Ashley K. Pringle

Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus

Adult dentate neurogenesis is important for certain types of hippocampal‐dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety‐related behaviour and learning and memory. We report that adult NPY receptor knock‐out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin‐positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1‐receptor mediated and involves extracellular signal‐regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY‐releasing interneurons may modulate dentate neurogenesis.

Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre- and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.

Differential vulnerability of the CA1 and CA3 subfields of the hippocampus to superoxide and hydroxyl radicals in vitro

Abstract: The relative roles of the superoxide and hydroxyl radicals in oxidative stress‐induced neuronal damage were investigated using organotypic hippocampal slice cultures. Cultures exposed to 100 µM duroquinone, a superoxide‐generating compound, for 3 h developed CA1‐selective lesions over a period of 24 h. The damage accounted for ∼64% of the CA1 subfield, whereas CA3 showed just 6% damage, a pattern of damage comparable to that observed following hypoxia/ischaemia. Duroquinone‐induced damage was attenuated by a spin‐trap agent. In contrast, hydroxyl radical‐mediated damage, generated by exposure to 30 µM ferrous sulphate for 1 h, resulted in a CA3‐dominant lesion. The damage developed over 24 h, similar to that observed with duroquinone, but with ∼45% damage in CA3 compared with only 7% in CA1. These data demonstrate a selective vulnerability of the CA1 pyramidal neurones to superoxide‐induced damage and suggest that of the free radicals generated following hypoxia/ischaemia, superoxide, rather than hydroxyl radical, is instrumental in producing neuronal damage.

Organotypic cultures as tools for functional screening in the CNS

A major challenge for the pharmaceutical industry is the development of relevant model systems in which knowledge gained from high-throughput, genomic and proteomic approaches can be integrated to study function. Animal models are still the main choice for such studies but over the past few years powerful new in vitro systems have begun to emerge as useful tools to study function. Organotypic cultures made from slices of explanted tissue represent a complex multi-cellular in vitro environment with the potential to assess biological function and are uniquely placed to act as an important link between high-throughput approaches and animal models.

A microdialysis method for the recovery of IL-1β, IL-6 and nerve growth factor from human brain in vivo

Intracerebral microdialysis is used extensively as a research tool in the investigation of the neurochemical and metabolic changes that occur following acute brain injury. Microdialysis has enabled elucidation of intra-cerebral levels of substances such as lactate, pyruvate and glycerol but, as yet, has not been used effectively to recover macromolecules from the human brain. Traumatic brain injury is known to result in the generation of cytokines and neurotrophins into extracellular fluid compartment of the brain, with effects on neuronal damage and repair. We have developed a technique of in vivo sampling of the interstitial fluid of the brain of patients with severe head injuries which has allowed the measurement of IL-1beta, IL-6 and nerve growth factor. This report confirms the safety and effectiveness of this modified microdialysis method in the clinical setting of a neurological intensive care unit. The technique provides a timely addition to the armamentarium of the clinical scientist and will potentially lead to a greater understanding of neuroinflammation following acute traumatic brain injury.

7‐Hydroxylated epiandrosterone (7‐OH‐EPIA) reduces ischaemia‐induced neuronal damage both in vivo and in vitro

Recent evidence suggests that steroids such as oestradiol reduce ischaemia‐induced neurodegeneration in both in vitro and in vivo models. A cytochrome P450 enzyme termed cyp7b that 7‐hydroxylates many steroids is expressed at high levels in brain, although the role of 7‐hydroxylated steroids is unknown. We have tested the hypothesis that the steroid‐mediated neuroprotection is dependent on the formation of 7‐hydroxy metabolites. Organotypic hippocampal slice cultures were prepared from Wistar rat pups and maintained in vitro for 14 days. Cultures were then exposed to 3 h hypoxia and neuronal damage assessed 24 h later using propidium iodide fluorescence as a marker of cell damage. Neurodegeneration occurred primarily in the CA1 pyramidal cell layer. The steroids oestradiol, dehydroepiandrosterone and epiandrosterone (EPIA) were devoid of neuroprotective efficacy when present at 100 nm pre‐, during and post‐hypoxia. The 7‐hydroxy metabolites of EPIA, 7α‐OH‐EPIA and 7β‐OH‐EPIA significantly reduced neurotoxicity at 100 nm and 10 nm. 7β‐OH‐EPIA was also neuroprotective in two in vivo rat models of cerebral ischaemia: 0.1 mg/kg 7β‐OH‐EPIA significantly reduced hippocampal cell loss in a model of global forebrain ischaemia, whereas 0.03 mg/kg was neuroprotective in a model of focal ischaemia even when administration was delayed until 6 h after the onset of ischaemia. Taken together, these data demonstrate that 7‐hydroxylation of steroids confers neuroprotective efficacy, and that 7β‐OH‐epiandrosterone represents a novel class of neuroprotective compounds with potential for use in acute neurodegenerative diseases.

Attenuation and augmentation of ischaemia-related neuronal death by tumour necrosis factor-alpha in vitro.

Upregulation of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF) occurs rapidly in the brain following ischaemia, although it is unclear whether this represents a neurotoxic or neuroprotective response. We have investigated whether TNF has different actions in the pre‐ and postischaemic periods in a tissue culture model of cerebral ischaemia. Organotypic hippocampal slice cultures were prepared from 8–10‐day‐old rats and maintained in vitro for 14 days. Neuronal damage was induced by either 1 h oxygen–glucose deprivation or 3 h exposure to NMDA or the superoxide generator duroquinone, and assessed after 24 h by propidium iodide fluorescence. TNF pretreatment was neuroprotective against both oxygen–glucose deprivation and duroquinone. This effect was associated with an activation of the transcription factor NFκB and upregulation of manganese superoxide dismutase, and was prevented by a free radical scavenger. When addition of TNF was delayed until the postinsult period, an exacerbation of neurotoxicity occurred, which was also prevented by a free radical scavenger. The actions of TNF are determined by whether TNF is present before or after an ischaemia‐related insult. Both actions are mediated through the production of free radicals, and the response to TNF is determined by whether a cell is metabolically competent to respond by synthesis of antioxidant defences.

Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures

We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not neuroprotective. These data suggest that BDNF may form part of an endogenous neuroprotective mechanism.

Ischaemic pre-conditioning in organotypic hippocampal slice cultures is inversely correlated to the induction of the 72 kDa heat shock protein (HSP72).

In vivo, preconditioning with a sublethal insult can confer resistance to normally lethal episodes of cerebral ischaemia. This phenomenon has been linked with the induction of the 72 kDa heat shock protein (HSP72), but this has not been clearly demonstrated in vitro. We have used organotypic hippocampal slice cultures to investigate whether tolerance to lethal ischaemia is dependent on HSP72. Cultures were maintained in vitro for 14 days, and neuronal damage assessed using propidium iodide fluorescence. Prolonged neuronal HSP72 upregulation occurred following exposure to 30 min ischaemia, 45 min hypoxia and 1 microM kainate, but not 1 microM NMDA or 20 min ischaemia, all sublethal insults. Preconditioning with ischaemia, kainate or hypoxia 24 h prior to lethal ischaemia (45 min) was not protective, and when the delay was increased to 48 h, damage in the CA3 pyramidal cell region was significantly increased compared to cultures exposed to 45 min ischaemia alone. Preconditioning with 20 min ischaemia had no effect on the severity of ischaemic damage. Preconditioning with 1 microM NMDA significantly reduced neuronal damage produced by either 45 or 60 min ischaemia when the delay between insults was 48 h. NMDA pre-treatment also prevented neurotoxicity produced by glutamate (5-10 mM) but not NMDA (10-30 microM). These data suggest that in vitro, the increased expression of HSP72 following some sublethal insults should be considered as a marker of cell stress prejudicial to the survival of neurones subsequently exposed to ischaemia, while tolerance can be produced through mechanisms independent of HSP72 induction.

L-arginyl-3,4-spermidine is neuroprotective in several in vitro models of neurodegeneration and in vivo ischaemia without suppressing synaptic transmission

Stroke is the third most common cause of death in the world, and there is a clear need to develop new therapeutics for the stroke victim. To address this need, we generated a combinatorial library of polyamine compounds based on sFTX‐3.3 toxin from which L‐Arginyl‐3,4‐Spermidine (L‐Arg‐3,4) emerged as a lead neuroprotective compound. In the present study, we have extended earlier results to examine the compounds neuroprotective actions in greater detail. In an in vitro ischaemia model, L‐Arg‐3,4 significantly reduced CA1 cell death when administered prior to induction of 60 min of ischaemia as well as when administered immediately after ischaemia. Surprisingly, L‐Arg‐3,4 continued to prevent cell death significantly when administration was delayed for as long as 60 min after ischaemia. L‐Arg‐3,4 significantly reduced cell death in excitotoxicity models mediated by glutamate, NMDA, AMPA, or kainate. Unlike glutamate receptor antagonists, 300 μM L‐Arg‐3,4 did not suppress synaptic transmission as measured by evoked responses in acute hippocampal slices. L‐Arg‐3,4 provided significant protection, in vitro, in a superoxide mediated injury model and prevented an increase of superoxide production after AMPA or NMDA stimulation. It also decreased nitric oxide production after in vitro ischaemia and NMDA stimulation, but did so without inhibiting nitric oxide synthase directly. Furthermore, L‐Arg‐3,4 was significantly neuroprotective in an in vivo model of global forebrain ischaemia, without any apparent neurological side‐effects. Taken together, these results demonstrate that L‐Arg‐3,4 is protective in several models of neurodegeneration and may have potential as a new therapeutic compound for the treatment of stroke, trauma, and other neurodegenerative diseases.