Relationships between selective neuronal loss and microglial activation after ischaemic stroke in man.

Modern ischaemic stroke management involves intravenous thrombolysis followed by mechanical thrombectomy, which allows markedly higher rates of recanalization and penumbral salvage than thrombolysis alone. However, <50% of treated patients eventually enjoy independent life. It is therefore important to identify complementary therapeutic targets. In rodent models, the salvaged penumbra is consistently affected by selective neuronal loss, which may hinder recovery by interfering with plastic processes, as well as by microglial activation, which may exacerbate neuronal death. However, whether the salvaged penumbra in man is similarly affected is still unclear. Here we determined whether these two processes affect the non-infarcted penumbra in man and, if so, whether they are inter-related. We prospectively recruited patients with (i) acute middle-cerebral artery stroke; (ii) penumbra present on CT perfusion obtained <4.5 h of stroke onset; and (iii) early neurological recovery as a marker of penumbral salvage. PET with 11C-flumazenil and 11C-PK11195, as well as MRI to map the final infarct, were obtained at predefined follow-up times. The presence of selective neuronal loss and microglial activation was determined voxel-wise within the MRI normal-appearing ipsilateral non-infarcted zone and surviving penumbra masks, and their inter-relationship was assessed both across and within patients. Dilated infarct contours were consistently excluded to control for partial volume effects. Across the 16 recruited patients, there was reduced 11C-flumazenil and increased 11C-PK11195 binding in the whole ipsilateral non-infarcted zone (P = 0.04 and 0.02, respectively). Within the non-infarcted penumbra, 11C-flumazenil was also reduced (P = 0.001), but without clear increase in 11C-PK11195 (P = 0.18). There was no significant correlation between 11C-flumazenil and 11C-PK11195 in either compartment. This mechanistic study provides direct evidence for the presence of both neuronal loss and microglial activation in the ipsilateral non-infarcted zone. Further, we demonstrate the presence of neuronal loss affecting the surviving penumbra, with no or only mild microglial activation, and no significant relationship between these two processes. Thus, microglial activation may not contribute to penumbral neuronal loss in man, and its presence in the ipsilateral hemisphere may merely reflect secondary remote degeneration. Selective neuronal loss in the surviving penumbra may represent a novel therapeutic target as an adjunct to penumbral salvage to further improve functional outcome. However, microglial activation may not stand as the primary therapeutic approach. Protecting the penumbra by acutely improving perfusion and oxygenation in conjunction with thrombectomy for example, may be a better approach. 11C-flumazenil PET would be useful to monitor the effects of such therapies

Assessing the Effects of Cytoprotectants on Selective Neuronal Loss, Sensorimotor Deficit and Microglial Activation after Temporary Middle Cerebral Occlusion.

Although early reperfusion after stroke salvages the still-viable ischemic tissue, peri-infarct selective neuronal loss (SNL) can cause sensorimotor deficits (SMD). We designed a longitudinal protocol to assess the effects of cytoprotectants on SMD, microglial activation (MA) and SNL, and specifically tested whether the KCa3.1-blocker TRAM-34 would prevent SNL. Spontaneously hypertensive rats underwent 15 min middle-cerebral artery occlusion and were randomized into control or treatment group, which received TRAM-34 intraperitoneally for 4 weeks starting 12 h after reperfusion. SMD was assessed longitudinally using the sticky-label test. MA was quantified at day 14 using in vivo [11C]-PK111195 positron emission tomography (PET), and again across the same regions-of-interest template by immunofluorescence together with SNL at day 28. SMD recovered significantly faster in the treated group (p = 0.004). On PET, MA was present in 5/6 rats in each group, with no significant between-group difference. On immunofluorescence, both SNL and MA were present in 5/6 control rats and 4/6 TRAM-34 rats, with a non-significantly lower degree of MA but a significantly (p = 0.009) lower degree of SNL in the treated group. These findings document the utility of our longitudinal protocol and suggest that TRAM-34 reduces SNL and hastens behavioural recovery without marked MA blocking at the assessed time-points

PET imaging of the neurovascular interface in cerebrovascular disease

Cerebrovascular disease encompasses a range of pathologies affecting different components of the cerebral vasculature and brain parenchyma. Large artery atherosclerosis, acute cerebral ischaemia, and intracerebral small vessel disease all demonstrate metabolic processes that are key to pathogenesis. Although structural imaging has been a mainstay of stroke clinical care and research, it has limited ability to detect these pathophysiological processes in vivo. Positron emission tomography (PET) provides a means to detect and quantify metabolic processes in each facet of cerebrovascular disease non-invasively. The use of PET has helped shape the understanding of key concepts in cerebrovascular medicine, including the vulnerable atherosclerotic plaque, salvageable ischaemic penumbra, neuroinflammation and selective neuronal loss after ischaemic insult, and the relationships between chronic hypoxia, neuroinflammation, and amyloid deposition in cerebral small vessel disease. This review considers how the ability to image these processes at the neurovascular interface has contributed to our understanding of cerebrovascular disease and facilitated translational research to advance clinical care.N.R.E. is supported by a research training fellowship from The Dunhill Medical Trust (grant number RTF44/0114). J.M.T. is supported by a Wellcome Trust research training fellowship (104492/Z/14/Z). J.H.F.R. is part-supported by the Higher Education Funding Council for England (HEFCE), the British Heart Foundation, and the Wellcome Trust. H.S.M. is supported by the Medical Research Council (MRC) as a National Institute for Health Research (NIHR) Senior Investigator. E.A.W. is supported by the British Heart Foundation. H.S.M., J.H.F.R., and E.A.W. are supported by the NIHR Cambridge Biomedical Research Centre

Effects of hyperoxia on 18F-fluoro-misonidazole brain uptake and tissue oxygen tension following middle cerebral artery occlusion in rodents: Pilot studies.

PURPOSE: Mapping brain hypoxia is a major goal for stroke diagnosis, pathophysiology and treatment monitoring. 18F-fluoro-misonidazole (FMISO) positron emission tomography (PET) is the gold standard hypoxia imaging method. Normobaric hyperoxia (NBO) is a promising therapy in acute stroke. In this pilot study, we tested the straightforward hypothesis that NBO would markedly reduce FMISO uptake in ischemic brain in Wistar and spontaneously hypertensive rats (SHRs), two rat strains with distinct vulnerability to brain ischemia, mimicking clinical heterogeneity. METHODS: Thirteen adult male rats were randomized to distal middle cerebral artery occlusion under either 30% O2 or 100% O2. FMISO was administered intravenously and PET data acquired dynamically for 3hrs, after which magnetic resonance imaging (MRI) and tetrazolium chloride (TTC) staining were carried out to map the ischemic lesion. Both FMISO tissue uptake at 2-3hrs and FMISO kinetic rate constants, determined based on previously published kinetic modelling, were obtained for the hypoxic area. In a separate group (n = 9), tissue oxygen partial pressure (PtO2) was measured in the ischemic tissue during both control and NBO conditions. RESULTS: As expected, the FMISO PET, MRI and TTC lesion volumes were much larger in SHRs than Wistar rats in both the control and NBO conditions. NBO did not appear to substantially reduce FMISO lesion size, nor affect the FMISO kinetic rate constants in either strain. Likewise, MRI and TTC lesion volumes were unaffected. The parallel study showed the expected increases in ischemic cortex PtO2 under NBO, although these were small in some SHRs with very low baseline PtO2. CONCLUSIONS: Despite small samples, the apparent lack of marked effects of NBO on FMISO uptake suggests that in permanent ischemia the cellular mechanisms underlying FMISO trapping in hypoxic cells may be disjointed from PtO2. Better understanding of FMISO trapping processes will be important for future applications of FMISO imaging

Inflammatory mechanisms in focal cerebral ischaemia

Stroke is a complex pathophysiological process and the role of inflammation in its initiation and resolution has been much debated. Inflammation is now emerging as a contributor in the development of ischaemic brain damage. The use of anti -inflammatory strategies to reduce damage and improve functional outcome of stroke patients may be valuable in the treatment for a condition that currently has no effective treatment. The exact contribution of the inflammatory response and the involvement of the various components of the immune system are still under investigationIn this thesis, focal cerebral ischaemia was induced in three animal models in an attempt to investigate the contribution of the inflammatory response. The rat monofilament model of middle cerebral artery (MCA) occlusion, considered by some to be a pro- inflammatory model, was set up for the first time in Edinburgh and validated as suitable model for further investigation. Permanent and transient models were established to allow the evaluation of possible reperfusion injury. Both monofilament models were compared with the Endothelin -1 model already established and routinely in use in the laboratory. Analysis of the volume of damage and distribution of the lesion revealed no differences between the three models. However, this observation did not in itself rule out the possibility of different pathophysiological mechanisms in the three models that ultimately resulted in apparently similar sized lesions.FK506, a potent neuroprotectant widely used experimentally, exhibited different neuroprotective efficacies. In all models, FK506 significantly reduced the overall volume of both damage and oedema. The majority of the neuroprotection was observed in the cortex although striatal protection was seen in the transient rat monofilament model. The neuroprotection observed in the transient monofilament model was approximately twice that seen in the permanent model and similar to that in the Endothelin -1 model suggesting distinct pathways that lead to cell death. Data for FK506 administration in the mouse monofilament model demonstrated neuroprotection for the first time in this species was included as an interesting comparison with the rat data.In keeping with the investigation of inflammation in cerebral ischaemia, it was proposed that FK506 neuroprotection was in part mediated through modulation of the inflammatory response. The response of the microglia, the resident immune cells of the central nervous system was examined following FK506 administration. Although the drug appeared to have a noticeable effect the activation state of the microglia, the response was not consistent and difficult to quantify by histological methods. Microglial cultures were established to investigate the effect of FK506 in a less complex system. Ramified microglial cultures were established but the analysis of microglia in vitro by morphology also proved difficult and another method of assessing activation of the cells was pursued. The microglia are known to secrete noxious stimuli when activated amongst which are the pro- inflammatory cytokines. IL-1P, IL -6 and TNFa gene expression was investigated to assess microglial activation. Lipopolysaccharide treated animals and treated microglial preparations were used initially to refine the use of multiplex polymerase chain reaction (MPCR) analysis of gene products. This was extended to tissue from both monofilament models. IL -1(3, IL -6 and TNFa were detected in the cortex and striatum when measured at 3 and 24 hrs post occlusion and showed an earlier cytokine response where reperfusion occurred. It is suggested that the early cytokine response is associated with the endogenous inflammatory cells. Western analysis experiments were performed to verify the presence of the corresponding cytokine proteins with little success. The recent availability of improved cytokine antibodies enabled the examination of cytokines (IL -1f3 and TNFa) in ischaemic by enzyme linked immunosorbant assay (ELISA). No TNFa response was detected despite the presence of mRNA. IL -lß was detected at 3 and 24 hrs post -insult with greater expression at 24 hrs. It may be speculated that this increased expression at the later time is related to the peripheral inflammatory cell infiltration. There was no difference in expression levels between models and FK506 had no affect on the cytokine expression.In summary, the re- introduction of blood to ischaemic tissue appears to alter the response of the individual cells although this does not change their ultimate fate. In instances where reperfusion is established, the tissue appears to be more amenable to neuroprotection by FK506. It is suggested that this is associated with the blockade of the endogenous inflammatory mechanisms that respond acutely to an ischaemic insult

Thinning, movement, and volume loss of residual cortical tissue occurs after stroke in the adult rat as identified by histological and magnetic resonance imaging analysis

ix, 162 leaves : ill. ; 28 cmPlasticity of residual cortical tissue has been identified as an important mediator of functional post-stroke recovery. After neonatal stroke the thickness of residual tissue can change, the tissue can move, and tissue can fill in the stroke core. Nevertheless, the majority of preclinical stroke research utilizes adult rats. Thus, the purpose of the present thesis was to systematically document such gross morphological changes in peri-infarct tissue after stroke in the adult rat. Morphological changes were assessed in pial strip devascularization, photothrombotic occlusion, and middle cerebral artery occlusion models of stroke using histological and magnetic resonance imaging. Decreases in cortical thickness, volume, and neural density were found to extend far beyond the stroke infarct and included the sensorimotor regions of the intact hemisphere. Movement of residual tissue towards the infarct was observed and confirmed using anatomical markers placed in intact cortical tissue at the time of stroke induction. I conclude that the extensive time-dependent morphological changes that occur in residual cortical tissue must be considered when evaluating plasticity-related cortical changes associated with post-stroke recovery of function

Longitudinal Assessments of Normal and Perilesional Tissues in Focal Brain Ischemia and Partial Optic Nerve Injury with Manganese-enhanced MRI

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SOD1 nanozyme salvages ischemic brain by locally protecting cerebral vasculature

Copper/zinc superoxide dismutase (CuZnSOD; SOD1) is widely considered as a potential therapeutic candidate for pathologies involving oxidative stress, but its application has been greatly hindered by delivery issues. In our previous study, nano-formulated SOD1 (cl-nanozyme) was shown to decrease infarct volume and improve sensorimotor functions after single intravenous (IV) injection in a rat middle cerebral artery occlusion (MCAO) model of ischemia/reperfusion (I/R) injury. However, it remained unclear how cl-nanozyme was able to deliver SOD1 to the brain and exert therapeutic efficacy. Present study aims to answer this question by exploring micro-distribution pattern of cl-nanozyme in the rat brain after stroke. Immunohistochemistry studies demonstrated cl-nanozyme co-localization with fibrin along damaged arteries and capillaries in the ischemic hemisphere. We further found that cl-nanozyme can be cross-linked into thrombi formed after I/R injury in the brain, and this effect is independent of animal species (rat/mouse) used for modeling I/R injury. This work is also the first report reinforcing therapeutic potential of cl-nanozyme in a well-characterized mouse MCAO model of I/R injury