Perihaematomal oedema evolution over two weeks after spontaneous intracerebral haemorrhage and association with outcome: prospective cohort study.

Introduction We know little about the evolution of perihaematomal oedema (PHO) >24 hours after ICH onset. We aimed to determine the trajectory of PHO after ICH onset and its association with outcome. Methods We did a prospective cohort study using a pre-specified scanning protocol in adults with first-ever spontaneous ICH and measured absolute PHO volumes on CT head scans at ICH diagnosis and 3±2, 7±2 and 14±2 days after ICH onset. We used the largest ICH if ICHs were multiple. The primary outcomes were (a) the trajectory of PHO after ICH onset and (b) the association between PHO (absolute volume at the time when most repeat CT head scans were obtained, and change in PHO volume at this time compared with the first CT head scan) and poor functional outcome (modified Rankin scale 3-6 at 90 days). We pre-specified multivariable logistic regression models of this association adjusting analyses for potential confounders: age, GCS, infratentorial ICH location and intraventricular extension. Results In 106 participants of whom forty nine (46%) were female, with a median ICH volume 7ml (interquartile range [IQR] 2-22ml), the trajectory of median PHO volume was an increase from 14ml (IQR 7-26ml) at diagnosis to 18ml (IQR 8-40ml) at 3±2 days (n=87), 20ml (IQR 8-48ml) at 7±2 days (n=93) and 21ml (IQR 10-54ml) at 14±2 days (n=78) (p=<0.001). PHO volume at each time point was collinear with ICH volume at diagnosis (│r│>0.7) but the change in PHO volume between diagnosis and each time point was not. Given collinearity, we used total lesion (i.e. ICH+PHO) volume instead of PHO volume in a logistic regression model of its association at each time point with outcome. Increasing total lesion (ICH+PHO) volume at day 7±2 was associated with poor functional outcome (adjusted OR per ml 1.02, 95% CI 1.00-1.03; p=0.036) but the increase in PHO volume between diagnosis and day 7±2 was not associated with poor functional outcome (adjusted OR per ml 1.03, 95% CI 0.99-1.07; p=0.132). Conclusion PHO volume increases throughout the first two weeks after onset of mild to moderate ICH. Total lesion (ICH+PHO) volume at day 7±2 was associated with poor functional outcome but the change in PHO volume between diagnosis and day 7±2 was not. Prospective cohort studies with larger sample sizes are needed to investigate these associations and their modifiers.

Hypoxia coordinates the spatial landscape of myeloid cells within glioblastoma to affect survival.

Myeloid cells are highly prevalent in glioblastoma (GBM), existing in a spectrum of phenotypic and activation states. We now have limited knowledge of the tumor microenvironment (TME) determinants that influence the localization and the functions of the diverse myeloid cell populations in GBM. Here, we have utilized orthogonal imaging mass cytometry with single-cell and spatial transcriptomic approaches to identify and map the various myeloid populations in the human GBM tumor microenvironment (TME). Our results show that different myeloid populations have distinct and reproducible compartmentalization patterns in the GBM TME that is driven by tissue hypoxia, regional chemokine signaling, and varied homotypic and heterotypic cellular interactions. We subsequently identified specific tumor subregions in GBM, based on composition of identified myeloid cell populations, that were linked to patient survival. Our results provide insight into the spatial organization of myeloid cell subpopulations in GBM, and how this is predictive of clinical outcome.

The NLRP3 inflammasome is essential for IL-18 production in a model of macrophage activation syndrome.

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin (IL)-18 and interferon (IFN)-γ. Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-DNA induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome, or downstream caspase-1, prevented MAS-mediated upregulation of plasma IL-18 but interestingly did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore IL-1 receptor blockade with IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that in the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18, a key cytokine in clinical cases of MAS, but was not a driving factor in the pathogenesis of CpG-induced MAS.

Selective deletion of interleukin-1 alpha in microglia regulates neuronal responses and neurorepair processes after experimental ischemic stroke.

Inflammation is a key contributor to stroke pathogenesis and drives exacerbated brain damage leading to poor outcome. Interleukin-1 (IL-1) is an important regulator of post-stroke inflammation, and blocking its actions is beneficial in pre-clinical stroke models and safe in the clinical setting. IL-1α and IL-1ß are the two major IL-1 type 1 receptor (IL-1R1) agonists from the IL-1 family. The distinct roles of both isoforms, and particularly that of IL-1α, remain largely unknown. Here we show that IL-1α and IL-1ß have different spatio-temporal expression profiles in the brain after experimental stroke, with early microglial IL-1α expression (4 h) and delayed IL-1ß expression in infiltrated neutrophils and a small microglial subset (24-72 h). We examined the specific contribution of microglial-derived IL-1α in experimental permanent and transient ischemic stroke through cell-specific tamoxifen-inducible Cre-loxP-mediated recombination. Microglial IL-1α deletion did not influence acute brain damage, cerebral blood flow, IL-1ß expression, neutrophil infiltration, microglial nor endothelial activation after ischemic stroke. However, microglial IL-1α knock out (KO) mice showed reduced peri-infarct vessel density and reactive astrogliosis at 14 days post-stroke, alongside a worse functional recovery. RNA sequencing analysis and subsequent pathway analysis on ipsilateral/contralateral cortex 4 h after stroke revealed a downregulation of the neuronal CREB signaling pathway in microglial IL-1α KO compared to WT mice. Our study identifies for the first time a critical role for microglial IL-1α on neuronal activity, neurorepair and functional recovery after stroke, highlighting the importance of targeting specific IL-1 mechanisms in brain injury to develop more effective therapies.

Baseline perihematomal edema, C-reactive protein, and 30-day mortality are not associated in intracerebral hemorrhage.

Background: The relationship between baseline perihematomal edema (PHE) and inflammation, and their impact on survival after intracerebral hemorrhage (ICH) are not well understood. Objective: Assess the association between baseline PHE, baseline C-reactive protein (CRP), and early death after ICH. Methods: Analysis of pooled data from multicenter ICH registries. We included patients presenting within 24 h of symptom onset, using multifactorial linear regression model to assess the association between CRP and edema extension distance (EED), and a multifactorial Cox regression model to assess the association between CRP, PHE volume and 30-day mortality. Results: We included 1,034 patients. Median age was 69 (interquartile range [IQR] 59-79), median baseline ICH volume 11.5 (IQR 4.3-28.9) mL, and median baseline CRP 2.5 (IQR 1.5-7.0) mg/L. In the multifactorial analysis [adjusting for cohort, age, sex, log-ICH volume, ICH location, intraventricular hemorrhage (IVH), statin use, glucose, and systolic blood pressure], baseline log-CRP was not associated with baseline EED: for a 50% increase in CRP the difference in expected mean EED was 0.004 cm (95%CI 0.000-0.008, p = 0.055). In a further multifactorial analysis, after adjusting for key predictors of mortality, neither a 50% increase in PHE volume nor CRP were associated with higher 30-day mortality (HR 0.97; 95%CI 0.90-1.05, p = 0.51 and HR 0.98; 95%CI 0.93-1.03, p = 0.41, respectively). Conclusion: Higher baseline CRP is not associated with higher baseline edema, which is also not associated with mortality. Edema at baseline might be driven by different pathophysiological processes with different effects on outcome.

The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome.

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin (IL)-18 and interferon (IFN)-γ. Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-DNA induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome, or downstream caspase-1, prevented MAS-mediated upregulation of plasma IL-18 but interestingly did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore IL-1 receptor blockade with IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that in the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18, a key cytokine in clinical cases of MAS, but was not a driving factor in the pathogenesis of CpG-induced MAS.

Hypoxia and interleukin-1-primed mesenchymal stem/stromal cells as novel therapy for stroke.

Promising preclinical stroke research has not yielded meaningful and significant success in clinical trials. This lack of success has prompted the need for refinement of preclinical studies with the intent to optimize the chances of clinical success. Regenerative medicine, especially using mesenchymal stem/stromal cells (MSCs), has gained popularity in the last decade for treating many disorders, including central nervous system (CNS), such as stroke. In addition to less stringent ethical constraints, the ample availability of MSCs also makes them an attractive alternative to totipotent and other pluripotent stem cells. The ability of MSCs to differentiate into neurons and other brain parenchymal and immune cells makes them a promising therapy for stroke. However, these cells also have some drawbacks that, if not addressed, will render MSCs unfit for treating ischaemic stroke. In this review, we highlighted the molecular and cellular changes that occur following an ischaemic stroke (IS) incidence and discussed the physiological properties of MSCs suitable for tackling these changes. We also went further to discuss the major drawbacks of utilizing MSCs in IS and how adequate priming using both hypoxia and interleukin-1 can optimize the beneficial properties of MSCs while eliminating these drawbacks.

Stroke and Translational Research - Review of Experimental Models with a Focus on Awake Ischaemic Induction and Anaesthesia.

Animal models are an indispensable tool in the study of ischaemic stroke with hundreds of drugs emerging from the preclinical pipeline. However, all of these drugs have failed to translate into successful treatments in the clinic. This has brought into focus the need to enhance preclinical studies to improve translation. The confounding effects of anaesthesia on preclinical stroke modelling has been raised as an important consideration. Various volatile and injectable anaesthetics are used in preclinical models during stroke induction and for outcome measurements such as imaging or electrophysiology. However, anaesthetics modulate several pathways essential in the pathophysiology of stroke in a dose and drug dependent manner. Most notably, anaesthesia has significant modulatory effects on cerebral blood flow, metabolism, spreading depolarizations, and neurovascular coupling. To minimise anaesthetic complications and improve translational relevance, awake stroke induction has been attempted in limited models. This review outlines anaesthetic strategies employed in preclinical ischaemic rodent models and their reported cerebral effects. Stroke related complications are also addressed with a focus on infarct volume, neurological deficits, and thrombolysis efficacy. We also summarise routinely used focal ischaemic stroke rodent models and discuss the attempts to induce some of these models in awake rodents.

A novel human iPSC model of COL4A1/A2 small vessel disease unveils a key pathogenic role of matrix metalloproteinases.

Cerebral small vessel disease (SVD) affects the small vessels in the brain and is a leading cause of stroke and dementia. Emerging evidence supports a role of the extracellular matrix (ECM), at the interface between blood and brain, in the progression of SVD pathology, but this remains poorly characterized. To address ECM role in SVD, we developed a co-culture model of mural and endothelial cells using human induced pluripotent stem cells from patients with COL4A1/A2 SVD-related mutations. This model revealed that these mutations induce apoptosis, migration defects, ECM remodeling, and transcriptome changes in mural cells. Importantly, these mural cell defects exert a detrimental effect on endothelial cell tight junctions through paracrine actions. COL4A1/A2 models also express high levels of matrix metalloproteinases (MMPs), and inhibiting MMP activity partially rescues the ECM abnormalities and mural cell phenotypic changes. These data provide a basis for targeting MMP as a therapeutic opportunity in SVD.

A multi-disciplinary commentary on preclinical research to investigate vascular contributions to dementia.

Although dementia research has been dominated by Alzheimer's disease (AD), most dementia in older people is now recognised to be due to mixed pathologies, usually combining vascular and AD brain pathology. Vascular cognitive impairment (VCI), which encompasses vascular dementia (VaD) is the second most common type of dementia. Models of VCI have been delayed by limited understanding of the underlying aetiology and pathogenesis. This review by a multidisciplinary, diverse (in terms of sex, geography and career stage), cross-institute team provides a perspective on limitations to current VCI models and recommendations for improving translation and reproducibility. We discuss reproducibility, clinical features of VCI and corresponding assessments in models, human pathology, bioinformatics approaches, and data sharing. We offer recommendations for future research, particularly focusing on small vessel disease as a main underpinning disorder.

Intracerebral Administration of a Novel Self-Assembling Peptide Hydrogel Is Safe and Supports Cell Proliferation in Experimental Intracerebral Haemorrhage.

Intracerebral haemorrhage (ICH) is the deadliest form of stroke, but current treatment options are limited, meaning ICH survivors are often left with life-changing disabilities. The significant unmet clinical need and socioeconomic burden of ICH mean novel regenerative medicine approaches are gaining interest. To facilitate the regeneration of the ICH lesion, injectable biomimetic hydrogels are proposed as both scaffolds for endogenous repair and delivery platforms for pro-regenerative therapies. In this paper, the objective was to explore whether injection of a novel self-assembling peptide hydrogel (SAPH) Alpha2 was feasible, safe and could stimulate brain tissue regeneration, in a collagenase-induced ICH model in rats. Alpha2 was administered intracerebrally at 7 days post ICH and functional outcome measures, histological markers of damage and repair and RNA-sequencing were investigated for up to 8 weeks. The hydrogel Alpha2 was safe, well-tolerated and was retained in the lesion for several weeks, where it allowed infiltration of host cells. The hydrogel had a largely neutral effect on functional outcomes and expression of angiogenic and neurogenic markers but led to increased numbers of proliferating cells. RNAseq and pathway analysis showed that ICH altered genes related to inflammatory and phagocytic pathways, and these changes were also observed after administration of hydrogel. Overall, the results show that the novel hydrogel was safe when injected intracerebrally and had no negative effects on functional outcomes but increased cell proliferation. To elicit a regenerative effect, future studies could use a functionalised hydrogel or combine it with an adjunct therapy.

Uncoupling of Ca2+ sparks from BK channels in cerebral arteries underlies hypoperfusion in hypertension-induced vascular dementia.

The deficit in cerebral blood flow (CBF) seen in patients with hypertension-induced vascular dementia is increasingly viewed as a therapeutic target for disease-modifying therapy. Progress is limited, however, due to uncertainty surrounding the mechanisms through which elevated blood pressure reduces CBF. To investigate this, we used the BPH/2 mouse, a polygenic model of hypertension. At 8 mo of age, hypertensive mice exhibited reduced CBF and cognitive impairment, mimicking the human presentation of vascular dementia. Small cerebral resistance arteries that run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to diminished activity of large-conductance Ca2+-activated K+ (BK) channels-key vasodilatory ion channels of cerebral vascular smooth muscle cells. Activation of BK channels by transient intracellular Ca2+ signals from the sarcoplasmic reticulum (SR), termed Ca2+ sparks, leads to hyperpolarization and vasodilation. Combining patch-clamp electrophysiology, high-speed confocal imaging, and proximity ligation assays, we demonstrated that this vasodilatory mechanism is uncoupled in hypertensive mice, an effect attributable to physical separation of the plasma membrane from the SR rather than altered properties of BK channels or Ca2+ sparks, which remained intact. This pathogenic mechanism is responsible for the observed increase in constriction and can now be targeted as a possible avenue for restoring healthy CBF in vascular dementia.

Preconditioning with interleukin-1 alpha is required for the neuroprotective properties of mesenchymal stem cells after ischemic stroke in mice.

Mesenchymal stem cell (MSC) pre-conditioning with interleukin-1 alpha (IL-1ɑ) drives MSCs toward a potent anti-inflammatory phenotype. The aim of this study was to assess the therapeutic potential of intra-arterially administered IL-1ɑ preconditioned MSCs, after experimental cerebral ischaemia in mice. After 3 h from the start of middle cerebral artery occlusion, animals were treated with vehicle, 9.1 × 104 non-conditioned or IL-1ɑ preconditioned MSCs by intra-arterial administration. Animals were allowed to recover for 1.5 h after treatment to measure cerebral blood flow (CBF), and 3 days or 14 days post-stroke to evaluate lesion volume and functional outcomes. At 3-days post-stroke preconditioned MSCs reduced (by 67%) lesion volume and increased CBF (by 32%) compared to vehicle, while non-conditioned MSCs had no effect. A separate cohort of animals recovered to 14 days post-stroke also showed reduced infarct volume (by 51%) at 48 h (assessed by MRI) and better functional recovery at 14 days when treated with preconditioned MSCs when compared to vehicle. Preconditioning MSCs with IL-1α increases their neuroprotective capability and improves functional recovery after delayed intra-arterial administration. With increasing use of thrombectomy, the adjunct use of preconditioned MSCs therefore represents a highly relevant therapy to improve outcomes in ischemic stroke.

Characterization of a mutant samhd1 zebrafish model implicates dysregulation of cholesterol biosynthesis in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS1-9) is a genetically determined encephalopathy that falls under the type I interferonopathy disease class, characterized by excessive type I interferon (IFN-I) activity, coupled with upregulation of IFN-stimulated genes (ISGs), which can be explained by the vital role these proteins play in self-non-self-discrimination. To date, few mouse models fully replicate the vast clinical phenotypes observed in AGS patients. Therefore, we investigated the use of zebrafish as an alternative species for generating a clinically relevant model of AGS. Using CRISPR-cas9 technology, we generated a stable mutant zebrafish line recapitulating AGS5, which arises from recessive mutations in SAMHD1. The resulting homozygous mutant zebrafish larvae possess a number of neurological phenotypes, exemplified by variable, but increased expression of several ISGs in the head region, a significant increase in brain cell death, microcephaly and locomotion deficits. A link between IFN-I signaling and cholesterol biosynthesis has been highlighted by others, but not previously implicated in the type I interferonopathies. Through assessment of neurovascular integrity and qPCR analysis we identified a significant dysregulation of cholesterol biosynthesis in the zebrafish model. Furthermore, dysregulation of cholesterol biosynthesis gene expression was also observed through RNA sequencing analysis of AGS patient whole blood. From this novel finding, we hypothesize that cholesterol dysregulation may play a role in AGS disease pathophysiology. Further experimentation will lend critical insight into the molecular pathophysiology of AGS and the potential links involving aberrant type I IFN signaling and cholesterol dysregulation.

In vivo methods for imaging blood-brain barrier function and dysfunction.

The blood-brain barrier (BBB) is the interface between the central nervous system and systemic circulation. It tightly regulates what enters and is removed from the brain parenchyma and is fundamental in maintaining brain homeostasis. Increasingly, the BBB is recognised as having a significant role in numerous neurological disorders, ranging from acute disorders (traumatic brain injury, stroke, seizures) to chronic neurodegeneration (Alzheimer's disease, vascular dementia, small vessel disease). Numerous approaches have been developed to study the BBB in vitro, in vivo, and ex vivo. The complex multicellular structure and effects of disease are difficult to recreate accurately in vitro, and functional aspects of the BBB cannot be easily studied ex vivo. As such, the value of in vivo methods to study the intact BBB cannot be overstated. This review discusses the structure and function of the BBB and how these are affected in diseases. It then discusses in depth several established and novel methods for imaging the BBB in vivo, with a focus on MRI, nuclear imaging, and high-resolution intravital fluorescence microscopy.

Systemic innate myeloid responses to acute ischaemic and haemorrhagic stroke.

Acute ischaemic and haemorrhagic stroke account for significant disability and morbidity burdens worldwide. The myeloid arm of the peripheral innate immune system is critical in the immunological response to acute ischaemic and haemorrhagic stroke. Neutrophils, monocytes, and dendritic cells (DC) contribute to the evolution of pathogenic local and systemic inflammation, whilst maintaining a critical role in ongoing immunity protecting against secondary infections. This review aims to summarise the key alterations to myeloid immunity in acute ischaemic stroke, intracerebral haemorrhage (ICH), and subarachnoid haemorrhage (SAH). By integrating clinical and preclinical research, we discover how myeloid immunity is affected across multiple organ systems including the brain, blood, bone marrow, spleen, and lung, and evaluate how these perturbations associate with real-world outcomes including infection. These findings are placed in the context of the rapidly developing field of human immunology, which offers a wealth of opportunity for further research.

Targeting firing rate neuronal homeostasis can prevent seizures.

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.

Treatment with IgM-enriched intravenous immunoglobulins enhances clearance of stroke-associated bacterial lung infection.

Post-stroke infection is a common complication of stroke that is associated with poor outcome. We previously reported that stroke induces an ablation of multiple sub-populations of B cells and reduces levels of immunoglobulin M (IgM) antibody, which coincides with the development of spontaneous bacterial pneumonia. The loss of IgM after stroke could be an important determinant of infection susceptibility and highlights this pathway as a target for intervention. We treated mice with a replacement dose of IgM-enriched intravenous immunoglobulin (IgM-IVIg) prior to and 24 h after middle cerebral artery occlusion (MCAO) and allowed them to recover for 2- or 5-day post-surgery. Treatment with IgM-IVIg enhanced bacterial clearance from the lung after MCAO and improved lung pathology but did not impact brain infarct volume. IgM-IVIg treatment induced immunomodulatory effects systemically, including rescue of splenic plasma B cell numbers and endogenous mouse IgM and IgA circulating immunoglobulin concentrations that were reduced by MCAO. Treatment attenuated MCAO-induced elevation of selected pro-inflammatory cytokines in the lung. IgM-IVIg treatment did not increase the number of lung mononuclear phagocytes or directly modulate macrophage phagocytic capacity but enhanced phagocytosis of Staphylococcus aureus bioparticles in vitro. Low-dose IgM-IVIg contributes to increased clearance of spontaneous lung bacteria after MCAO likely via increasing availability of antibody in the lung to enhance opsonophagocytic activity. Immunomodulatory effects of IgM-IVIg treatment may also contribute to reduced levels of damage in the lung after MCAO. IgM-IVIg shows promise as an antibacterial and immunomodulatory agent to use in the treatment of post-stroke infection.

Selective brain entry of lipid nanoparticles in haemorrhagic stroke is linked to biphasic blood-brain barrier disruption.

Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site. Methods: Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI. Results: Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (Ktrans [min-1]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI. Discussion: Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules.

Functionally linked potassium channel activity in cerebral endothelial and smooth muscle cells is compromised in Alzheimer's disease.

The brain microcirculation is increasingly viewed as a potential target for disease-modifying drugs in the treatment of Alzheimer's disease patients, reflecting a growing appreciation of evidence that cerebral blood flow is compromised in such patients. However, the pathogenic mechanisms in brain resistance arteries underlying blood flow defects have not yet been elucidated. Here we probed the roles of principal vasodilatory pathways in cerebral arteries using the APP23 mouse model of Alzheimer's disease, in which amyloid precursor protein is increased approximately sevenfold, leading to neuritic plaques and cerebrovascular accumulation of amyloid-ß similar to those in patients with Alzheimer's disease. Pial arteries from APP23 mice (18 mo old) exhibited enhanced pressure-induced (myogenic) constriction because of a profound reduction in ryanodine receptor-mediated, local calcium-release events ("Ca2+ sparks") in arterial smooth muscle cells and a consequent decrease in the activity of large-conductance Ca2+-activated K+ (BK) channels. The ability of the endothelial cell inward rectifier K+ (Kir2.1) channel to cause dilation was also compromised. Acute application of amyloid-ß 1-40 peptide to cerebral arteries from wild-type mice partially recapitulated the BK dysfunction seen in APP23 mice but had no effect on Kir2.1 function. If mirrored in human Alzheimer's disease, these tandem defects in K+ channel-mediated vasodilation could account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and crippled functional hyperemia. These data direct future research toward approaches that reverse this dual vascular channel dysfunction, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.