ1-42) application in vitro, using neuroglial and astrocytes, has also been used to screen various neuroprotective drugs. The injection of synthetic Aβ peptides β12-28, β25-35, and β1-40 into the septum of adult rats induced a marked decrease in basal and potassium-evoked acetylcholine release in the hippocampus. These findings confirmed in vivo the neurotoxic effects of Aβ observed in primary neuronal cell cultures exposed to Aβ peptides.

The multiple mechanisms through which Ap peptides, involving oxidative stress, loss of cellular calcium homeostasis and mitochondrial dysfunction have been reviewed and, in both in-vitro and in-vivo experiments, it has been shown that Aβ neurotoxicity depends on its fibrillary aggregation forming a sheet. These preclinical experiments give support to the hypothesis of the pivotal pathogenetic role of AS deposit in Alzheimer’s disease, throw some light on the molecular mechanisms of AS toxicity, and offer an experimental model for testing potentially useful drugs. Undoubtedly, there are limitations in the validity of intracerebral Ap injections as a model of Alzheimer’s disease.

Amyloid precursor protein transgenic mouse model

The development of transgenic mice, mimicking the genetic mutations occurring in familial Alzheimer’s disease and showing some of the neurochemical and morphological alterations of the disease, is another example of the interactions between clinical and preclinical investigations into this disease. The clinical and genetic investigations have identified the early-onset, familial forms of the disease, and the genes in which autosomal dominant mutations take place. There is a clear recognition of autosomal dominant cases induced by mutations of APP (chromosome 21), presenilin 1 (chromosome 14), or presenilin 2 (chromosome 1), which allowed development of transgenic mouse models of Alzheimer’s disease. These transgenic animals exhibit some of the pathological hallmarks of the disease, including neuritic plaques, although they have not evidenced neurofibrillary tangles and have limited cell death. These models facilitate investigation of the relationship of amyloid deposition to other aspects of the pathology of Alzheimer’s disease, including inflammation, hormonal levels, trophic factor influences, calcium metabolism, amino acid toxicity and apoptosis. However, there has been limited behavioural testing of transgenic mice, but impairments of memory have been reported on the Morris water maze, spatial reference memory and Y-maze alternation tasks.

Apolipoprotein knockout models

Cognitive tasks analogous to the deficits observed in human Alzheimer’s disease need to be developed for application to transgenic, knockout and other models currently used to investigate pathogenesis. Tests of language are obviously not applicable but assessment of attention, memory, spatial orientation and executive function are feasible. A variety of transgenic and knockout apolipoprotein models are available to screen novel molecules for the treatment of the disease. The E-4 allele of apolipoprotein (ApoE-4) confers an increased risk for Alzheimer’s disease and a decreased age of onset. Review of the cognitive testing and behavioural measures of the various available animal models reveals the impoverished state of these assessments and the need to develop new evaluation technologies.

Brain inflammation models

Epidemiological and clinical studies, reporting the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in reducing the incidence and progression of Alzheimer’s disease, provided strong support for the critical involvement of inflammatory processes in the pathogenesis of the disease. Brain inflammation is considered to be a pathogenetic link in many neurodegenerative diseases, including Alzheimer’s disease. The long-term lipopolysaccharide (LPS) infusion into the fourth ventricle was followed by astrocyte activation, an increase in microglia cells, an increase in the levels of interleukin (IL)-1β, tumour necrosis factor (TNF)-α, APP mRNA and the degeneration of hippocampal neurones.

In-vitro screening methods for acetylcholinesterase inhibition

Several methods have been reported for the screening of acetylcholinesterase inhibitory activity from herbal medicinal products (HMPs). Acetylcholinesterase inhibition was initially detected by the use of gut-bath pharmacological methods with isolated tissue preparations such as guinea pig ileum. These methods are costly in several respects, including time, animal tissue and amounts of compound needed, so they have been replaced by more sensitive chemical methods. Consideration of the relative merits of various methods that might be useful in studying the time course of acetylcholinesterase activity in very small tissue samples use a combined method reported by Koelle (1951) with a sulphydryl reagent studied by Ellman (1959). This Ellman method is extremely sensitive and is applicable to either small amounts of tissue or to low concentrations of enzyme.

Figure: The detection of acetylcholinesterase activity by Ellman’s method.

The principle of this colorimetric method is the measurement of the rate of production of thio-choline, as acetylthiocholine is hydrolysed by the acetylcholinesterase enzyme. Thiocholine reacts with Ellman reagent [5,5'-dithiobis-(2-nitro-benzoic acid) (DTNB)] to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate (Figure: The detection of acetylcholinesterase activity by Ellman’s method). This product has a yellow chromophore that can be detected at 405 nm. The reaction with the thiol has been shown to be sufficiently rapid so as not to be rate limiting in the measurement of the enzyme, and in the concentrations used does not inhibit the enzymatic hydrolysis. The absorbance obtained using a standard volume of a known concentration of the substrate with a fixed dose of acetylcholinesterase is compared with that in the presence of an added compound or extract, a significant reduction indicating an inhibitory role for the substance added.

This visible spectroscopy procedure requires several millilitres of reaction mixture and it was sometimes difficult to obtain enough material to show an effect. The development of the method for use on a smaller scale, using microtitre well plates and a microplate reader, has been introduced and has enabled determinations to be performed with a much higher throughput. Microtitre plate assay method requires smaller amounts of reagents and test substances.

The Ellman reaction has also been adapted for thin-layer chromatography (TLC) bioautography assay for acetylcholinesterase inhibitory activity. The TLC plate is developed in the usual way. After development, enzyme-inhibitory activities of the developed spots were detected by spraying the substrate, dye and enzyme based on Ellman’s method. After incubating the plates, white spots on a yellow background showed inhibition of acetylcholinesterase. A false-positive test is carried out, to confirm any acetylcholinesterase-inhibiting activity arising from inhibition of thiocholine hydrolysis caused by the enzyme. To detect false-positive reactions, the plate is sprayed with substrate, dye and enzyme without test compounds. After a few minutes incubation, a yellow background appeared, the occurrence of white spots indicating false-positive reactions.

A similar method for TLC detection has been introduced by Marston et al. (2002), which uses acetylnaphthol as the substrate and measures the amount of naphthol, the reaction product formed, by its chromogenic reaction with Fast Blue B salt.

High-performance liquid chromatography (HPLC) with online coupled ultraviolet, mass spectrometric and biochemical detection methods for the identification of acetylcholinesterase inhibitors has also been developed. This uses a reverse-phase column with the column eluate being split into two streams, one with an ultraviolet detector and the other connected to a biochemical detection system. This latter system consisted of the eluant being mixed with acetylcholinesterase and DTNB before the online introduction of acetylthiocholine. The intensity of the reaction product was measured at 405 nm by means of a spectrophotometer as an indication of the amount of thiocholine-DTNB product formed. A HPLC method for the detection of acetylcholinesterase inhibition on immobilised acetylcholinesterase column and high-performance liquid chromatography with online coupled ultraviolet, mass spectrometric and biochemical detection for acetylcholinesterase inhibitory activity has also been reported.

Related posts:

1. STRATEGY FOR SEARCHING THE LITERATURE The following sources of information were consulted to identify relevant Ginkgo trials: 1. Searching of electronic bibliographic databases: Medline, 1966-1997 (National Library of Medicine; Index Medicus; by means of SilverPlatter 3.0; emphasis on publications in medical journals); Embase, 1974-1997 (Excerpta Medica; emphasis on publications in medical journals); PsycLIT, 1972-1997 (by means of SilverPlatter 3.0; emphasis...
2. DESIGN OF THE CURRENT REVIEW Review Strategy: General Considerations For the current review neither the principles of criterion-based quality assessment, nor the rules of statistical pooling were applied. Instead a middle course was followed between the narrative approach and the criterion-based quality assessment approach. Through several tables, organized according to the type of health problem of interest, relevant information will...
3. GINKGO BILOBA EXTRACTS FOR THE TREATMENT OF CEREBRAL INSUFFICIENCY AND DEMENTIA   Ginkgo biloba special extract preparations are classified as antidementia drugs or nootropics. Nootropics are a heterogeneous group of drugs which, by various pharmacodynamic mechanisms, improve as final outcome disturbances of higher integrative noetic functions (e.g. memory, concentration, comprehension, attention, thinking and orientation) and impairment of vigilance. The main indication is dementia according to the...
4. CLINICAL TRIALS Impairment of Cerebral Function/Cerebral Insuffiency Impairment of cerebral function and cerebral insuffiency are not well-defined syndromes (in contrast to dementia or major depression). These terms are mainly used for arbitrarily naming a cluster of symptoms associated with ageing and were characteristic for early to moderate dementia at times when commonly accepted criteria for dementia did...