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Background: Cannabis use suppresses the endocannabinoid system in healthy individuals. However, the association between cannabis use with the endocannabinoid system is understudied in individuals with psychosis despite the high rate of cannabis use in these individuals. Methods: We enrolled 83 individuals who were admitted to an inpatient psychiatric unit with psychotic presentations, and measured their plasma levels of main endocannabinoids, Anandamide (AEA) and 2-Acylglycerol (2-AG), and endocannabinoid related compounds, Palmitoylethanolamine, and N-oleoylethanolamine. Cannabis use was assessed with urine toxicology and frequency of cannabis use was assessed using self-reported questionnaires. The Positive and Negative Syndrome Scale was used to assess the severity of psychotic symptoms. Results: Overall, we had 38 individuals in cannabis positive group (CN+) and 45 individuals in cannabis negative group (CN−). Compared to CN−, CN+ group had lower plasma levels of AEA, which remained significant after controlling for age, gender, race/ethnicity, and use of other drugs. Conclusion: Cannabis use is associated with low plasma AEA levels in individuals with psychosis, which is in the same line with reported suppressive effects of cannabis on the endocannabinoid system in healthy individuals. Further studies are needed to investigate the clinical significance of this finding.

The successful treatment of depressive disorders critically depends on adherence to prescribed treatment regimens. Despite increasing rates of antidepressant medication prescription, adherence to the full treatment course remains poor. Rates of antidepressant non-adherence are higher for uninsured patients and members of some marginalized racial and ethnic communities due to factors such as inequities in healthcare and access to insurance. Among patients treated in a free, student-run and faculty-supervised clinic serving uninsured patients in a majority Hispanic community in East Harlem, adherence rates are lower than those observed in patients with private or public New York State health insurance coverage. A prior study of adherence in these patients revealed that difficulty in obtaining medications from an off-site hospital pharmacy was a leading factor that patients cited for non-adherence. To alleviate this barrier to obtaining prescriptions, we tested the effectiveness of on-site, in-clinic medication dispensing for improving antidepressant medication adherence rates among uninsured patients. We found that dispensing medications directly to patients in clinic was associated with increased visits at which patients self-reported proper adherence and increased overall adherence rates. Furthermore, we found evidence that higher rates of antidepressant medication adherence were associated with more favorable treatment outcomes. All patients interviewed reported increased satisfaction with on-site dispensing. Overall, this study provides promising evidence that on-site antidepressant dispensing in a resource-limited setting improves medication adherence rates and leads to more favorable treatment outcomes with enhanced patient satisfaction.

Hypertension is an important risk factor for age-related cognitive decline and neuronal pathologies. Studies have shown a correlation between hypertension, disruption in neurovascular coupling and cerebral autoregulation, and cognitive decline. However, the mechanisms behind this are unclear. To further understand this, it is advantageous to study neurovascular coupling as hypertension progresses in a rodent model. Here, we use a longitudinal functional MRI (fMRI) protocol to assess the impact of hypertension on neurovascular coupling in spontaneously hypertensive rats (SHRs). Eight female SHRs were studied at 2, 4, and 6 months of age, as hypertension progressed. Under an IV infusion of propofol, animals underwent fMRI, functional MR spectroscopy, and cerebral blood flow (CBF) quantification to study changes in neurovascular coupling over time. Blood pressure significantly increased at 4 and 6 months (P < .0001). CBF significantly increased at 4 months old (P < .05), in the acute stage of hypertension. The size of the active region decreased significantly at 6 months old (P < .05). Change in glutamate signal during activation, and N-acetyl-aspartate (NAA) signal, remained constant. This study shows that, while cerebral autoregulation is impaired in acute hypertension, the blood oxygenation-level-dependent (BOLD) response remains unaltered until later stages. At this stage, the consistent NAA and glutamate signals show that neuronal death has not occurred, and that neuronal activity is not affected at this stage. This suggests that neuronal activity and viability is not lost until much later, and changes observed here in BOLD activity are due to vascular effects.

Cerebral ischemic stroke is a leading cause of death and disability, but current pharmacological therapies are limited in their utility and effectiveness. In vitro and in vivo models of ischemic stroke have been developed which allow us to further elucidate the pathophysiological mechanisms of injury and investigate potential drug targets. In vitro models permit mechanistic investigation of the biochemical and molecular mechanisms of injury but are reductionist and do not mimic the complexity of clinical stroke. In vivo models of ischemic stroke directly replicate the reduction in blood flow and the resulting impact on nervous tissue. The most frequently used in vivo model of ischemic stroke is the intraluminal suture middle cerebral artery occlusion (iMCAO) model, which has been fundamental in revealing various aspects of stroke pathology. However, the iMCAO model produces lesion volumes with large standard deviations even though rigid surgical and data collection protocols are followed. There is a need to refine the MCAO model to reduce variability in the standard outcome measure of lesion volume. The typical approach to produce vessel occlusion is to induce an obstruction at the origin of the middle cerebral artery and reperfusion is reliant on the Circle of Willis (CoW). However, in rodents the CoW is anatomically highly variable which could account for variations in lesion volume. Thus, we developed a refined approach whereby reliance on the CoW for reperfusion was removed. This approach improved reperfusion to the ischemic hemisphere, reduced variability in lesion volume by 30%, and reduced group sizes required to determine an effective treatment response by almost 40%. This refinement involves a methodological adaptation of the original surgical approach which we have shared with the scientific community via publication of a visualised methods article and providing hands-on training to other experimental stroke researchers.

As the population ages, the incidence of age-related neurological diseases and cognitive decline increases. To further understand disease-related changes in brain function it is advantageous to examine brain activity changes in healthy aging rodent models to permit mechanistic investigation. Here, we examine the suitability, in rodents, of using a novel, minimally invasive anaesthesia protocol in combination with a functional MRI protocol to assess alterations in neuronal activity due to physiological aging. 11 Wistar Han female rats were studied at 7, 9, 12, 15 and 18 months of age. Under an intravenous infusion of propofol, animals underwent functional magnetic resonance imaging (fMRI) and functional magnetic resonance spectroscopy (fMRS) with forepaw stimulation to quantify neurotransmitter activity, and resting cerebral blood flow (CBF) quantification using arterial spin labelling (ASL) to study changes in neurovascular coupling over time. Animals showed a significant decrease in size of the active region with age (P ​, Highlights • Describe a novel anaesthetic protocol to examine age-related alterations in neuronal activity in rodents. • Size of the BOLD signal in the somatosensory cortex decreased with age. • Reduction in glutamate turnover with age. • No change in resting CBF with age.

Overdose of stimulant drugs has been associated with increased risk of adverse cardiovascular events (ACVE), some of which may be ascribed to endothelial dysfunction. The aims of this study were to evaluate biomarkers of endothelial dysfunction in emergency department (ED) patients with acute cocaine overdose and to assess the association between in-hospital ACVE in ED patients with any acute drug overdose. This was a prospective consecutive cohort study over 9 months (2015-2016) at two urban, tertiary-care hospital EDs. Consecutive adults (≥18 years) presenting with suspected acute drug overdose were eligible and separated into three groups: cocaine (n = 47), other drugs (n = 128), and controls (n = 11). Data were obtained from medical records and linked to waste serum specimens, sent as part of routine clinical care, for biomarker analysis. Serum specimens were collected and analyzed using enzyme-linked immunosorbent assay kit for three biomarkers of endothelial dysfunction: (a) endothelin-1 (ET-1), (b) regulated upon activation normal T cell expressed and secreted (RANTES), and (c) soluble intercellular adhesion molecule-1 (siCAM-1). Mean siCAM was elevated for cocaine compared with controls and other drugs (p < .01); however, mean RANTES and ET-1 levels were not significantly different for any drug exposure groups. Receiver operating characteristics curve analysis for prediction of in-hospital ACVE revealed excellent performance of siCAM-1 (area under curve, 0.86; p < .001) but lack of predictive utility for either RANTES or ET-1. These results suggest that serum siCAM-1 is a viable biomarker for acute cocaine overdose and that endothelial dysfunction may be an important surrogate for adverse cardiovascular events following any drug overdose.

Background Cerebral ischemic stroke is a significant cause of morbidity and mortality. Sex differences exist following stroke in terms of incidence, symptoms, outcomes and response to some treatments. Importantly, molecular mechanisms of injury, activated following ischemia may differ between the sexes and if so may account, at least in part, for sex differences seen in treatment response. Here we aimed to determine, using single-sex organotypic hippocampal slice cultures, whether the effectiveness of a potential treatment option, i.e. sex steroids, exhibited any sexual dimorphism and whether sex affected the mechanisms of apoptosis activated following ischemia. Results Following exposure to ischemia, male-derived tissue exhibited higher levels of cell death than female-derived tissue. Various sex steroid hormones, i.e. progesterone, allopregnanolone, and estradiol, were protective in terms of reducing the amount of cell death in male- and female-derived tissue whereas medoxyprogesterone acetate (MPA) was only protective in female-derived tissue. The protective effect of progesterone was abolished in the presence of finasteride, a 5α-reductase inhibitor, suggesting it was largely mediated via its conversion to allopregnanolone. To test the hypothesis that sex differences exist in the activation of specific elements of the apoptotic pathway activated following ischemia we administered Q-VD-OPH, a caspase inhibitor, or PJ34, an inhibitor of poly (ADP ribose) polymerase (PARP). Caspase inhibition was only effective, in terms of reducing cell death, in female-derived tissue, whereas PARP inhibition was only protective in male-derived tissue. However, in both sexes, the protective effects of progesterone and estradiol were not observed in the presence of either caspase or PARP inhibition. Conclusions Sex differences exist in both the amount of cell death produced and those elements of the cell death pathway activated following an ischemic insult. There are also some sex differences in the effectiveness of steroid hormones to provide neuroprotection following an ischemic insult—namely MPA was only protective in female-derived tissue. This adds further support to the notion sex is an important factor to consider when investigating future drug targets for CNS disorders, such as ischemic stroke.

Dementia continues to be the most burdening neurocognitive disorder, having a negative impact on the lives of millions. The search for biomarkers to improve the clinical diagnosis of dementia is ongoing, with the focus on effective use of readily accessible peripheral markers. In this review, we concentrate on platelets as biomarkers of dementia and analyze their potential as easily-accessible clinical biomarkers for various subtypes of dementia. Current platelet protein biomarkers that have been investigated for their clinical utility in the diagnosis of dementia, in particular Alzheimer's disease, include amyloid-β protein precursor (AβPP), the AβPP secretases (BACE1 and ADAM10), α-synuclein, tau protein, serotonin, cholesterol, phospholipases, clusterin, IgG, surface receptors, MAO-B, and coated platelets. Few of them, i.e., platelet tau, AβPP (particularly with regards to coated platelets) and secreted ADAM10 and BACE1 show the most promise to be taken forward into clinical setting to diagnose dementia. Aside from protein biomarkers, changes in factors such as mean platelet volume have the potential to play a very specific role in both the dementia diagnosis and prognosis. This review raises a number of research questions for consideration before application of the above biomarkers to routine clinical setting. It is without doubt that there is a need for more clarification on the effects of dementia on platelet morphology and protein content before these changes can be clinically applied as dementia biomarkers and explored further in differentiating distinct dementia subtypes.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Combined therapy with epidermal growth factor (EGF) and growth hormone-releasing peptide 6 (GHRP-6) in stroke models has accumulated evidence of neuroprotective effects from several studies, but needs further support before clinical translation. Comparing EGF + GHRP-6 to hypothermia, a gold neuroprotection standard, may contribute to this purpose.The aims of this study were to compare the neuroprotective effects of a combined therapy based on EGF + GHRP-6 with hypothermia in animal models of (a) global ischemia representing myocardial infarction and (b) focal brain ischemia representing ischemic stroke.(a) Global ischemia was induced in Mongolian gerbils by a 15-min occlusion of both carotid arteries, followed by reperfusion. (b) Focal brain ischemia was achieved by intracerebral injection of endothelin 1 in Wistar rats. In each experiment, three ischemic treatment groups - vehicle, EGF + GHRP-6, and hypothermia - were compared to each other and to a sham-operated control group. End points were survival, neurological scores, and infarct volume.(a) In global ischemia, neurological score at 48-72 h, infarct volume, and neuronal density of hippocampal CA1 zone in gerbils treated with EGF + GHRP-6 were similar to the hypothermia-treated group. (b) In focal ischemia, the neurologic score and infarct volume of rats receiving EGF + GHRP-6 were also similar to animals in the hypothermia group.With hypothermia being a good standard neuroprotectant reference, these results provide additional proof of principle for EGF and GHRP-6 co-administration as a potentially neuroprotective stroke therapy.

Stroke continues to be a leading cause of mortality and morbidity worldwide, and novel therapeutic options for ischaemic stroke are urgently needed. In this context, drug combination therapies seem to be a viable approach, which has not been fully explored in preclinical studies.In this work, we assessed the dose-response relationship and therapeutic time window, in global brain ischaemia, of a combined therapeutic approach of recombinant human epidermal growth factor (EGF) and growth hormone-releasing peptide-6 (GHRP-6).Mongolian gerbils underwent 15 minutes occlusion of both common carotid arteries. Four different doses of rhEGF, GHRP-6 and these combined agents were intraperitoneally administered immediately after the onset of reperfusion. Having identified a better response with both agents, rhEGF+GHRP-6 were administered at 2, 4, 6, 8 or 24 hours after the onset of reperfusion to assess the time window of effectiveness. Animals were evaluated daily for neurological deficits. Three days post-occlusion, the animals were sacrificed and 2,3,5-triphenyltetrazolium chloride was used to quantify infarcted tissues.The coadministration of rhEGF and GHRP-6 at doses of 100 and 600 μg/kg, respectively, administered up to 4 hours following the ischaemic insult, significantly improved survival and neurological outcome, and reduced infarct volume compared with vehicle treatment. These results are considered as an additional proof of concept as supporting a combined therapeutic approach and justify the further development of this preclinical research.

Inflammatory abnormalities are well-documented in individuals with chronic psychotic disorders. Particular attention has focused on interleukin-6 (IL-6) and its correlation with psychotic symptom severity. Cannabis use is associated with an increased risk of psychosis and also has immunomodulating properties. It has been hypothesized that inflammatory disturbances are a common underlying pathology between cannabis use and psychosis. We measured inflammatory markers in individuals admitted to a psychiatric unit with acute psychosis who had toxicology positive for natural and/or synthetic cannabinoids (n = 59) compared to patients with negative cannabinoid toxicology (n = 60). Psychosis severity was assessed using the Positive and Negative Syndrome Scale (PANSS). While PANSS scores were similar between groups, cannabinoid-positive participants were more likely to receive pro re nata (PRN or as-needed) medications for agitation in the psychiatric emergency room, particularly synthetic cannabinoid-positive participants. In unadjusted models, cannabinoid-positive participants had lower interferon-γ (IFN-γ) levels (p = 0.046), but this finding was not significant after adjusting for covariates and multiple comparisons. Among cannabinoid-positive participants, IL-6 levels negatively correlated with PANSS total score (p = 0.040), as well as positive (p = 0.035) and negative (p = 0.024) subscales. Results suggest inflammatory alterations among psychotic individuals with comorbid cannabinoid use.

Background: Following ischemic stroke, recanalisation and restoration of blood flow to the affected area of the brain is critical and directly correlates with patient recovery. In vivo models of ischemic stroke show high variability in outcomes, which may be due to variability in reperfusion. We previously reported that a surgical refinement in the middle cerebral artery occlusion (MCAO) model of stroke, via repair of the common carotid artery (CCA), removes the reliance on the Circle of Willis for reperfusion and reduced infarct variability. Here we further assess this refined surgical approach on reperfusion characteristics following transient MCAO in mice. Methods: Mice underwent 60 min of MCAO, followed by either CCA repair or ligation at reperfusion. All mice underwent laser speckle contrast imaging at baseline, 24 h and 48 h post-MCAO. Results: CCA ligation reduced cerebral perfusion in the ipsilateral hemisphere compared to baseline (102.3 ± 4.57%) at 24 h (85.13 ± 16.09%; P < 0.01) and 48 h (75.04 ± 12.954%; P < 0.001) post-MCAO. Repair of the CCA returned perfusion to baseline (94.152 ± 2.44%) levels and perfusion was significantly improved compared to CCA ligation at both 24 h (102.83 ± 8.41%; P < 0.05) and 48 h (102.13 ± 9.34%; P < 0.001) post-MCAO. Conclusions: Our findings show CCA repair, an alternative surgical approach for MCAO, results in improved ischemic hemisphere perfusion during the acute phase.

Excitotoxicity is the principle mechanism of acute injury during stroke. It is defined as the unregulated accumulation of excitatory neurotransmitters such as glutamate within the extracellular space, leading to over-activation of receptors, ionic disruption, cell swelling, cytotoxic Ca2+ elevation and a feed-forward loop where membrane depolarisation evokes further neurotransmitter release. Glutamate-mediated excitotoxicity is well documented in neurons and oligodendrocytes but drugs targeting glutamate excitotoxicity have failed clinically which may be due to their inability to protect astrocytes. Astrocytes make up ~50% of the brain volume and express high levels of P2 adenosine triphosphate (ATP)-receptors which have excitotoxic potential, suggesting that glutamate and ATP may mediate parallel excitotoxic cascades in neurons and astrocytes, respectively. Mono-cultures of astrocytes expressed an array of P2X and P2Y receptors can produce large rises in [Ca2+]i; mono-cultured neurons showed lower levels of functional P2 receptors. Using high-density 1:1 neuron:astrocyte co-cultures, ischemia (modelled as oxygen-glucose deprivation: OGD) evoked a rise in extracellular ATP, while P2 blockers were highly protective of both cell types. GluR blockers were only protective of neurons. Neither astrocyte nor neuronal mono-cultures showed significant ATP release during OGD, showing that cell type interactions are required for ischemic release. P2 blockers were also protective in normal-density co-cultures, while low doses of combined P2/GluR blockers where highly protective. These results highlight the potential of combined P2/GluR block for protection of neurons and glia.

Experimental stroke in rodents, via middle cerebral artery occlusion (MCAO), can be associated with a negative impact on wellbeing and mortality. In hypertensive rodents, pre-stroke craniotomy increased survival and decreased body weight loss post-MCAO. Here we determined the effect, in normotensive Sprague-Dawley rats following 60 minutes MCAO, with or without pre-surgical craniotomy, on post-stroke outcomes in terms of weight loss, neurological deficit, lesion volume and functional outcomes. There was no effect of pre-stroke craniotomy on indicators of wellbeing including survival rate (P = 0.32), body weight loss (P = 0.42) and neurological deficit (P = 0.75). We also assessed common outcome measures following experimental stroke and found no effect of pre-stroke craniotomy on lesion volume as measured by T2-weighted MRI (P = 0.846), or functional performance up to 28 days post-MCAO (staircase test, P = 0.32; adhesive sticker test, P = 0.49; cylinder test, P = 0.38). Thus, pre-stroke craniotomy did not improve animal welfare in terms of body weight loss and neurological deficit. However, it is important, given that a number of drug delivery studies utilise the craniotomy procedure, to note that there was no effect on lesion volume or functional outcome following experimental stroke.

Two multi-centre phase III clinical trials examining the protective potential of progesterone following traumatic brain injury have recently failed to demonstrate any improvement in outcome. Thus, it is timely to consider how this impacts on the translational potential of progesterone treatment for ischaemic stroke. A wealth of experimental evidence supports the neuroprotective properties of progesterone, and associated metabolites, following various types of central nervous system injury. In particular, for ischaemic stroke, studies have also begun to reveal possible mechanisms of such neuroprotection. However, the results in traumatic brain injury now question whether further clinical development of progesterone for ischaemic stroke is relevant.

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information)., The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Meetings of the working group were funded by the NC3Rs. Dr. Michael J. O Neill is a full time employee of Eli Lilly & Co.

Sex is shown to affect various aspects of the clinical spectrum of ischaemic stroke, from how an individual patient may present with ischaemic stroke through to how they respond to treatment strategies. In this chapter, we review the evidence for the effect of sex on ischaemic stroke and focus on how the pathological mechanisms of ischaemic stroke, in particular apoptosis, may differ between the sexes. Understanding sex-specific consequences of ischaemic stroke may enable the development of relevant and effective treatment strategies for an individual suffering an ischaemic stroke.

Excitotoxicity is a major contributor to cell death during the acute phase of ischemic stroke but aggressive pharmacological targeting of excitotoxicity has failed clinically. Here we investigated whether pretreatment with low doses of memantine, within the range currently used and well tolerated for the treatment of Alzheimer's disease, produce a protective effect in stroke. A coculture preparation exposed to modeled ischemia showed cell death associated with rapid glutamate rises and cytotoxic Ca2+ influx. Cell death was significantly enhanced in the presence of high memantine concentrations. However, low memantine concentrations significantly protected neurons and glia via excitotoxic cascade interruption. Mice were systemically administered a range of memantine doses (0.02, 0.2, 2, 10, and 20 mg/kg/day) starting 24 hours before 60 minutes reversible focal cerebral ischemia and continuing for a 48-hour recovery period. Low dose (0.2 mg/kg/day) memantine treatment significantly reduced lesion volume (by 30% to 50%) and improved behavioral outcomes in stroke lesions that had been separated into either small/striatal or large/striatocortical infarcts. However, higher doses of memantine (20 mg/kg/day) significantly increased injury. These results show that clinically established low doses of memantine should be considered for patients ‘at risk’ of stroke, while higher doses are contraindicated.

Ischaemic strokes evoke blood–brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Cerebral stroke is a leading cause of long-term disability and a major cause of death in the developed world. The total incidence of stroke is projected to rise substantially over the next 20 years as a result of the rising elderly population. Although age is one of the most significant prognostic markers for poor outcome after stroke, very few experimental studies have been conducted in aged animals. Importantly, sex differences in both vulnerability to stroke and outcome after cerebral ischaemia have frequently been reported and attributed to the action of steroid hormones. Progesterone is a candidate neuroprotective factor for stroke, although the majority of pre-clinical studies have focused on using young, healthy adult animals. In terms of cerebral stroke, males and postmenopausal females represent the groups at highest risk of cerebral stroke and these categories can be modelled using either aged or ovariectomised female animals. In this review, we discuss the importance of conducting experimental studies in aged animals compared to young, healthy animals, as well as the impact this has on experimental outcomes. In addition, we focus on reviewing the studies that have been conducted to date examining the neuroprotective potential of progesterone in aged animals. Importantly, the limited studies that have been conducted in aged animals do lend further support to progesterone as a therapeutic option after ischaemic stroke that warrants further investigation.

Preclinical studies suggest progesterone is neuroprotective after cerebral ischemia. The gold standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data (IAD). Preclinical studies of progesterone in experimental stroke were identified from searches of electronic databases and reference lists. Corresponding authors of papers of interest were contacted to obtain IAD and, if unavailable, summary data were obtained from the publication. Data are given as standardized mean differences (SMDs, continuous data) or odds ratios (binary data), with 95% confidence intervals (95% CIs). In an unadjusted analysis of IAD and summary data, progesterone reduced standardized lesion volume (SMD −0.766, 95% CI −1.173 to −0.358, P < 0.001). Publication bias was apparent on visual inspection of a Begg’s funnel plot on lesion volume and statistically using Egger’s test ( P = 0.001). Using individual animal data alone, progesterone was associated with an increase in death in adjusted analysis (odds ratio 2.64, 95% CI 1.17 to 5.97, P = 0.020). Although progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death after experimental stroke, particularly in young ovariectomized female animals. Experimental studies must report the effect of interactions on death and on modifiers, such as age and sex.

Cerebral stroke continues to be a major cause of death and the leading cause of long-term disability in developed countries. Evidence reviewed here suggests that gender influences various aspects of the clinical spectrum of ischemic stroke, in terms of influencing how a patients present with ischemic stroke through to how they respond to treatment. In addition, this review focuses on discussing the various pathologic mechanisms of ischemic stroke that may differ according to gender and compares how intrinsic and hormonal mechanisms may account for such gender differences. All clinical trials to date investigating putative neuroprotective treatments for ischemic stroke have failed, and it may be that our understanding of the injury cascade initiated after ischemic injury is incomplete. Revealing aspects of the pathophysiological consequences of ischemic stroke that are gender specific may enable gender relevant and effective neuroprotective strategies to be identified. Thus, it is possible to conclude that gender does, in fact, have an important role in ischemic stroke and must be factored into experimental and clinical investigations of ischemic stroke.

Although COX-2 inhibition in animal models of ischaemia has shown neuroprotection, clinical trials revealed long term side effects with COX-2 inhibitors. A more focussed approach is necessary to retain the therapeutic effects of prostaglandins. This study investigated the role of the PGE(2) EP(4) receptor using both in vitro and in vivo models of ischaemia. To demonstrate whether targeting the EP(4) receptor is as neuroprotective as COX-2 inhibition, simultaneous experiments were carried out using a selective COX-2 inhibitor. Organotypic hippocampal sliced cultures, exposed to 2 h of oxygen glucose deprivation, were treated with; DMSO only, COX-2 inhibitor (NS-398), EP(4) agonist (L-902688) or EP(4) antagonist (GW627368X) and cell death was assessed. The EP(4) agonist and the COX-2 inhibitor significantly reduced cell death following in vitro ischaemia, whereas treatment with the EP(4) antagonist significantly increased cell death in hippocampal cultures. Following a 1 h occlusion of middle cerebral artery, mice were treated with the COX-2 inhibitor (10 mg kg, I.P), EP(4) agonist (0.75 μg/kg, I.P) or vehicle (I.P), at the onset of reperfusion and again at 24 h post stroke. The COX-2 inhibitor and EP(4) agonist treated animals showed a significant reduction in infarct volume (P < .05) at 48 h post stroke compared to the vehicle treated group. These results show that selective activation of the EP(4) receptor following acute ischaemic damage is neuroprotective, and support the concept of targeting protective prostaglandin receptor signalling as a potential therapeutic target for cerebral stroke.

As a predictor of potential clinical outcome, we performed a systematic review of controlled studies that assessed experimental stroke outcome in rodents maintained on special diets (calorie restriction and ketogenic diet) or following the direct administration of ketone bodies. Pre-clinical studies were identified by searching web databases and the reference lists of relevant original articles and reviews. Sixteen published studies (a total of 733 experimental animals) met specific criteria and were analyzed using Cochrane Review Manager software. This resulted in objective evidence to suggest beneficial effects of the ketogenic pathway on pathological and functional outcomes following experimental stroke.

The learning abilities of planarian worms (Dugesia tigrina) were assessed by using a number of Pavlovian conditioning paradigms. Experiment 1 showed that planaria were susceptible to basic conditioning in that they readily developed a conditioned response to a change in ambient luminance when it was consistently paired with an electric shock over a number of trials. In Experiment 2, the change in luminance was presented in a compound with a vibration stimulus during conditioning. Subsequent tests revealed poor conditioning of the elements compared with control groups in which the animals were conditioned in the presence of the elements alone, an instance of overshadowing. In Experiment 3, pre-training of one of the elements before compound conditioning resulted in blocking of learning about the other element. These results add to other studies that have reported cue competition effects in animal species belonging to different phyla (chordate, mollusk, arthropod), suggesting that learning in these phyla could be ruled by similar principles. The results are discussed adopting an evolutionary-comparative approach.

Cerebral stroke is a major cause of death and the leading cause ofneurological disability in the Western world (1). Unfortunately,despite its continuing impact on the health system, very few effec-tive therapies are available. Thrombolytic therapy, in the form oftissue plasminogen activator, remains one of the few agents in useclinically, although it has a narrow time window (2) and unresolvedissues regarding its many side-effects (3). There is a need for basicresearch exploring potential neuroprotective candidates to deter-mine whether clinical investigation is justified. Previous epidemio-logical studies have established that pre-menopausal women, incomparison with aged-matched men, have a lower risk of strokeoccurrence (4,5) and experience improved recovery after stroke (6).However, vascular events increase in frequency after the meno-pause (7), which is coincident with a decline in circulating steroidhormones, such as oestrogens and progesterone. It is suggestedthat endogenous steroid hormones may account for the naturalbenefit that females experience in terms of reduced stroke occur-rence and improved outcome.Recent clinical trials have questioned the potential of oestrogensas a prospective neuroprotective agent after ischaemic stroke (8,9),whereas progesterone-only clinical trials have yet to be conducted.However, after traumatic brain injury (TBI), two recent clinical trialssupport progesterone as a candidate neuroprotective factor becauseprogesterone treatment after TBI resulted in a lower 30-day mortal-ity rate versus placebo (10), and improved neurological outcome6 months after TBI (11). In terms of cerebral ischaemia, progester-one has been reported both to reduce the pathological damage andimprove functional outcome (12). A wealth of experimental evi-dence supports the neuroprotective role of progesterone, in particu-lar after cerebral ischaemia (13,14), and the mechanisms of

Current pharmacological interventions for acute stroke are largely ineffective or confounded by adverse effects, emphasising the need to develop new pharmacological treatments for neuroprotection. We have developed a robust in vitro model previously used in rats to assess dopamine release in mouse caudate nucleus brain slices, measured by fast cyclic voltammetry, during oxygen and glucose deprivation (OGD) as a model for cerebral ischaemia: this model will allow the study of transgenic mouse strains. During the pre-OGD equilibration period we found that a temperature of 33 °C, with solution containing 10 mM glucose provided the optimum baseline conditions from which reliable OGD-induced changes in dopamine efflux could be measured, without being susceptible to spontaneous release events. During OGD we found no significant difference in any of the parameters measured between perfusion with glucose-free solution, and perfusion with solution containing 2 mM glucose. We therefore suggest, in agreement with previous work, that using 2 mM glucose during OGD is appropriate, and using these conditions we were able to reliably produce OGD-evoked dopamine release.

Fast cyclic voltammetry can be used to measure dopamine release after oxygen and glucose deprivation (OGD) induced anoxic depolarization in vitro. Here we measure dopamine efflux with 1s time resolution, which is appropriate to measure OGD-evoked dopamine efflux accurately. In the present study, we examined whether OGD-evoked dopamine efflux could be used to show pre-ischaemic conditioning in the rat caudate brain slice. Caudate slices were exposed to 0, 2, or 10 min OGD pre-ischaemic conditioning, then 60 min later exposed to a second OGD event of 15 min duration. We measured the OGD-evoked dopamine efflux using fast cyclic voltammetry and in some experiments caudate dopamine and DOPAC tissue levels were measured using HPLC and 20 μm cryostat sections were Nissl stained to indicate neuronal loss. We found that 10 but not 2 min OGD pre-ischaemic conditioning resulted in a longer time to onset of OGD-evoked dopamine efflux on the main OGD event (475 ± 31 and 287 ± 30 s for 10 Vs 0 min pre-ischaemic conditioning respectively). Further, 10 min OGD pre-ischaemic conditioning resulted in less dopamine efflux on the second OGD event (4.23 ± 1.12 and 8.14 ± 0.82 μM for 10 Vs 0 min pre-ischaemic conditioning respectively), despite these slices having similar tissue dopamine content and DOPAC/DA ratio, and the rate of dopamine release was slower in the main OGD event (21 ± 5 and 74 ± 8 nM/s for 10 Vs 0 min pre-ischaemic conditioning respectively). These data suggest that 10 min OGD pre-ischaemic conditioning can evoke tolerance to a second OGD event and that voltammetric recording of OGD-evoked dopamine efflux is a useful model of pre-ischaemic conditioning in neuronal tissue.

Drugs that inhibit histone deacetylase (HDAC) activities have enormous potential as neuroprotective agents. We performed a systematic review of controlled animal studies that administered known inhibitors of the zinc-dependent HDACs before and/or after acute cerebral injury and assessed anatomic/functional outcomes. Relevant studies were found by searching PubMed, Embase and Web of Science. From more than 100 identified publications, those data meeting specific criteria were analyzed using the Cochrane Review Manager software. A beneficial effect of administering HDAC inhibitors was seen in studies involving cerebral ischemia or non-ischemic models of acute cerebral injury. Specific studies assessed efficacy when drug was administered up to 14 days prior to, and 14 days following, the onset of cerebral injury. This systematic review provides objective evidence of a neuroprotective role for drugs that inhibit HDACs and highlights particular areas that require further experimental investigation.

Background\ud Granulocyte-colony stimulating factor (G-CSF) shows promise as a treatment for stroke. This systematic review assesses G-CSF in experimental ischaemic stroke.\ud \ud Methods\ud Relevant studies were identified with searches of Medline, Embase and PubMed. Data were extracted on stroke lesion size, neurological outcome and quality, and analysed using Cochrane Review Manager using random effects models; results are expressed as standardised mean difference (SMD) and odds ratio (OR).\ud \ud Results\ud Data were included from 19 publications incorporating 666 animals. G-CSF reduced lesion size significantly in transient (SMD -1.63, p4 weeks post ischaemia) was not (SMD 0.76, p=0.35). Death (OR 0.27, p

Steroid sex hormones are potential neuroprotective candidates following CNS injury. All clinical trials to date have examined the effects of oestrogen alone or oestrogen-progestin combination therapy. Experimental studies have suggested that progesterone, in its own right, is a potential neuroprotective agent following acute cerebral injury. We performed a systematic review of controlled animal studies that administered progesterone before, or after, acute cerebral injury and measured lesion volume. Relevant studies were found from searching PubMed, Embase and Web of Science. From 119 identified publications, data from 18 studies using 480 experimental subjects met specific criteria and were analysed using the Cochrane Review Manager software. Following cerebral ischaemia, a significant benefit of progesterone was observed regardless of the assigned study quality score ( P = 0.0002) whereas, following traumatic brain injury (TBI) a significant benefit of progesterone was only observed in studies that obtained the highest quality score of 5 ( P = 0.02). Progesterone reduced lesion volume in a dose-dependent manner following either cerebral ischaemia ( P < 0.001) or TBI ( P = 0.03) with the most effective progesterone dose varying according to experimental injury model used. Progesterone treatment was only effective at reducing lesion volume when administered immediately following (i.e. 0–2 h) cerebral ischaemia ( P = 0.0008). No studies using models of cerebral ischaemia or TBI assessed efficacy when progesterone was administered at later than 6 h following the onset of cerebral injury. Limited data were available for different groups of animals according to age/hormonal status and the full dose–response relationship was not available in all experimental groups. Although this systematic review provides some supporting evidence for a neuroprotective role of progesterone following either cerebral ischaemia or TBI importantly it highlights areas which need further pre-clinical investigation.

BACKGROUND AND PURPOSE: Although CB(1) receptor activation evokes neuroprotection in response to cannabinoids, some cannabinoids have been reported to be peroxisome proliferator activated receptor (PPAR) ligands, offering an alternative protective mechanism. We have, therefore, investigated the ability of a range of cannabinoids to activate PPAR alpha and for N-oleoylethanolamine (OEA), an endogenous cannabinoid-like compound (ECL), to evoke neuroprotection. EXPERIMENTAL APPROACH: Assays of PPAR alpha occupancy and gene transactivation potential were conducted in cell-free and transfected HeLa cell preparations, respectively. In vivo estimates of PPAR alpha activation through fat mobilization and gene transcription were conducted in mice. Neuroprotection in vivo was investigated in wild-type and PPAR alpha gene-disrupted mice. KEY RESULTS: The ECLs OEA, anandamide, noladin ether and virodhamine were found to bind to the purified PPAR alpha ligand binding domain and to increase PPAR alpha-driven transcriptional activity. The high affinity synthetic CB(1/2) cannabinoid agonist WIN 55212-2 bound to PPAR alpha equipotently with the PPARalpha agonist fenofibrate, and stimulated PPARalpha-mediated gene transcription. The phytocannabinoid delta 9 tetrahydrocannabinol was without effect. OEA and WIN 55212-2 induced lipolysis in vivo, while OEA pre-treatment reduced infarct volume from middle cerebral artery occlusion in wild-type, but not in PPAR alpha-null mice. OEA treatment also led to increased expression of the NFkappa B-inhibitory protein, Ikappa B, in mouse cerebral cortex, while expression of the NFkappa B-regulated protein COX-2 was inhibited. CONCLUSIONS AND IMPLICATIONS: These data demonstrate the potential for a range of cannabinoid compounds, of diverse structures, to activate PPAR alpha and suggest that at least some of the neuroprotective properties of these agents could be mediated by nuclear receptor activation.

Estrogens are believed to provide females with endogenous protection against cerebrovascular events although clinical trials studying long-term hormone replacement have yielded disappointing results. In contrast, estrogens might be neuroprotective after experimental ischemia. We performed a systematic review of controlled experimental studies that administered estrogens before, or after, cerebral ischemia and measured lesion volume. Relevant studies were found from searching PubMed, Embase and Web of Science. From 161 identified publications, 27 studies using 1304 experimental subjects were analyzed using the Cochrane Review Manager software. Estrogens reduced lesion volume in a dose-dependent manner, after either transient ( P < 0.001) or permanent ( P < 0.001) ischemia and whether administered before or up to 4 h after ischemia onset; no studies assessed efficacy for later time periods. The effect size for estrogens decreased with increasing quality scores for studies of transient ischemia. Estrogens reduced lesion volume when administered to ovariectomized females and young adult males, but had no effect in intact females. Limited data were present for aged animals and the full dose-response relationship was not available in all experimental groups. On the basis of these data, estrogens are a candidate treatment for ischemic stroke, although further preclinical studies are also warranted.

Granulocyte-colony stimulating factor (G-CSF) is reported to be neuroprotective after transient cerebral ischemia with respect to decreasing lesion volume and enhancing functional recovery. We investigated whether G-CSF is neuroprotective after permanent ischemia and the possible mechanisms underlying this neuroprotection. Mice underwent permanent or 60-minute middle cerebral artery occlusion (MCAO) and received G-CSF (50 microg/kg) or vehicle at the onset or 1 hour post-MCAO. Forty-eight hours after transient MCAO, structural magnetic resonance imaging revealed a significant reduction (50%) in the amount of edematous tissue present in G-CSF-treated mice (p < 0.05). G-CSF treatment also prevented a significant increase in ipsilateral brain water content that was present in vehicle-treated mice after transient (p < 0.05) and permanent (p < 0.001) MCAO. Forty-eight hours after permanent MCAO, G-CSF decreased (50%) the cortical lesion volume (p < 0.05). Using real-time polymerase chain reaction, we found that G-CSF treatment significantly suppressed (p < 0.05) the injury-induced upregulation of IL-1beta mRNA while having no effect on TNFalpha and NOS-2 mRNA expression. This suggests that part of the neuroprotection may be attributed to the ability of G-CSF to reduce the inflammatory response.

Nitric oxide produced by the neuronal or inducible isoform of nitric oxide synthase (nNOS, iNOS) is detrimental in acute ischemic stroke (IS), whereas that derived from the endothelial isoform is beneficial. However, experimental studies with nitric oxide synthase inhibitors have given conflicting results. Relevant studies were found from searches of EMBASE, PubMed, and reference lists; of 456 references found, 73 studies involving 2321 animals were included. Data on the effects of NOS inhibition on lesion volume (mm3, %) and cerebral blood flow (CBF; %, ml * min(-1) * g(-1)) were analyzed using the Cochrane Review Manager software. NOS inhibitors reduced total infarct volume in models of permanent (standardized mean difference (SMD) -0.56, 95% confidence interval (95% CI) -0.86, -0.26) and transient (SMD -0.99, 95% CI -1.25, -0.72) ischemia. Cortical CBF was reduced in models of permanent but not transient ischemia. When assessed by type of inhibitor, total lesion volume was reduced in permanent models by nNOS and iNOS inhibitors, but not by nonselective inhibitors. All types of NOS inhibitors reduced infarct volume in transient models. NOS inhibition may have negative effects on CBF but further studies are required. Selective nNOS and iNOS inhibitors are candidate treatments for acute IS.

Gender differences in outcome following cerebral ischemia have frequently been observed and attributed to the actions of steroid hormones. Progesterone has been shown to possess neuroprotective properties following transient ischemia, with respect to decreasing lesion volume and improving functional recovery. The present study was designed to determine the mechanisms of progesterone neuroprotection, and whether these relate to the inflammatory response. Male mice underwent either 60 min or permanent middle cerebral artery occlusion (MCAO) and received progesterone (8 mg/kg ip) or vehicle 1 h, 6 h and 24 h post-MCAO. Forty-eight hours following transient MCAO, structural magnetic resonance imaging revealed a significant decrease in the amount of edematous tissue present in progesterone-treated mice as compared with vehicle. Using real-time PCR we found that progesterone treatment significantly suppressed the injury-induced upregulation of interleukin (IL)-1beta, transforming growth factor (TGF)beta2, and nitric oxide synthase (NOS)-2 mRNAs in the ipsilateral hemisphere while having no effect on tumor necrosis factor (TNF)-alpha mRNA expression. Progesterone treatment following permanent MCAO also resulted in a significant decrease in lesion volume. This was not apparent in mice lacking a functional NOS-2 gene. Thus, progesterone is neuroprotective in both permanent and transient ischemia, and this effect is related to the suppression of specific aspects of the inflammatory response.

One of the responses to cerebral ischemia is an increase in the production of nitric oxide, catalyzed by enzymes expressed in both resident and infiltrating cells. The nitric oxide that is generated does contribute to the ensuing pathology, but it can also be beneficial. The effects of nitric oxide depend on the cell site of production, the amount generated, and the chemical nature of the products of further oxidation. Understanding how nitric oxide production from microglia and astrocytes contributes to ischemic pathology is important for the development and application of future therapeutics based on inhibiting or amplifying its production in the injured brain.