Cognitive decline and diabetes: a systematic review of the neuropathological correlates accounting for cognition at death.

Given conflicting findings in epidemiologic studies, we determined the relative contributions of different neuropathologies to the excess risk of cognitive decline in diabetes mellitus (DM) through a systematic review of the literature. Included studies compared subjects with and without DM and reported neuropathological outcomes accounting for cognition at death. Data on Alzheimer's disease (AD) pathology, cerebrovascular disease and non-vascular, non-AD pathology were extracted from each study. Eleven studies (n=6 prospective cohorts, n=5 retrospective post-mortem series, total n=6330) met inclusion criteria. All 11 studies quantified AD changes and 10/11 measured cerebrovascular disease: macroscopic lesions (n=9), microinfarcts (n=8), cerebral amyloid angiopathy (CAA, n=7), lacunes (n=6), white matter disease (n=5), haemorrhages (n=4), microbleeds (n=1), hippocampal microvasculature (n=1). Other pathology was infrequently examined. No study reported increased AD pathology in DM, three studies showed a decrease (n=872) and four (n= 4018) showed no difference, after adjustment for cognition at death. No study reported reduced cerebrovascular pathology in DM. Three studies (n=2345) reported an increase in large infarcts, lacunes and microinfarcts. One study found lower cognitive scores in DM compared to non-DM subjects despite similar cerebrovascular and AD-pathology load suggesting contributions from other neuropathological processes. In conclusion, lack of an association between DM and AD-related neuropathology was consistent across studies, irrespective of methodology. In contrast to AD, DM was associated with increased large and small vessel disease. Data on other pathologies such as non-AD neurodegeneration, and blood-brain-barrier breakdown were lacking. Further studies evaluating relative contributions of different neuropathologies to the excess risk of DM are needed.

Neuroprotection in stroke: the importance of collaboration and reproducibility.

Acute ischaemic stroke accounts for 6.5 million deaths per year, and by 2030 will result in the annual loss of over 200 million disability-adjusted life years globally. There have been considerable recent advances in the gold standard of acute ischaemic stroke treatment, some aspects of which-aspirin to prevent recurrence, and treating patients in specialized stroke wards-are widely applicable. Recanalization of the occluded artery through thrombolysis and/or endovascular thrombectomy is restricted to only a small proportion of patients, due to contra-indications and the costs associated with establishing the infrastructure to deliver these treatments. The use of neuroprotective agents in stroke has been a notable failure of translation from medical research into clinical practice. Yet, with the advent of endovascular thrombectomy and the ability to investigate patients in much greater detail through advanced imaging modalities, neuroprotective agents can and should be re-examined as adjunct therapies to recanalization. In parallel, this requires appropriate planning on behalf of the preclinical stroke research community: there is a need to reinvestigate these therapies in a more collaborative manner, to enhance reproducibility through reduced attrition, improved reporting, and adopting an approach to target validation that more closely mimics clinical trials. This review will describe some of the novel strategies being used in stroke research, and focus on a few key examples of neuroprotective agents that are showing newfound promise in preclinical models of stroke therapy. Our primary aim is to give an overview of some of the challenges faced by preclinical stroke research, and suggest potential ways to improve translational success.

The exact science of stroke thrombolysis and the quiet art of patient selection.

The science of metric-based patient stratification for intravenous thrombolysis, revolutionized by the landmark National Institute of Neurological Disorders and Stroke trial, has transformed acute ischaemic stroke therapy. Recanalization of an occluded artery produces tissue reperfusion that unequivocally improves outcome and function in patients with acute ischaemic stroke. Recanalization can be achieved mainly through intravenous thrombolysis, but other methods such as intra-arterial thrombolysis or mechanical thrombectomy can also be employed. Strict guidelines preclude many patients from being treated by intravenous thrombolysis due to the associated risks. The quiet art of informed patient selection by careful assessment of patient baseline factors and brain imaging could increase the number of eligible patients receiving intravenous thrombolysis. Outside of the existing eligibility criteria, patients may fall into therapeutic 'grey areas' and should be evaluated on a case by case basis. Important factors to consider include time of onset, age, and baseline blood glucose, blood pressure, stroke severity (as measured by National Institutes of Health Stroke Scale) and computer tomography changes (as measured by Alberta Stroke Programme Early Computed Tomography Score). Patients with traditional contraindications such as wake-up stroke, malignancy or dementia may have the potential to receive benefit from intravenous thrombolysis if they have favourable predictors of outcome from both clinical and imaging criteria. A proportion of patients experience complications or do not respond to intravenous thrombolysis. In these patients, other endovascular therapies or a combination of both may be used to provide benefit. Although an evidence-based approach to intravenous thrombolysis for acute ischaemic stroke is pivotal, it is imperative to examine those who might benefit outside of protocol-driven practice.

A role for vessel-associated extracellular matrix proteins in multiple sclerosis pathology.

Multiple sclerosis (MS) is unsurpassed for its clinical and pathological hetherogeneity, but the biological determinants of this variability are unknown. HLA-DRB1*15, the main genetic risk factor for MS, influences the severity and distribution of MS pathology. This study set out to unravel the molecular determinants of the heterogeneity of MS pathology in relation to HLA-DRB1*15 status. Shotgun proteomics from a discovery cohort of MS spinal cord samples segregated by HLA-DRB*15 status revealed overexpression of the extracellular matrix (ECM) proteins, biglycan, decorin, and prolargin in HLA-DRB*15-positive cases, adding to established literature on a role of ECM proteins in MS pathology that has heretofore lacked systematic pathological validation. These findings informed a neuropathological characterisation of these proteins in a large autopsy cohort of 41 MS cases (18 HLA-DRB1*15-positive and 23 HLA-DRB1*15-negative), and seven non-neurological controls on motor cortical, cervical and lumbar spinal cord tissue. Biglycan and decorin demonstrate a striking perivascular expression pattern in controls that is reduced in MS (-36.5%, p = 0.036 and - 24.7%, p = 0.039; respectively) in lesional and non-lesional areas. A concomitant increase in diffuse parenchymal accumulation of biglycan and decorin is seen in MS (p = 0.015 and p = 0.001, respectively), particularly in HLA-DRB1*15-positive cases (p = 0.007 and p = 0.046, respectively). Prolargin shows a faint parenchymal pattern in controls that is markedly increased in MS cases where a perivascular deposition pattern is observed (motor cortex +97.5%, p = 0.001; cervical cord +49.1%, p = 0.016). Our findings point to ECM proteins and the vascular interface playing a central role in MS pathology within and outside the plaque area. As ECM proteins are known potent pro-inflammatory molecules, their parenchymal accumulation may contribute to disease severity. This study brings to light novel factors that may contribute to the heterogeneity of the topographical variation of MS pathology.

Transdermal fentanyl for cancer pain.

BACKGROUND: Opioid drugs have been used for many years to relieve pain. Transdermal fentanyl offers one option for delivering and maintaining pain relief in patients with moderate or severe cancer pain. OBJECTIVES: To determine the analgesic efficacy of transdermal fentanyl for relief of cancer pain, and to assess the adverse events associated with the use of transdermal fentanyl for relief of cancer pain. SEARCH METHODS: The following databases were searched: Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2013, Issue 4 of 12); MEDLINE (1966 to May 2013); EMBASE (1974 to May 2013; CANCERLIT (PubMED) (November 2012); and ClinicalTrials.gov (May 2013). SELECTION CRITERIA: Published randomised controlled trials (RCTs) using placebo or active comparators reporting on the analgesic effect of transdermal fentanyl in adults and children with cancer pain. Studies with fewer than 10 participants were excluded. DATA COLLECTION AND ANALYSIS: Data were extracted independently by two review authors. We extracted any available data on the number or proportion of patients with 'no worse than mild pain' or treatment success (very satisfied, or very good or excellent on patient global impression scales), together with information about adverse events and withdrawals. MAIN RESULTS: We identified nine studies meeting the inclusion criteria, including a Turkish study that is awaiting formal translation. There were 1244 participants randomised in classically designed RCTs, of whom 1197 had evaluable data, and 138 patients enrolled in an enriched enrolment, randomised withdrawal (EERW) trial. Overall, 600 participants were treated with transdermal fentanyl patches, 382 with various formulations of morphine, 36 with methadone, and 221 with paracetamol plus codeine. There were major sources of potential bias, including lack of blinding, small size, high levels of attrition, and inconsistent reporting.We could not compare data in a meaningful analysis regarding adverse events such as nausea, abdominal pain, gastrointestinal bleeding, and confusion. These events may have been attributable to the underlying disease process.There were insufficient comparable data for meta-analysis to be undertaken or to produce numbers needed to treat (NNT) for the analgesic effect. In seven studies with 461 participants reporting pain intensity results after about two weeks, the mean or median pain scores were on the borderline of mild and moderate pain. Most participants would have had no worse than mild pain on treatment. Another reported that 77% of participants using transdermal fentanyl had an undefined successful outcome. Fewer participants experienced constipation with transdermal fentanyl (28%) than with oral morphine (46%), giving a risk ratio of 0.61 (95% CI 0.47 to 0.78); the NNT to prevent constipation was 5.5 (95% CI 3.8 to 10). AUTHORS' CONCLUSIONS: The randomised trial literature for effectiveness of transdermal fentanyl is limited, but it is an important medicine. Most studies recruited fewer than 100 participants and did not provide data appropriate for meta-analysis. Only a few reported how many patients had good pain relief but, where data were reported, a majority had no worse than mild pain within a reasonably short time period. The evidence pointed to a useful and significant reduction in complaints about constipation for transdermal fentanyl compared with oral morphine.

Regional contribution of vascular dysfunction in white matter dementia: clinical and neuropathological insights.

The maintenance of adequate blood supply and vascular integrity is fundamental to ensure cerebral function. A wide range of studies report vascular dysfunction in white matter dementias, a group of cerebral disorders characterized by substantial white matter damage in the brain leading to cognitive impairment. Despite recent advances in imaging, the contribution of vascular-specific regional alterations in white matter dementia has been not extensively reviewed. First, we present an overview of the main components of the vascular system involved in the maintenance of brain function, modulation of cerebral blood flow and integrity of the blood-brain barrier in the healthy brain and during aging. Second, we review the regional contribution of cerebral blood flow and blood-brain barrier disturbances in the pathogenesis of three distinct conditions: the archetypal white matter predominant neurocognitive dementia that is vascular dementia, a neuroinflammatory predominant disease (multiple sclerosis) and a neurodegenerative predominant disease (Alzheimer's). Finally, we then examine the shared landscape of vascular dysfunction in white matter dementia. By emphasizing the involvement of vascular dysfunction in the white matter, we put forward a hypothetical map of vascular dysfunction during disease-specific progression to guide future research aimed to improve diagnostics and facilitate the development of tailored therapies.

Posterior circulation ischaemic stroke diagnosis and management.

This narrative review provides an overview of the posterior circulation and the clinical features of common posterior circulation stroke (PCS) syndromes in the posterior arterial territories and how to distinguish them from mimics. We outline the hyperacute management of patients with suspected PCS with emphasis on how to identify those who are likely to benefit from intervention based on imaging findings. Finally, we review advances in treatment options, including developments in endovascular thrombectomy (EVT) and intravenous thrombolysis (IVT), and the principles of medical management and indications for neurosurgery. Observational and randomised clinical trial data have been equivocal regarding EVT in PCS, but more recent studies strongly support its efficacy. There have been concomitant advances in imaging of posterior stroke to guide optimal patient selection for thrombectomy. Recent evidence suggests that clinicians should have a heightened suspicion of posterior circulation events with the resultant implementation of timely, evidence-based management.

Suppression of the inflammatory response by diphenyleneiodonium after transient focal cerebral ischemia.

Diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, reduces production of reactive oxygen species (ROS) and confers neuroprotection to focal cerebral ischemia. Our objective was to investigate whether the neuroprotective action of DPI extends to averting the immune response. DPI-induced gene changes were analyzed by microarray analysis from rat brains subjected to 90 min of middle cerebral artery occlusion, treated with NaCl (ischemia), dimethylsulfoxide (DMSO), or DMSO and DPI (DPI), and reperfused for 48 h. The genomic expression profile was compared between groups using ingenuity pathway analysis at the pathway and network level. DPI selectively up-regulated 23 genes and down-regulated 75 genes more than twofold compared with both DMSO and ischemia. It significantly suppressed inducible nitric oxide synthase signaling and increased the expression of methionine adenosyltransferasesynthetase 2A and adenosylmethionine decarboxylase 1 genes, which are involved in increasing the production of the antioxidant glutathione. The most significantly affected gene network comprised genes implicated in the inflammatory response with an expression change indicating an overall suppression. Both integrin- and interleukin-17A-signaling pathways were also significantly associated and suppressed. In conclusion, the neuroprotective effects of DPI are mediated not only by suppressing ischemia-triggered oxidative stress but also by limiting leukocyte migration and infiltration.

Equity for excellence in academic institutions: a manifesto for change.

Higher academic institutions in the UK need to drive improvements in equity, diversity, and inclusion (EDI) through sustainable practical interventions. A broad view of inclusivity is based on an intersectional approach that considers race, geographical location, caring responsibilities, disability, neurodiversity, religion, and LGBTQIA+ identities. We describe the establishment of a diverse stakeholder group to develop practical grass-roots recommendations through which improvements can be advanced. We have developed a manifesto for change, comprising six domains through which academic institutions can drive progress through setting short, medium, and long-term priorities. Interventions will yield rewards in recruitment and retention of a diverse talent pool, leading to enhanced impact and output.

Growth Differentiation Factor-11 Causes Neurotoxicity During Ischemia in vitro.

Age-related neuronal dysfunction can be overcome by circulating factors present in young blood. Growth differentiation factor-11 (GDF-11), a systemic factor that declines with age, can reverse age-related dysfunction in brain, heart and skeletal muscle. Given that age increases susceptibility to stroke, we hypothesized that GDF-11 may be directly protective to neurons following ischemia. Primary cortical neurons were isolated from E18 Wistar rat embryos and cultured for 7-10 days. Neurons were deprived of oxygen and glucose (OGD) to simulate ischemia. Neuronal death was assessed by lactate dehydrogenase, propidium iodide or CellTox™ green cytotoxicity assays. 40 ng/mL GDF-11 administration during 2 h OGD significantly increased neuronal death following 24 h recovery. However, GDF-11 pre-treatment did not affect neuronal death during 2 h OGD. GDF-11 treatment during the 24 h recovery period after 2 h OGD also did not alter death. Real-time monitoring for 24 h revealed that by 2 h OGD, GDF-11 treatment had increased neuronal death which remained raised at 24 h. Co-treatment of 1 µM SB431542 (ALK4/5/7 receptor inhibitor) with GDF-11 prevented GDF-11 neurotoxicity after 2 h OGD and 24 h OGD. Transforming growth factor beta (TGFß) did not increase neuronal death to the same extent as GDF-11 following OGD. GDF-11 neurotoxicity was also exhibited following neuronal exposure to hydrogen peroxide. These results reveal for the first time that GDF-11 is neurotoxic to primary neurons in the acute phase of simulated stroke through primarily ALK4 receptor signaling.

Investigation of the novel mTOR inhibitor AZD2014 in neuronal ischemia.

INTRODUCTION: Hamartin, a component of the tuberous sclerosis complex (TSC) that actively inhibits the mammalian target of rapamycin (mTOR), may mediate the endogenous resistance of Cornu Ammonis 3 (CA3) hippocampal neurons following global cerebral ischemia. Pharmacological compounds that selectively inhibit mTOR may afford neuroprotection following ischemic stroke. We hypothesize that AZD2014, a novel mTORC1/2 inhibitor, may protect neurons following oxygen and glucose deprivation (OGD). METHODS: Primary neuronal cultures from E18 Wistar rat embryos were exposed to 2 h OGD or normoxia. AZD2014 was administered either during OGD, 24 h prior to OGD or for 24 h following OGD. Cell death was quantified by lactate dehydrogenase assay. We characterized the expression of mTOR pathway proteins following exposure to AZD2014 using western blotting. RESULTS: Following 2 h OGD +24 h recovery, AZD2014 increased neuronal death when present during OGD. Rapamycin, the archetypal mTOR inhibitor, had no effect on cell death. Treatment with AZD2014 24 h prior to OGD and 24 h after OGD also enhanced cell death. While Western blotting showed a trend towards decreased expression levels of phospho-Akt relative to total Akt with increasing AZD2014 concentration, hamartin expression was also significantly decreased leading to activation of mTOR. CONCLUSION: AZD2014 was detrimental to neurons that underwent ischemia. AZD2014 appeared to reduce hamartin, a known neuroprotective mediator, thereby preventing any beneficial effects of mTOR inhibition. Further characterization of the role of individual mTOR complexes (mTORC1 and mTORC2) and their upstream and downstream regulators are necessary to reveal whether these pathways are neuroprotective targets for stroke.

The effect of rapamycin treatment on cerebral ischemia: A systematic review and meta-analysis of animal model studies.

BACKGROUND: Amplifying endogenous neuroprotective mechanisms is a promising avenue for stroke therapy. One target is mammalian target of rapamycin (mTOR), a serine/threonine kinase regulating cell proliferation, cell survival, protein synthesis, and autophagy. Animal studies investigating the effect of rapamycin on mTOR inhibition following cerebral ischemia have shown conflicting results. AIM: To conduct a systematic review and meta-analysis evaluating the effectiveness of rapamycin in reducing infarct volume in animal models of ischemic stroke. SUMMARY OF REVIEW: Our search identified 328 publications. Seventeen publications met inclusion criteria (52 comparisons: 30 reported infarct size and 22 reported neurobehavioral score). Study quality was modest (median 4 of 9) with no evidence of publication bias. The point estimate for the effect of rapamycin was a 21.6% (95% CI, 7.6%-35.7% p < 0.01) improvement in infarct volume and 30.5% (95% CI 17.2%-43.8%, p < 0.0001) improvement in neuroscores. Effect sizes were greatest in studies using lower doses of rapamycin. CONCLUSION: Low-dose rapamycin treatment may be an effective therapeutic option for stroke. Modest study quality means there is a potential risk of bias. We recommend further high-quality preclinical studies on rapamycin in stroke before progressing to clinical trials.

Rapamycin in ischemic stroke: Old drug, new tricks?

The significant morbidity that accompanies stroke makes it one of the world's most devastating neurological disorders. Currently, proven effective therapies have been limited to thrombolysis and thrombectomy. The window for the administration of these therapies is narrow, hampered by the necessity of rapidly imaging patients. A therapy that could extend this window by protecting neurons may improve outcome. Endogenous neuroprotection has been shown to be, in part, due to changes in mTOR signalling pathways and the instigation of productive autophagy. Inducing this effect pharmacologically could improve clinical outcomes. One such therapy already in use in transplant medicine is the mTOR inhibitor rapamycin. Recent evidence suggests that rapamycin is neuroprotective, not only via neuronal autophagy but also through its broader effects on other cells of the neurovascular unit. This review highlights the potential use of rapamycin as a multimodal therapy, acting on the blood-brain barrier, cerebral blood flow and inflammation, as well as directly on neurons. There is significant potential in applying this old drug in new ways to improve functional outcomes for patients after stroke.

How useful are systematic reviews for informing palliative care practice? Survey of 25 Cochrane systematic reviews.

BACKGROUND: In contemporary medical research, randomised controlled trials are seen as the gold standard for establishing treatment effects where it is ethical and practical to conduct them. In palliative care such trials are often impractical, unethical, or extremely difficult, with multiple methodological problems. We review the utility of Cochrane reviews in informing palliative care practice. METHODS: Published reviews in palliative care registered with the Cochrane Pain, Palliative and Supportive Care Group as of December 2007 were obtained from the Cochrane Database of Systematic Reviews, issue 1, 2008. We reviewed the quality and quantity of primary studies available for each review, assessed the quality of the review process, and judged the strength of the evidence presented. There was no prior intention to perform any statistical analyses. RESULTS: 25 published systematic reviews were identified. Numbers of included trials ranged from none to 54. Within each review, included trials were heterogeneous with respect to patients, interventions, and outcomes, and the number of patients contributing to any single analysis was generally much lower than the total included in the review. A variety of tools were used to assess trial quality; seven reviews did not use this information to exclude low quality studies, weight analyses, or perform sensitivity analysis for effect of low quality. Authors indicated that there were frequently major problems with the primary studies, individually or in aggregate. Our judgment was that the reviewing process was generally good in these reviews, and that conclusions were limited by the number, size, quality and validity of the primary studies.We judged the evidence about 23 of the 25 interventions to be weak. Two reviews had stronger evidence, but with limitations due to methodological heterogeneity or definition of outcomes. No review provided strong evidence of no effect. CONCLUSION: Cochrane reviews in palliative care are well performed, but fail to provide good evidence for clinical practice because the primary studies are few in number, small, clinically heterogeneous, and of poor quality and external validity. They are useful in highlighting the weakness of the evidence base and problems in performing trials in palliative care.

The role of the endoplasmic reticulum stress response following cerebral ischemia.

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Imiquimod for actinic keratosis: systematic review and meta-analysis.

Benefit and harm associated with treating actinic keratosis (AK) with the immune response modifier imiquimod was assessed using published randomized-controlled trials. Five randomized double-blind trials lasted 12-16 weeks and treated 1,293 patients. Complete clearance occurred in 50% of patients treated with imiquimod, compared to 5% treated with vehicle, and the number needed to treat (NNT) for one patient to have their keratosis completely cleared after 12-16 weeks was 2.2 (95% confidence interval 2.0-2.5). For partial (>/=75%) clearance the NNT was 1.8 (1.7-2.0). The proportion of patients with any adverse event, any local adverse event, or any treatment-related adverse event was substantially higher with imiquimod than with vehicle, and numbers needed to harm for one additional adverse event with imiquimod over 12-16 weeks ranged from 3.2 to 5.9. Particular local adverse events with imiquimod included erythema (27%), scabbing or crusting (21%), flaking (9%), erosion (6%), edema (4%), and weeping (3%). Imiquimod 5% cream was effective in the treatment of AK, preventing potential development of squamous cell carcinoma. Future investigation might be aimed at elucidating optimal dosing to minimize adverse events without detriment to efficacy, and evaluating long-term recurrence.

The transient intraluminal filament middle cerebral artery occlusion model as a model of endovascular thrombectomy in stroke.

The clinical relevance of the transient intraluminal filament model of middle cerebral artery occlusion (tMCAO) has been questioned due to distinct cerebral blood flow profiles upon reperfusion between tMCAO (abrupt reperfusion) and alteplase treatment (gradual reperfusion), resulting in differing pathophysiologies. Positive results from recent endovascular thrombectomy trials, where the occluding clot is mechanically removed, could revolutionize stroke treatment. The rapid cerebral blood flow restoration in both tMCAO and endovascular thrombectomy provides clinical relevance for this pre-clinical model. Any future clinical trials of neuroprotective agents as adjuncts to endovascular thrombectomy should consider tMCAO as the model of choice to determine pre-clinical efficacy.