Where will the next generation of stroke treatments come from?

Remarkable progress has occurred over the last two decades in stroke interventions. Many have been developed on the basis of their efficacy in other disorders. This "inheritance" approach should continue, but two areas where completely novel therapeutic targets might emerge are the stimulation of neuroplasticity and unraveling the genetic code of stroke heterogeneity (Table 2). For the former, the next steps are to identify small-molecule, nontoxic compounds that most effectively enhance plasticity in animal models, and then subject them to clinical trial in humans. For the latter, more and larger-scale cooperative GWASs in carefully phenotyped stroke populations are required to better understand the polygenic nature of cerebrovascular disease. Then, the physiological relevance of genetic abnormalities can be determined in in vitro and in vivo systems before candidate compounds are developed.

Analysis of sequence contexts flanking T.G mismatches leads to predictions about reactivity of the mismatched T to osmium tetroxide.

Osmium tetroxide and hydroxylamine are used to detect mutations in DNA and RNA after hybridization of mutant and wild-type DNA. Mismatched T and C bases, respectively, are modified by these reagents and the DNA strand cleaved at the mismatched bases by subsequent treatment with piperidine. This allows detection and location of the mutation. Although most T.G mismatches have been reported to be reactive to osmium tetroxide, some have been reported to be unreactive. The aim of this study was to collect and analyze the reactive and unreactive T.G mismatches. We have collected sequence contexts of all reactive and unreactive T.G mismatches for analysis. This involves 10 unreactive T.G mismatches (plus one T.C) and 19 reactive T.G mismatches. Sequence effects of bases surrounding these mismatches must influence this reactivity. There must be many types of such sequence effects. We postulate that because of the dominance of 5' G bases near the T of unreactive T.G mismatches and the absence of 5' G bases in reactive T.G mismatches that the stacking of the 5' G on the mismatched T is the reason for this lack of reactivity in the majority of the cases studied here.

Leukaemia inhibitory factor prevents injury induced proliferation of striatal dopamine uptake sites.

The injury associated with implantation of an inert gelatin matrix (gel foam) into normal mouse striatum induces a long-lived increase in binding of [3H]mazindol to presynaptic dopamine uptake sites, probably due to proliferation of striatal dopaminergic terminals. Because of the known effects of leukaemia inhibitory factor (LIF) on catecholaminergic cells, we tested the hypothesis that LIF may alter the striatal dopaminergic response to injury in vivo. Application of LIF to mouse striatum in a gel foam implant abolished the usual injury induced proliferation of dopamine uptake sites. The ability of LIF to prevent proliferation of dopamine terminals may have important implications for our understanding of neural regeneration, the aetiology of Parkinson's disease and its treatment by intrastriatal grafting.

Expression of glial cell line-derived neurotrophic factor (GDNF) mRNA following mechanical injury to mouse striatum.

Although glial cell line-derived neurotrophic factor (GDNF) expression is low in the adult brain, its administration protects dopaminergic neurons against a range of insults, leading to the suggestion of a role in dopaminergic regeneration. If locally produced GDNF is to fulfil a role in dopaminergic regeneration after injury, it seems reasonable to hypothesize that its expression will increase after mechanical trauma. We have demonstrated that GDNF mRNA expression increases within 6 h of using a wire knife to injure adult mouse striatum. Expression doubles after 1 week and remains elevated for at least 1 month. Most GDNF expression is associated with haemosiderin-containing cells, indicating production by brain macrophages. GDNF production by macrophages may be essential for neural regeneration following CNS trauma.

Intrastriatal angiotensin II induces turning behaviour in 6-hydroxydopamine lesioned rats.

In rats with unilateral 6-hydroxydopamine lesions in the nigrostriatal pathway, injection of angiotensin II (2 nmol) into the unlesioned striatum elicited dose-related tight rotations ipsilateral to the lesion. This rotation was suppressed by coadministration of the angiotensin AT1 receptor antagonist, losartan (2 nmol), which had no significant effect when injected alone. Preadministration of the dopamine antagonist, haloperidol (2 mg/kg i.p.) completely blocked angiotensin II-induced turning at doses of 0.3-3 nmol, and partially at 10 nmol. These results further confirm the hypothesis that Ang II is intrinsically involved in modulating dopamine release in the striatum, an effect which is mediated predominantly by AT1 receptors.

Surgical damage stimulates proliferation of dopamine uptake sites in normal mouse brain.

To test the hypothesis that proliferation of host dopaminergic tissue in response to injury plays an important role in the response to intrastriatal grafting, we transplanted autologous adrenal medullary to striatum in normal C57-black mice and compared this procedure with transplantation of non-dopaminergic tissue (frontal cortex) or a non-cellular matrix (Gelfoam). [3H]Mazindol autoradiography revealed that all three protocols resulted in a marked proliferation of dopamine uptake sites 10 months after transplantation.

Dopaminergic responses to striatal damage.

The improvements obtained by grafting dopamine-rich tissues into the striatum of patients with Parkinson's disease are generally attributed to production and release of dopamine by the graft. However, it is becoming increasingly clear that grafting also stimulates the host dopaminergic system. We provide evidence in a mouse model of striatal damage that surgical cavitation induces a concerted response from the dopaminergic system with proliferation of striatal presynaptic dopamine uptake sites, increased tyrosine hydroxylase activity, increased concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid. The response increases with time and ultimately includes contralateral stimulation of striatal tyrosine hydroxylase activity and elevation of dihydroxyphenylacetic acid and homovanillic acid concentrations. The time course and extent of the host dopaminergic response suggests that it may make a significant contribution to observed clinical improvements after intrastriatal transplantation in human parkinsonism.

Direct analysis of tetrahydrobiopterin in cerebrospinal fluid by high-performance liquid chromatography with redox electrochemistry: prevention of autoxidation during storage and analysis.

Measurement of tetrahydrobiopterin in cerebrospinal fluid requires careful handling of samples during storage and analysis. Addition of dithioerythritol and deoxygenation of the mobile phase with helium prevents breakdown of tetrahydrobiopterin during chromatography. Tetrahydrobiopterin in cerebrospinal fluid is unstable at room temperature, 100% being lost within 3.5 h, this breakdown does not generate equivalent quantities of dihydrobiopterin and biopterin. Addition of dithioerythritol and diethylenetriaminepenta-acetic acid to cerebrospinal fluid prevents breakdown of tetrahydrobiopterin for 6 mth at -70 degrees C and for up to 5 h at 4 degrees C. At room temperature less than 5% of tetrahydrobiopterin was lost after 2 h.

A simple automated injection technique for the high-pressure liquid chromatographic determination of plasma retinol.

We describe a rapid automated method for determination of retinol in 100 microliters plasma. A lipid extract of plasma is analysed by reverse phase high pressure liquid chromatography using methanol-water as eluent. Automation of injection is achieved by adaptation of a Technicon AutoAnalyzer Sampler 2 coupled with a Micromedic dilution system and a pneumatically actuated loop injector. Twenty patients' specimens can be analysed in 4 hours, including extraction time, standards, and quality controls, with one extract injected every 4 minutes.

Meta-analysis of data from animal studies: a practical guide.

Meta-analyses of data from human studies are invaluable resources in the life sciences and the methods to conduct these are well documented. Similarly there are a number of benefits in conducting meta-analyses on data from animal studies; they can be used to inform clinical trial design, or to try and explain discrepancies between preclinical and clinical trial results. However there are inherit differences between animal and human studies and so applying the same techniques for the meta-analysis of preclinical data is not straightforward. For example preclinical studies are frequently small and there is often substantial heterogeneity between studies. This may have an impact on both the method of calculating an effect size and the method of pooling data. Here we describe a practical guide for the meta-analysis of data from animal studies including methods used to explore sources of heterogeneity.

Inhibiting BDNF expression by antisense oligonucleotide infusion causes loss of nigral dopaminergic neurons.

Brain derived neurotrophic factor (BDNF) expression is significantly reduced in the Parkinson's disease substantia nigra. This neurotrophin has potent affects on dopaminergic neuron survival protecting them from the neurotoxins MPTP and 6-hydroxydopamine (6-OHDA) commonly used to create animal models of Parkinson's disease and also promoting dopaminergic axonal sprouting. In this study, we demonstrate that an antisense oligonucleotide infusion (200 nM for 28 days) to prevent BDNF production in the substantia nigra of rats mimics many features of the classical animal models of Parkinson's disease. 62% of antisense treated rats rotate (P < or = 0.05) in response to dopaminergic receptor stimulation by apomorphine. 40% of substantia nigra pars compacta tyrosine hydroxylase immunoreactive neurons are lost (P < or = 0.00001) and dopamine uptake site density measured by (3)H-mazindol autoradiography is reduced by 34% (P < or = 0.005). Loss of haematoxylin and eosin stained nigral neurons is significant (P < or = 0.0001) but less extensive (34%). These observations indicate that loss of BDNF expression leads both to down regulation of the dopaminergic phenotype and to dopaminergic neuronal death. Therefore, reduced BDNF mRNA expression in Parkinson's disease substantia nigra may contribute directly to the death of nigral dopaminergic neurons and the development of Parkinson's disease.

Analysis and clinical significance of pterins.

This review briefly describes the biochemistry of pterins, their involvement in pathological processes and the use of pterin measurement in diagnosis and monitoring of disease. Chromatographic and other methods of pterin analysis are detailed with particular emphasis being placed on the need for correct sample collection and handling.

A fluorimetric assay for succinic semialdehyde dehydrogenase activity suitable for prenatal diagnosis of the enzyme deficiency.

Succinic semialdehyde dehydrogenase (SSAD) is an enzyme involved in the turnover of the neurotransmitter 4-aminobutyrate (GABA). Deficiency of SSAD results in developmental delay, ataxia, seizures and 4-hydroxybutyric aciduria. We have developed a simple fluorimetric assay for the enzyme and applied it to measurement of SSAD activity in a range of cell types often used for prenatal and postnatal diagnosis of enzyme defects. Lymphocytes from children with SSAD deficiency were found to have < 5% of the activity found in lymphocytes from normal children. Heterozygotes are asymptomatic and have intermediate enzyme activities. Although SSAD activity has been detected previously in uncultured chorionic villi, we found that SSAD was not expressed in cultured chorionic villus cells nor in some fibroblast-like amniocytes from control fetuses. Lymphocytes from fetal blood and non-fibroblastic amniocytes have high SSAD activities, and should be suitable for prenatal diagnosis of SSAD deficiency.

Estimation of tetrahydrobiopterin and other pterins in cerebrospinal fluid using reversed-phase high-performance liquid chromatography with electrochemical and fluorescence detection.

We describe an isocratic, reversed-phase high-performance liquid chromatographic method for the simultaneous measurement of fully oxidised, dihydro- and tetrahydropterins in cerebrospinal fluid. Tetrahydrobiopterin is detected electrochemically using an ESA Coulochem detector in the redox mode. Dihydropterins are detected by fluorescence following post-column electrochemical oxidation, and fully oxidised pterins by their natural fluorescence. Apart from addition of antioxidants, no sample preparation is required. Comparison is made with methods requiring chemical oxidation for detection of tetrahydrobiopterin. Some results on children with neurological disease are presented.

Pteridines and mono-amines: relevance to neurological damage.

Patients with phenylalanine hydroxylase deficiency show increased concentrations of biopterins and neopterins, and reduced concentrations of serotonin and catecholamines, when phenylalanine concentrations are raised. The pterin rise reflects increased synthesis of dihydroneopterin and tetrahydrobiopterin, and the amine fall a reduction in amine synthesis due to inhibition by phenylalanine of tyrosine and tryptophan transport into neurones. The pterin and amine changes appear to be independent of each other and are present in the central nervous system as well as the periphery; they disappear when phenylalanine concentrations are reduced to normal. Patients with arginase deficiency show a similar amine disturbance but have normal pterin levels. The amine changes probably contribute neurological symptoms but pterin disturbance is not known to affect brain function. Patients with defective biopterin metabolism exhibit severely impaired amine synthesis due to tetrahydrobiopterin deficiency. Pterin concentrations vary with the site of the defect. Symptoms include profound hypokinesis and other features of basal ganglia disease. Neither symptoms nor amine changes are relieved by controlling phenylalanine concentrations. Patients with dihydropteridine reductase (DHPR) deficiency accumulate dihydrobiopterins and develop secondary folate deficiency which resembles that occurring in patients with defective 5,10-methylene tetrahydrofolate reductase activity. The latter disorder is also associated with Parkinsonism and defective amine and pterin turnover in the central nervous system, and a demyelinating illness occurs in both disorders. In DHPR deficiency cerebral calcification may develop in a similar distribution to that seen in congenital folate malabsorption and methotrexate toxicity. Symptoms are ameliorated by therapy with 5-formyltetrahydrofolate but exacerbated by folic acid.

Identification and in vitro expression of mutations causing dihydropteridine reductase deficiency.

Six mutations resulting in the recessive inherited disorder dihydropteridine reductase deficiency are reported, five of which are previously unknown. Two are nonsense mutations, resulting in premature termination of the protein, with the remaining four being missense mutations. The mutations found lie in the middle to 3' end of the dihydropteridine reductase reading frame, with the exception of one mutation which lies at codon 23, which is the only mutation found in more than one patient. The mutation pattern can be described as heterogeneous. The wild type and several of the mutant DHPR cDNA's were expressed in E. coli and the proteins purified and examined by a variety of techniques, including calculation of kinetic constants. One mutation (Gly23-->Asp) results in completely inactive protein, while a second (Trp108-->Gly) has substantial activity but does not completely dimerize. Both this mutant and a third, His158-->Tyr, are extremely susceptible to in vitro protease digestion, indicating that their three-dimensional structure has been altered. The protein studies underline the heterogeneous nature of DHPR mutations, in that the effects of different amino acid substitutions on the DHPR enzyme are varied.