Until the beginning of this decade the antioxidant, melatonin, had been considered as little more than a tranquilizing hormone, responsible for regulating certain circadian and circannual rhythms. However, it is the discovery of melatonin as a free radical scavenger that has generated the most interest in recent years. The reduction of melatonin with age has been associated with neurodegenerative diseases such as Alzheimer’s disease (AD)and therefore, melatonin has been implicated to have an important clinical role in neuroprotection. Thus, for several years melatonin has attracted increasing attention from the general press with many advertisements touting this indoleamine to act as an aphrodisiac, rejuvenator, protector against diseases and a general wonder drug. However, melatonin formulations appear with no labelling for the correct storage conditions, dosage and side effects, as well as no control for purity and self-medicating with an unregulated product. In addition, there is much controversy surrounding the antioxidative properties of the indolemaine, 6-hydroxymelatonin (6-OHM). Therefore, the first part of this study aims to elucidate the physico-chemical and various stability characteristics of the pineal antioxidant, melatonin, while the second part is devoted to investigating the neuroprotective properties of the primary hepatic metabolite of melatonin, 6-OHM. The physical properties of melatonin were determined using various chemical techniques. This information served to both characterize and confirm the identity of melatonin raw material used in this study. In addition, this information serves to be essential as the physical properties of melatonin have not been reported in detail in literature, to date. Thereafter, using a validated high performance liquid chromatography (HPLC) method, the various physico-chemical and stability characteristics of melatonin were determined. Melatonin was shown to be extremely lipophilic, while the hygroscopic study indicates that melatonin raw material is extremely hygroscopic at temperatures above 40°C, whereas melatonin tablets are hygroscopic when left out of the original container. This study highlights the need for consumers to be aware of the proper storage of melatonin tablets to improve the stability and ensure long term integrity of the compound. Since, melatonin is most often administered orally, thus exposing it to a large variations in pH, within the gastrointestinal tract, it was decided to investigate the stability of melatonin over a range of pH’s and temperatures. The findings imply that melatonin is relatively stable at body temperature when ingested orally and that orally administered slow release preparations of melatonin should be relatively stable and therefore exhibit favourable bioavailability. However melatonin was shown to be unstable in solution. This provides important information and a challenge to the formulators of this drug substance in a liquid dosage form. An assessment of the photostability of melatonin dosage forms using International Committee on Harmonization (ICH) conditions revealed melatonin to be light sensitive and thus indicates a need for careful consideration of the packaging of these drug products. In addition a detailed assessment of the photochemistry and photoproducts formed during the UV photodegradation of melatonin is reported. Melatonin is shown to rapidly degrade in the presence of UV light, with the presence of oxygen accelerating the photodegradation. N1-acetyl-N2-formyl-5-methoxykynurenamine(AFMK) and 6-OHM were identified as the major photoproducts formed and these agents have been shown previously to retain antioxidant activity. One of the concerns of using melatonin in sunscreens is its photostability. However, it is reported in this study that the degraded solution of melatonin still possesses equipotent free radical scavenging ability as melatonin, despite the absence of melatonin in solution. In addition, melatonin is shown to reduce UV-induced oxidative stress in rat skin homogenate. Thus, these results make melatonin a likely candidate for inclusion in sunscreen preparations. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. 6-OHM is not only formed as the major hepatic metabolite of melatonin, but also when melatonin reacts with toxic radicals as well as UV light. Thus the second part of the study aims to elucidate and further characterize the mechanism behind 6-OHM’s neuroprotection. The results show 6-OHM to be a more potent singlet oxygen and superoxide anion scavenger than melatonin. In addition, the results show 6-OHM to offer protection against, oxidative stress and lipid peroxidation induced by several neurotoxins in the rat brain and hippocampus. The hippocampus is an important region of the brain responsible for the formation of memory and any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include quinolinic acid (QA) and iron (II). Histological studies undertaken reveal that 6-OHM is able to protect hippocampal neurons against QA and iron (II) induced necrotic cell death. Immunohistochemical investigations showed that QA moderately induces apoptotic cell death in the hippocampus which is inhibited by both melatonin and 6-OHM. The study sought to elucidate possible mechanisms by which 6-OHM exerts its neuroprotective capabilities and the results show 6-OHM to inhibit the action of cyanide on the mitochondrial electron transport chain (ETC), one of the most common sources of free radicals. In addition, 6-OHM treatment alone, increased ETC activity above basal control levels and the results show 6-OHM to increase complex I activity in the mitochondrial ETC. Electrochemical, ultraviolet/visible spectroscopy (UV/Vis) and HPLC assessment show that an interaction exists between 6-OHM and iron (III) and 6-OHM is able to reduce iron (III) to a more biologically usable form viz. iron (II) which can be incorporated into important biomolecules such as heme. One dire consequence of this interaction is the ready provision of iron (II) to drive the Fenton reaction. However the biological and histological assessments show 6-OHM to prevent iron (II)-induced lipid peroxidation and necrotic cell death and thus, provide evidence of its antioxidant properties. The results also show 6-OHM to promote Hsp70 induction in the hippocampus. Heat shock proteins, especially Hsp 70 plays a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. 6-OHM treatment alone and together with QA was shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that 6-OHM helps to protect against cellular protein damage induced by any form of stress the cell may encounter. Melatonin treatment alone and in combination with QA was shown to prevent increases in the level of Hsp70 in the hippocampus, indicating that melatonin was able to reduce oxidative stress induced by QA such that Hsp70 expression was not required. The discovery of neuroprotective agents, such as melatonin and 6-OHM, is becoming important considering the rapid rise in the elderly population and the proportionate increase in neurological disorders. The findings of this study indicate the need for important information regarding the correct storage conditions and stability characteristics of melatonin dosage forms. In addition, the results indicate that 6-OHM has a definite role to play as an antioxidant. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders.

Copper is an extremely toxic metal in biological systems and thus, its availability to the system, must be effectively and efficiently controlled. Copper is vital for life, as it is essential for critical enzymes in biological systems. It is free copper in the biological systems that is toxic, as free copper induces free radical generation, which disrupts lipid membranes, interacts with DNA causing mutations, and eventually leads to cell death. Wilson’s disease is a inherited copper disease, which results in hepatolenticular disease. Copper is unable to be excreted, and thus accumulates, eventually spilling over into the bloodstream from the liver, and “poisons” the patient. The Wilson’s disease patient leads a short life, due to neurological and hepatological problems. There is no cure for Wilson’s disease, only chelation therapy using potent chelators such as penicillamine and EDTA. Zinc, in high doses, can be used to compete with copper absorption. This has proved to be the only successful therapy at present. This study investigates the possible use of melatonin as a copper binder/chelator. Melatonin has been shown to interact with copper in vitro. By binding/chelating to copper, melatonin may inhibit copper-induced free radical generation, and thus prevent copper from interacting with DNA to cause mutations and act as a cytotoxin. In vivo studies on copper (2mg/kg) administered for 2-weeks and 6-weeks were carried out on Wistar rats. The potential of melatonin (12mg/kg) to prevent copper-induced cellular damage was investigated. The results indicate that melatonin does not protect the lipid membranes from copper-induced lipid peroxidation. In vitro investigations using 1mM, 5mM and 10mM copper and 5mM melatonin, show that melatonin prevents copper-induced lipid peroxidation at a copper concentration of 1mM (p<0.001). The 5mM and 10mM copper induces less lipid peroxidation, compared to the 1mM copper. It has been reported that metal ions, antioxidants and chelating agents can influence peroxide decomposition during the assay. Melatonin (5mM) administration does not significantly prevent copper-induced lipid peroxidation at 5mM and 10mM copper. It is possible that due to melatonin’s relatively low concentration, it is unable to inhibit lipid peroxidation induced by the copper. The chemical nature of the interaction between melatonin and copper was also investigated, using NMR, IR and electrochemistry techniques. The NMR and IR techniques show that melatonin coordinates with Cu²⁺ and not Cu¹⁺, at the carbonyl group of melatonin. The electrochemistry experiments using cyclic voltammetry and adsorptive stripping voltammetry, show that melatonin forms a strong bond with Cu¹⁺. Cu²⁺ prefers binding to oxygen, and that is clearly seen in the NMR and IR. Cu¹⁺ prefers binding to nitrogen and then oxygen, and this is seen in the electrochemistry, as Cu¹⁺ is forced to bind through one of the nitrogens on the melatonin. Previously, it has been shown that melatonin binds/chelates with Cu²⁺. Histochemical investigations show that copper administration for 2-weeks and 6-weeks, causes extensive mitochondrial damage in liver and kidney’s proximal convoluted tubule epithelium cells. Melatonin (12mg/kg) co-administration with copper for 2-weeks and 6-weeks did not significantly protect the mitochondria from copper-induced damage. Copper-specific stains (rhodanine, silver sulphide and rubeanic acid) were used to stain liver, brain and kidney tissue samples. Rhodanine and silver sulphide were equally sensitive in staining copper in the 2-week samples, but not at all in the 6-week samples. This could not be explained. Rubeanic acid was ineffective in all samples tested. Thus, it appears that specific copper stains cannot be used in making a definitive diagnosis in cases of copper overload, and that specific copper stains do not always correlate with a high concentration of copper present in tissues. Pineal organ culture was used to determine the effect of copper administration on pineal indole synthesis. Exogenous (³H) tryptophan was administered to the pineal organ cultures, and the level of (³H) pineal indoles synthesised, were measured. Pineals from 2-week and 6-week copper/melatonin treated animals exhibited paradoxical 5- methoxytryptophol (ML) levels, as compared to the 2-week and 6-week copper treated animals. The 2-week copper/melatonin administered animals, showed a decrease in the ML level (p<0.01), and the copper/melatonin administered for 6-weeks, showed an increase in the ML levels (p<0.01). This indicates that melatonin interacts with the HIOMT enzyme. Pineals from 6-week copper/melatonin treated animals, as compared to the 6-week copper treated animals, showed an increase in N-acetylserotonin levels. This indicates that melatonin prevents the inhibition of the NAT enzyme. The final experiment was to determine in vitro, the effect of Cu²⁺ and Cu¹⁺ administration, on mitochondrial electron transport chain. Rat liver homogenate was incubated with and solutions of Cu²⁺ (10mM) and Cu¹⁺ (10mM) and melatonin (10mM). Cu²⁺ administration caused an inhibition of the electron transport at t=0 and t=60, whereas Cu¹⁺ administration at t=0 caused an inhibition of electron transport, but at t=60, Cu¹⁺ administration stimulated electron transport. Melatonin administered with Cu²⁺, resulted in an inhibition of the electron transport chain at t=0 and t=60. The findings of this study indicate that melatonin might have a potentially beneficial effect in copper overloading, by binding/chelating copper.

Parkinson’s disease, one of the most common neurodegenerative disorders associated with ageing, is characterised by abnormal and profound loss of nigrostriatal dopaminergic neurons. The cause of Parkinson’s disease is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate oxidative damage and mitochondrial impairment, particularly at the level of complex I enzyme. Recently, rotenone, a commonly used organic pesticide and a classical inhibitor of mitochondrial complex I has been reported to reproduce the specific features of Parkinson’s disease in rodents. The mitochondrial respiratory chain is one of the most important sites of reactive oxygen species production under physiological conditions. Toxic free radicals have been implicated in a variety of neurodegenerative diseases as well as ageing itself. Melatonin, a secretory product of the pineal gland is a multifaceted free radical scavenger and natural antioxidant. In the present study, the neuroprotective effects of melatonin against the environmental neurotoxin, rotenone was investigated. Initial studies showed that inhibition of mitochondrial complex I enzyme by rotenone induced superoxide radical generation. Melatonin, administered to the rat in vivo and in vitro was able to offer neuroprotection by curtailing the production of superoxide radicals induced by rotenone. Mitochondria, being the major target of rotenone, the effects of melatonin were investigated at the mitochondrial level. Melatonin was able to increase the electron transport chain activity thus preventing the respiratory inhibition by rotenone. The pineal hormone also counteracted the action of rotenone on complex I enzyme. These results suggest melatonin’s ability to potentially limit the free radical generation and thereby modulate the mitochondrial functions. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. Melatonin also offered significant protection in vivo and in vitro against rotenone induced lipid peroxidation. Since iron plays a major role in oxidative damage and in the progression of Parkinson’s disease, the effect of melatonin on both rotenone and iron induced lipid peroxidation was investigated, the results of which show that melatonin affords protection and this was suggested to be due to its interaction with the rotenone-iron complex that might have formed. Electrochemical studies were further used to characterise the interactions between melatonin, rotenone and iron (III). Melatonin was shown to bind with iron and thus reducing their toxicity. Histological studies were undertaken to assess the effects of melatonin on rotenone induced toxicity on the dopaminergic neurons in the rat brain. Rotenone treated brains showed extensive neuronal damage whereas with melatonin less damage was observed. Rotenone induces apoptosis via reactive oxygen species production and apoptotic cell death has been identified in PD brains. Furthermore, the apoptotic cell death was detected and quantified by the TUNEL staining. Rotenone treated sections showed signs of apoptosis whereas with melatonin, less apoptotic damage was observed. The findings of this study indicate that the neurohormone, melatonin may protect against rotenone-induced neurodegeneration. Since melatonin production falls substantially during ageing, the loss of this antioxidant is theorized to be instrumental in the degenerative processes associated with advanced age. Considering how devastating diseases such as Parkinson’s disease, are to a patient and the patient’s families, the discovery of protective agents are a matter of urgency. Further investigations using the pesticide model will help to determine the involvement of environmental exposure in the pathogenesis of human diseases as well as to test therapeutic strategies for the treatment of such diseases.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Alzheimer's disease is a progressive neurodegenerative disorder, in which there is a marked decline in neurotransmitters, especially those of the cholinergic pathways. One of the approaches to the symptomatic treatment of Alzheimer's disease is the inhibition of the breakdown of the neurotransmitter acetylcholine, using an acetylcholinesterase inhibitor. One such drug tested, is the organophosphate, metrifonate. Any drug used for the treatment of neurodegenerative disorders should preferably not induce further neurological damage. Thus, in the present study, we investigated whether or not metrifonate is neuroprotective. The in vivo and in vitro effect of this drug on free radicals generation shows that metrifonate increases the level ofthese reactive species. Lipid peroxidation induced using quinolinic acid (QA) and iron (II) and show that metrifonate increased the peroxidative damage induced by using quinolinic acid. Metrifonate is also able to induce lipid peroxidation both in vivo and in vitro. This was reduced in vitro in the presence of melatonin. Using iron (II), in vi/ro, there was no significant difference in the level of lipid peroxidation in the presence of this drug. An investigation of the activity of the mitochondrial electron transport chain and complex I of the electron transport chain in the presence of metrifonate revealed that metrifonate reduces the activity of the electron transport chain at the level of complex I. The activity of the mitochondrial electron transport chain was restored in the presence of melatonin. Pineal organ culture showed that metrifonate does not increase melatonin production. Histological and apoptosis studies show that tissue necrosis and apoptosis respectively, occur in the presence of this agent, which is reduced in the presence of melatonin. Metal binding studies were performed USing ultraviolet spectroscopy, and electrochemical analysis to examine the interaction of metrifonate with iron (II) and iron (III). No shift in the peak was observed in the ultraviolet spectrum when iron (ll) was added to metrifonate. Electrochemical studies show that there may be a very weak or no ligand formed between the metal and drug. This study shows that while drugs such as metrifonate may be beneficial in restoring cognitive function in Alzheimer's disease, it could also have the potential to enhance neurodegeneration, thus worsening the condition, in the long term. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Stroke is one of the most common causes of disability and death worldwide. The most commonly experienced stroke in the clinical setting is focal ischaemia in which the middle cerebral artery (MCA) is occluded and leads to a complex series of various pathophysiological pathways that ultimately lead to neuronal cell death. Several studies have been conducted on various therapeutic agents in the search for a neuroprotective drug and various animal models have been used to carry out this research. While theanine, a component of green tea and minocycline, a tetracycline antibiotic, have been shown to possess some neuroprotective properties, the mechanisms by which these two agents carry out these effects still remains unclear. The objectives of this study were to investigate the mechanisms by which these drugs carry out these neuroprotective effects and their neuroprotective ability in a MCA occlusion model of focal ischaemia. Ischaemia leads to oxidative stress due to the imbalance of free radicals and the endogenous antioxidant defence system. An antioxidant assay using the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH●) radical was used to assess the antiradical properties of each drug. It was found that minocycline showed superior antioxidant activity in vitro when compared to theanine. Further studies on the drugs‟ ability to attenuate the Fenton reaction (in which iron catalyses the formation of reactive species) were elucidated using electrochemical analysis, UV/VIS studies, ferrozine and ferritin assays. It was found that minocycline, in contrast to theanine, was able to bind to iron ions and thus potentially prevent the participation of iron in metal catalysed radical reaction. The antioxidant activity of both drugs was further investigated by assessing their effect on cyanide-induced superoxide generation and quinolinic acid (QA)-induced lipid peroxidation (LP). Experimental evidence shows that both drugs had no significant effect on the generation of superoxide in vitro and that there was a significant decrease in LP for minocycline in vitro and theanine in vivo. The metal binding and antioxidant properties were postulated to be a possible mechanism through which these agents reduced lipid peroxidation. A study was conducted to determine the effects of the drugs on the biosynthesis of the neurotoxin, QA and it was found that minocycline increases the levels of holoenzyme activity of tryptophan-2, 3-dioxygenase (TDO) in vitro and that theanine reduces the levels of the same enzyme in vivo after treatment for 10 days. TDO is the enzyme that converts tryptophan to other products that enable enzymatic activity to change it to QA. Minocycline was thought to bring about this effect as it has been shown from preceding experimental studies that it is an effective reducing agent. Theanine on the other hand is hypothesised to bring about a reduction in holoenzyme activity by changing the binding of tryptophan to the enzyme or affecting the radicals that participate in the enzymatic degradation of tryptophan. A focal ischaemic model of stroke was induced by occluding the MCA. Histological examination of the hippocampus post -ischaemia shows a reduction in the size of the infarct after pre-treatment with minocycline only. A further study into the effects of the drugs on the generation of superoxide and on the levels of the endogenous glutathione after a stroke was carried out. Pre-treatment of the animals with either theanine or minocycline showed no significant effects on the generation of the radical species or of the endogenous antioxidant which ruled out these as a mechanism of neuroprotection of both drugs, post-ischaemia.The findings of this study provide novel information on the possible mechanisms by which both theanine and minocycline act to bring about neuroprotection. In particular in this study, pre-treatment with minocycline has shown promise in the focal ischaemic model of stroke.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a profound loss of dopaminergic neurons from the substantia nigra (SN). Among the many pathogenic mechanisms thought to be responsible for the demise of these cells, dopamine (DA)-dependent oxidative stress and oxidative damage has taken center stage due to extensive experimental evidence showing that DA-derived reactive oxygen species (ROS) and oxidized DA metabolites are toxic to SN neurons. Despite its being the most efficacious drug for symptom reversal in PD, there is concern that levodopa (LD) may contribute to the neuronal degeneration and progression of PD by enhancing DA concentrations and turnover in surviving dopaminergic neurons. The present study investigates the potential neurotoxic and neuroprotective effects of DA in vitro. These effects are compared to the toxicity and neuroprotective effects observed in the rat striatum after the administration of LD and selegiline (SEL), both of which increase striatal DA levels. The effects of exogenous LD and/or SEL administration on both the oxidative stress caused by increased striatal iron (II) levels and its consequences have also been investigated. 6-Hydroxydopamine (6-OHDA) is a potent neurotoxin used to mimic dopaminergic degeneration in animal models of PD. The formation of 6-OHDA in vivo could destroy central dopaminergic nerve terminals and enhance the progression of PD. Inorganic studies using high performance liquid chromatography with electrochemical detection (HPLC-ECD) show that hydroxyl radicals can react with DA to form 6-OHDA in vitro. SEL results in a significant decrease in the formation of 6-OHDA in vitro, probably as a result of its antioxidant properties. However, the exogenous administration of LD, with or without SEL, either does not lead to the formation of striatal 6-OHDA in vivo or produces concentrations below the detection limit of the assay. This is despite the fact that striatal DA levels in these rats are significantly elevated (two-fold) compared to the control group. The auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA causes an increase in the production of superoxide anions in whole rat brain homogenate in vitro. In addition to this, DA is able to enhance the production of hydroxyl radicals by Fenton chemistry (Fe(III)-EDTA/H2O2) in a cell free environment. Treatment with systemic LD elevates the production of striatal superoxide anions, but does not lead to a detectable increase in striatal hydroxyl radical production in vivo. The co-adminstration of SEL with LD is able to prevent the LD induced rise in striatal superoxide levels. It has been found that the presence of DA or 6-OHDA is able to reduce lipid peroxidation in whole rat brain homogenate induced by Fe(II)-EDTA/H2O2 and ascorbate (Fenton system). However, DA and 6-OHDA increase protein oxidation in rat brain homogenate, which is further increased in the presence of the Fenton system. In addition to this, the incubation of rat brain homogenate with DA or 6-OHDA is also accompanied by a significant reduction in the total GSH content of the homogenate. The exogenous administration of LD and/or SEL was found to have no detrimental effects on striatal lipids, proteins or total GSH levels. Systemic LD administration actually had a neuroprotective effect in the striatum by inhibiting iron (II) induced lipid peroxidation. Inorganic studies, including electrochemistry and the ferrozine assay show that DA and 6-OHDA are able to release iron from ferritin, as iron (II), and that DA can bind iron (III), a fact that may easily impede the availability of this metal ion for participation in the Fenton reaction. The binding of iron (III) by DA appears to discard the involvement of the Fenton reaction in the increased production of hydroxyl radicals induced by the addition of DA to mixtures containing Fe(II)-EDTA and hydrogen peroxide. 6-OHDA did not form a metal-ligand complex with iron (II) or iron (III). In addition to the antioxidant activity and MAO-B inhibitory activity of SEL, the iron binding studies show that SEL has weak iron (II) chelating activity and that it can also form complexes with iron (III). This may therefore be another mechanism involved in the neuroprotective action of SEL. The results of the pineal indole metabolism study show that the systemic administration of SEL increases the production of N-acetylserotonin (NAS) by the pineal gland. NAS has been demonstrated to be a potent antioxidant in the brain and protects against 6-OHDA induced toxicity. The results of this study show that DA displays antioxidant properties in relation to lipid eroxidation and exhibits pro-oxidant properties by causing an increase in the production of hydroxyl radicals and superoxide anions, as well as protein oxidation and a loss of total GSH content. Despite the toxic effects of DA in vitro, the treatment of rats with exogenous LD does not cause oxidative stress or oxidative damage. The results also show that LD and SEL have some neuroprotective properties which make these agents useful in the treatment of PD.

Accumulating evidence suggests that quinolinic acid has a role to play in disorders involving impairment of learning and memory. In the present study, the effect of the guanosine analogue antiherpetic, acyclovir, on quinolinic acid-induced spatial memory deficits was investigated, as well as some of the mechanisms which underlie this effect. Behavioural studies using a Morris water maze show that post-treatment of rats with acyclovir significantly improves spatial memory deficits induced by intrahippocampal injections of quinolinic acid. Histological analysis of the hippocampi show that the effect of acyclovir is related to its ability to alleviate quinolinic acid-induced necrotic cell death, through interference with some of the mechanisms of neurodegeneration. However, acyclovir is unable to alter a quinolinic acid-induced increase in glutamate release in the rat hippocampus, even though it alleviates quinolinic acid induced oxidative stress by scavenging the superoxide anion in vitro and in vivo in whole rat brain and hippocampus respectively. Due to the inverse relationship which exists between superoxide anion and glutathione levels, acyclovir also curtails the quinolinic acid-induced decrease in hippocampal glutathione levels. Acyclovir suppresses quinolinic acid-induced lipid peroxidation in vitro and in vivo, in whole rat brain and hippocampus respectively, through its alleviation of oxidative stress and possibly through the binding of iron (II) and / or iron (III), preventing the participation and redox recycling of iron (II) in the Fenton reaction, which quinolinic acid is thought to enhance by weak binding of ferrous ions. This argument is further strengthened by the ability of the drug to suppress iron (II)-induced lipid peroxidation in vitro directly. Inorganic studies including ultraviolet and visible spectroscopy, electrochemistry and the ferrozine assay show that acyclovir binds to iron (II) and iron (III) and that quinolinic acid forms an easily oxidisable association with iron (II). Acyclovir inhibits the endogenous biosynthesis of quinolinic acid by inhibiting the activity of liver tryptophan-2,3-dioxygenase, intestinal indoleamine-2,3-dioxygenase and rat liver 3-hydroxyanthranillic acid oxygenase in vitro and in vivo, possibly through competitive inhibition of haeme, scavenging of superoxide anion and binding of iron (II) respectively. An inverse relationship exists between tryptophan-2,3-dioxygenase activity and brain serotonin levels. Acyclovir administration in rats induces a rise in forebrain serotonin and 5-hydroxyindole acetic acid and reduces the turnover of forebrain serotonin to 5-hydroxyindole acetic acid. Furthermore, it shows that acyclovir does not alter forebrain norepinephrine levels. The results of the pineal indole metabolism study show that acyclovir increases 5-hydroxytryptophol, N-acetylserotonin and the neurohormone melatonin, but decreases 5-hydroxyindole acetic acid. The results of this study show that acyclovir has some neuroprotective properties which may make it useful in the alleviation of the anomalous neurobiology in neurodegenerative disorders.

Hybrid neural-microelectronic systems, systems composed of biological neural networks and neuronal models, have great potential for the treatment of neural injury and disease. The utility of such systems will be ultimately determined by the ability of the engineered component to correctly replicate the function of biological neural networks. These models can take the form of mechanistic models, which reproduce neural function by describing the physiologic mechanisms that produce neural activity, and empirical models, which reproduce neural function through more simplified mathematical expressions. We present our research into the role of model complexity in creating robust and flexible behaviors in hybrid systems. Beginning with a complex mechanistic model of a leech heartbeat interneuron, we create a series of three systematically reduced models that incorporate both mechanistic and empirical components. We then evaluate the robustness of these models to parameter variation, and assess the flexibility of the models activities. The modeling studies are validated by incorporating both mechanistic and semi-empirical models in hybrid systems with a living leech heartbeat interneuron. Our results indicate that model complexity serves to increase both the robustness of the system and the ability of the system to produce flexible outputs.

AIDS Dementia Complex (ADC) is a neurodegenerative disorder implicated in HIV-1 infection that is associated with elevated levels of the neurotoxin, quinolinic acid (QA) which causes a cascade of events to occur, leading to the production of reactive oxygen species (ROS), these being ultimately responsible for oxidative neurotoxicity. In clinical studies, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP) have been shown to potentially delay the progressive degeneration of neurons, thus reducing the frequency and neurological deficits associated with ADC. Despite these neuroprotective implications, there is still no biochemical data to demonstrate the mechanisms through which these agents offer neuroprotection. The present study aims to elucidate and further characterize the possible antioxidant and neuroprotective mechanisms of NVP and EFV in vitro and in vivo, using QA-induced neurotoxicity as a model. Research has demonstrated that antioxidants and metal chelators have the ability to offer neuroprotection against free radical induced injury and may be beneficial in the prevention or treatment of neurodegeneration. Hence the antioxidant and metal binding properties of these agents were investigated respectively. Inorganic studies, including the 1, 1-diphenyl-2 picrylhydrazyl (DPPH) assay, show that these agents readily scavenge free radicals in vitro, thus postulating the antioxidant property of these agents. The enhancement of superoxide radical generation and iron mediated Fenton reaction by QA is related to lipid peroxidation in biological systems, the extent of which was assayed using the nitroblue tetrazolium and thiobarbituric acid method respectively. Both agents significantly curtail QA-induced lipid peroxidation and potentially scavenge superoxide anions generated by cyanide in vitro. Furthermore, in vivo results demonstrate the ability of NVP and EFV to protect hippocampal neurons against lipid peroxidation induced by QA and superoxide radicals generated as a consequence thereof. The alleviation of QA-induced oxidative stress in vitro possibly occurs through the binding of iron (II) and / or iron (III), and this argument is further strengthened by the ability of EFV and not NVP to reduce iron (II)-induced lipid peroxidation in vitro directly. In addition the ferrozine and electrochemistry assay were used to measure the extent of iron (II) Fe[superscript 2+] and iron (III) Fe[superscript 3+] chelation activity. Both assays demonstrate that these agents bind iron (II) and iron (III), and prevent redox recycling of iron and subsequent complexation of Fe[superscript 2+] with QA which enhances neuronal damage. Both NNRTIs inhibit the endogenous biosynthesis of QA by inhibiting liver tryptophan 2, 3-dioxygenase activity in vivo and subsequently increasing hippocampal serotonin levels. Furthermore, these agents reduce the turnover of hippocampal serotonin to 5-hydroxyindole acetic acid. NVP and not EFV increase 5-hydroxyindole acetic acid and norepinephrine levels in the hippocampus. The results of the pineal indole metabolism study show that NVP increases the synthesis of melatonin, but decreases N-acetylserotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol levels. Furthermore, it shows that EFV decreases 5-hydroxyindole acetic acid and melatonin synthesis. Behavioural studies using a Morris water maze show that the post-treatment of rats with NVP and EFV significantly improves QA-induced spatial memory deficits in the hippocampus. This study therefore provides novel information regarding the neuroprotective mechanisms of NVP and EFV. These findings strengthen the argument that these NNRTIs not only have antiviral effects but possess potential neuroprotective properties, which may contribute to the effectiveness of these drugs in the treatment of ADC.