Jan Bert Gramsbergen

Organotypic Hippocampal Slice Cultures for Studies of Brain Damage, Neuroprotection and Neurorepair

Slices of developing brain tissue can be grown for several weeks as so-called organotypic slice cultures. Here we summarize and review studies using hippocampal slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia), Alzheimers disease (AD) and epilepsia. Studies of non-excitotoxic neurotoxic compounds and the experimental use of slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimers disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section on repair is focused on neurogenesis and cell migration. The culturing techniques, set-up of models, and analytical tools, including markers for neurodegeneration, like the fluorescent dye propidium iodide (PI), are reviewed and discussed. Comparisons are made between hippocampal slice cultures and other in vitro models using dispersed cell cultures, experimental in vivo models, and in some instances, clinical trials. New techniques including slice culturing of hippocampal tissue from transgenic mice as well as more mature brain tissue, and slice cultures coupled to microelectrode arrays (MEAs), on-line biosensor monitoring, and time-lapse fluorescence microscopy are also presented.

Age-related changes in kynurenic acid production in rat brain

Two separate in vitro assays were used to examine the biosynthesis of the broad spectrum excitatory amino acid receptor antagonist kynurenic acid (KYNA) during the life span of the adult rat. Assessment of KYNAs anabolic enzyme kynurenine aminotransferase revealed steady increases between 3 and 24 months of age in all five brain regions examined. No changes were observed in the liver. The changes were particularly pronounced in the cortex and in the striatum where enzyme activity increased three-fold during the period studied. KYNA production from its bioprecursor L-kynurenine was also investigated in tissue slices and was found to be significantly enhanced in the cortex and hippocampus of old animals. The effect of depolarizing agents or sodium replacement was virtually identical in tissues from young and old rats. These data, which are in excellent agreement with reports on an age-dependent increase of KYNA concentration in brain tissue, suggest an enhanced KYNA tone in the aged brain. Together with the reported decline in cerebral excitatory amino acid receptor densities with age, increased production of KYNA may play a role in cognitive and memory dysfunction in old animals.

Colchicine induces apoptosis in organotypic hippocampal slice cultures

The microtubule-disrupting agent colchicine is known to be particular toxic for certain types of neurons, including the granule cells of the dentate gyrus. In this study we investigated whether colchicine could induce such neuron-specific degeneration in developing (1 week in vitro) and mature (3 weeks in vitro) organotypic hippocampal slice cultures and whether the induced cell death was apoptotic and/or necrotic. When applied to 1-week-old cultures for 48 h, colchicine induced primarily apoptotic, but also a minor degree of necrotic cell death in the dentate granule cells, as investigated by cellular uptake of the fluorescent dye propidium iodide (PI), immunostaining for active caspase 3 and c-Jun/AP-1 (N) and fragmentation of nuclei as seen in Hoechst 33342 staining. All four markers appeared after 12 h of colchicine exposure. Two of them, active caspase 3 and c-Jun/AP-1 (N) displayed a similar time course and reached a maximum after 24 h of exposure, 24 h ahead of both PI uptake and Hoechst 33342 staining, which together displayed similar time profiles and a close correlation. In 3-week-old cultures, colchicine did not induce apoptotic or necrotic cell death. Attempts to interfere with the colchicine-induced apoptosis in 1-week-old cultures showed that colchicine-induced PI uptake and formation of apoptotic nuclei were temporarily prevented by coapplication of the protein synthesis inhibitor cycloheximide. Application of the pancaspase inhibitor z-VAD-fmk almost completely abolished the formation of active caspase 3 protein and apoptotic nuclei induced by colchicine, but the formation of necrotic nuclei increased correspondingly and the PI uptake was unaffected. We conclude that colchicine induces caspase 3-dependent apoptotic cell death of dentate granule cells in hippocampal brain slice cultures, but the apoptotic cell death is highly dependent on the developmental stage of the cultures.

Pharmacological activation of KCa3.1/KCa2.3 channels produces endothelial hyperpolarization and lowers blood pressure in conscious dogs.

BACKGROUND AND PURPOSE In rodents, the endothelial KCa channels, KCa3.1 and KCa2.3, have been shown to play a crucial role in initiating endothelium‐derived hyperpolarizing factor (EDHF) vasodilator responses. However, it is not known to what extent these channels are involved in blood pressure regulation in large mammals, which would also allow us to address safety issues. We therefore characterized canine endothelial KCa3.1 and KCa2.3 functions and evaluated the effect of the KCa3.1/KCa2.3 activator SKA‐31 on blood pressure and heart rate in dogs.

Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures.

The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model for neurotoxicological studies, including further studies of neurotoxic mechanisms of TMT. Four-week-old cultures, derived from 7-day-old donor rats and grown in serum-free medium, were exposed to TMT (0.5-100 microM) for 24 h followed by 24 h in normal medium. TMT-induced neurodegeneration was then monitored by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux into the culture medium, (c) cellular cobalt uptake as an index of calcium influx, (d) ordinary Nissl cell staining, and (e) immunohistochemical staining for microtubule-associated protein 2 (MAP-2). Cellular degeneration as assessed by densitometric measurements of PI uptake displayed a dose and time-dependent increase, with the following ranking of vulnerability of the hippocampal subfields: FD>CA4>/=CA3c>CA1>CA3ab. This differential neuronal vulnerability observed by PI uptake was confirmed by MAP-2 immunostaining and corresponded to in vivo cell stain observations of rats acutely exposed to TMT. The mean PI uptake of the cultures and the LDH efflux into the medium were highly correlated. The combined results obtained by the different markers indicate that the hippocampal slice culture method is a feasible model for further studies of TMT neurotoxicity.

Genetic KCa3.1-deficiency produces locomotor hyperactivity and alterations in cerebral monoamine levels.

Background The calmodulin/calcium-activated K+ channel KCa3.1 is expressed in red and white blood cells, epithelia and endothelia, and possibly central and peripheral neurons. However, our knowledge about its contribution to neurological functions and behavior is incomplete. Here, we investigated whether genetic deficiency or pharmacological activation of KCa3.1 change behavior and cerebral monoamine levels in mice. Methodology/Principal Findings In the open field test, KCa3.1-deficiency increased horizontal activity, as KCa3.1−/− mice travelled longer distances (≈145% of KCa3.1+/+) and at higher speed (≈1.5-fold of KCa3.1+/+). Working memory in the Y-maze was reduced by KCa3.1-deficiency. Motor coordination on the rotarod and neuromuscular functions were unchanged. In KCa3.1−/− mice, HPLC analysis revealed that turn-over rates of serotonin were reduced in frontal cortex, striatum and brain stem, while noradrenalin turn-over rates were increased in the frontal cortex. Dopamine turn-over rates were unaltered. Plasma catecholamine and corticosterone levels were unaltered. Intraperitoneal injections of 10 mg/kg of the KCa3.1/KCa2-activator SKA-31 reduced rearing and turning behavior in KCa3.1+/+ but not in KCa3.1−/− mice, while 30 mg/kg SKA-31 caused strong sedation in 50% of the animals of either genotypes. KCa3.1−/− mice were hyperactive (≈+60%) in their home cage and SKA-31-administration reduced nocturnal physical activity in KCa3.1+/+ but not in KCa3.1−/− mice. Conclusions/Significance KCa3.1-deficiency causes locomotor hyperactivity and altered monoamine levels in selected brain regions, suggesting a so far unknown functional link of KCa3.1 channels to behavior and monoaminergic neurotransmission in mice. The tranquilizing effects of low-dose SKA-31 raise the possibility to use KCa3.1/KCa2 channels as novel pharmacological targets for the treatment of neuropsychiatric hyperactivity disorders.

Cerebrospinal fluid biomarkers for Parkinson's disease - a systematic review.

Diagnosis of Parkinsons disease (PD) relies on clinical history and physical examination, but misdiagnosis is common in early stages. Identification of biomarkers for PD may allow early and more precise diagnosis and monitoring of dopamine replacement strategies and disease modifying treatments. Developments in analytical chemistry allow the detection of large numbers of molecules in plasma or cerebrospinal fluid, associated with the pathophysiology or pathogenesis of PD. This systematic review includes cerebrospinal fluid biomarker studies focusing on different disease pathways: oxidative stress, neuroinflammation, lysosomal dysfunction and proteins involved in PD and other neurodegenerative disorders, focusing on four clinical domains: their ability to (1) distinguish PD from healthy subjects and other neurodegenerative disorders as well as their relation to (2) disease duration after initial diagnosis, (3) severity of disease (motor symptoms) and (4) cognitive dysfunction. Oligomeric alpha‐synuclein might be helpful in the separation of PD from controls. Through metabolomics, changes in purine and tryptophan metabolism have been discovered in patients with PD. Neurofilament light chain (NfL) has a significant role in distinguishing PD from other neurodegenerative diseases. Several oxidative stress markers are related to disease severity, with the antioxidant urate also having a prognostic value in terms of disease severity. Increased levels of amyloid and tau‐proteins correlate with cognitive decline and may have prognostic value for cognitive deficits in PD. In the future, larger longitudinal studies, corroborating previous research on viable biomarker candidates or using metabolomics identifying a vast amount of potential biomarkers, could be a good approach.

Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model.

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na+/K+-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2+/G301R) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2G301R/G301R E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2+/G301R male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2+/G301R behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2.

Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures

Complex‐I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinsons disease and the toxicity of MPTP and its metabolite MPP+. Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP+ toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 µm MPP+ for 2 days, followed by 8 days ‘recovery’ in control medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase‐immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4‐diamino‐6‐hydroxypyrimidine had no significant synergistic effect on MPP+ toxicity. In contrast, the tetrahydrobiopterin precursor l‐sepiapterin attenuated the MPP+‐induced dopamine depletion and loss of tyrosine hydroxylase‐positive cells in a dose‐dependent manner with 40 µm l‐sepiapterin providing maximal protection. Accordingly, increasing intracellular tetrahydrobiopterin levels may protect against oxidative stress by complex‐I inhibition.

On-line monitoring of striatum glucose and lactate in the endothelin-1 rat model of transient focal cerebral ischemia using microdialysis and flow-injection analysis with biosensors

In vivo studies on cerebral glucose and lactate metabolism following a brain insult require fast and sensitive monitoring techniques. Here we report on-line monitoring of ischemic events and metabolic changes following reperfusion in striatum of freely moving rats subjected to endothelin-1 (60-240 pmol) induced, transient focal cerebral ischemia using slow microdialysis (0.5 microl/min), fast sampling (every minute) and flow-injection analysis with biosensors for glucose and lactate. The high-time resolution provides detailed information on lactate rise times and duration of low glucose. In rats, developing large striatal lesions, lactate increased from 1.0 +/- 0.1 to 4.2 +/- 0.7 mM within 37 +/- 1 min, whereas glucose dropped from 0.3 +/- 0.1 mM to below detection levels (<0.05 mM) for a period of 80 +/- 18 min. The lactate increase measured over a 2-h period after endothelin-1 infusion was highly correlated with striatal infarct size. In some rats oscillatory changes are observed which cannot be detected in traditional assays. The here-described monitoring technique applied in a clinically relevant rat model is a sensitive tool to study post-ischemic energy metabolism, effects of therapeutic interventions and its relationship with histological outcome.