Late changes in blood-brain barrier permeability in a rat tMCAO model of stroke detected by gadolinium-enhanced MRI.

OBJECTIVES: After cerebral ischaemia the blood-brain barrier (BBB) may be compromised and this has been observed in both clinical and preclinical studies. The timing of BBB disruption after ischaemia has long been considered to be biphasic, however some groups contest this view. Therefore, the purpose of this study was to characterize the BBB permeability timecourse in a rat model at both acute and chronic time points. METHODS: Unilateral transient middle cerebral artery occlusion (tMCAO) was performed in 15 male Sprague Dawley rats. Change in T1-weighted MR signal before and after an injection of gadolinium-based contrast agent was calculated voxelwise to derive a BBB permeability index (BBBPI) at both early (6 h, 12 h, and 24 h) and late (7 and 14 days) time points. RESULTS: As expected, BBBPI in the non-lesioned ROI was not significantly different from pre-occlusion baseline at any time point. However, BBBPI in the ipsilateral (lesioned) ROI was statistically different to baseline at day 7 (p < 0.001) and day 14 (p < 0.01) post-tMCAO. There was a small, but not-significant increase in BBBPI in the earlier phase (at 6 hours). DISCUSSION: Our results indicate a significant late opening of the BBB. This is important as the majority of previous studies have only characterised an early acute BBB permeability in ischemia. However, the later period of increased permeability may indicate an optimal time for drug delivery across the BBB, when it is especially suited to drugs targeting delayed processes.

Immunolocalization of Kisspeptin Associated with Amyloid-ß Deposits in the Pons of an Alzheimer's Disease Patient.

The pons region of the Alzheimer's disease (AD) brain is one of the last to show amyloid-ß (Aß) deposits and has been suggested to contain neuroprotective compounds. Kisspeptin (KP) is a hormone that activates the hypothalamic-pituitary-gonadal axis and has been suggested to be neuroprotective against Aß toxicity. The localization of KP, plus the established endogenous neuroprotective compounds corticotropin releasing hormone (CRH) and catalase, in tissue sections from the pons region of a male AD subject has been determined in relation to Aß deposits. Results showed Aß deposits also stained with KP, CRH, and catalase antibodies. At high magnification the staining of deposits was either KP or catalase positive, and there was only a limited area of the deposits with KP-catalase colocalization. The CRH does not bind Aß, whilst both KP and catalase can bind Aß, suggesting that colocalization in Aß deposits is not restricted to compounds that directly bind Aß. The neuroprotective actions of KP, CRH, and catalase were confirmed in vitro, and fibrillar Aß preparations were shown to stimulate the release of KP in vitro. In conclusion, neuroprotective KP, CRH, and catalase all colocalize with Aß plaque-like deposits in the pons region from a male AD subject.

Kisspeptin prevention of amyloid-ß peptide neurotoxicity in vitro.

Alzheimer's disease (AD) onset is associated with changes in hypothalamic-pituitary-gonadal (HPG) function. The 54 amino acid kisspeptin (KP) peptide regulates the HPG axis and alters antioxidant enzyme expression. The Alzheimer's amyloid-ß (Aß) is neurotoxic, and this action can be prevented by the antioxidant enzyme catalase. Here, we examined the effects of KP peptides on the neurotoxicity of Aß, prion protein (PrP), and amylin (IAPP) peptides. The Aß, PrP, and IAPP peptides stimulated the release of KP and KP 45-54. The KP peptides inhibited the neurotoxicity of Aß, PrP, and IAPP peptides, via an action that could not be blocked by kisspeptin-receptor (GPR-54) or neuropeptide FF (NPFF) receptor antagonists. Knockdown of KiSS-1 gene, which encodes the KP peptides, in human neuronal SH-SY5Y cells with siRNA enhanced the toxicity of amyloid peptides, while KiSS-1 overexpression was neuroprotective. A comparison of the catalase and KP sequences identified a similarity between KP residues 42-51 and the region of catalase that binds Aß. The KP peptides containing residues 45-50 bound Aß, PrP, and IAPP, inhibited Congo red binding, and were neuroprotective. These results suggest that KP peptides are neuroprotective against Aß, IAPP, and PrP peptides via a receptor independent action involving direct binding to the amyloid peptides.

Neither in vivo MRI nor behavioural assessment indicate therapeutic efficacy for a novel 5HT(1A) agonist in rat models of ischaemic stroke.

BACKGROUND: 5HT1A agonists have previously been shown to promote recovery in animal models of stroke using ex vivo outcome measures which have raised the hopes for a potential clinical implementation. The purpose of this study was to evaluate the potential neuroprotective properties of a novel 5HT1A agonist DU123015 in 2 different models of transient focal ischaemic stroke of varying severities using both in vivo neuroimaging and behavioural techniques as primary outcome measures. For these studies, the NMDA receptor antagonist MK-801 was also utilized as a positive control to further assess the effectiveness of the stroke models and techniques used. RESULTS: In contrast to MK-801, no significant therapeutic effect of DU123015 on lesion volume in either the distal MCAo or intraluminal thread model of stroke was found. MK-801 significantly reduced lesion volume in both models; the mild distal MCAo condition (60 min ischaemia) and the intraluminal thread model, although it had no significant impact upon the lesion size in the severe distal MCAo condition (120 min ischaemia). These therapeutic effects on lesion size were mirrored on a behavioural test for sensory neglect and neurological deficit score in the intraluminal thread model. CONCLUSION: This study highlights the need for a thorough experimental design to test novel neuroprotective compounds in experimental stroke investigations incorporating: a positive reference compound, different models of focal ischaemia, varying the duration of ischaemia, and objective in vivo assessments within a single study. This procedure will help us to minimise the translation of less efficacious compounds.

Multi-modal characterisation of the neocortical clip model of focal cerebral ischaemia by MRI, behaviour and immunohistochemistry.

The neocortical clip model of focal cerebral ischaemia has previously been used with success in neuroprotection studies. To further improve its translational qualities, we have characterised this model using a combination of serial Magnetic Resonance Imaging (MRI), neurological assessment, the bilateral asymmetry test (BAT) and immunohistochemistry. The right MCA was occluded in spontaneously hypertensive rats for 0, 60 and 120 min. MRI was performed pre-surgery, 1, 3 and 7 days post-surgery. Behavioural assessment was performed 2 days before and 3 and 7 days post-surgery whilst neurological deficits were monitored daily. Neuroimaging results showed that 0 min of MCA occlusion did not produce a lesion, whereas occlusion for 60 min produced a lesion that remained stable over time. Occlusion for 120 min caused a more severe lesion 1 day post-surgery, but decreased by 7 days. Behaviour, neurological scores and histological lesion volumes correlated strongly with MRI lesion volume. Immunohistochemistry revealed neuronal loss, astrogliosis and macrophage infiltration in lesioned cortices. The neocortical clip model produced ischaemic lesions that are restricted to cortical territories of the MCA. The duration of occlusion dictates lesion severity which may prove useful for probing therapeutic interventions at different stages of stroke progression. The correlation of MRI with two different behavioural measures and post-mortem histology strengthens the basis for MRI providing an in vivo surrogate marker for structural and behavioural deficits caused by a cortical stroke.