Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

<div><p>The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (<i>Ns^−/−</i>) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in <i>Ns^−/−</i> mice. Our results show excessive microglial activation in <i>Ns^−/−</i> mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.</p></div

Activation of microglia is increased in the absence of neuroserpin.

<p>(<b>A</b>) Absolute numbers of brain microglia in the ischemic hemisphere of wild type, and <i>Ns^−/−</i> mice 3 days after MCAO. Cell counts were determined by flow cytometry analysis of the CNS-infiltrating cells using TrueCount tubes. Brain microglia cells were identified as CD11b⁺ CD45^intermediate. Representative dot plots show CD11b⁺ CD45^high and CD11b⁺ CD45^intermediate-gated populations identifying macrophages and microglia respectively. The graphs show means±SD of 9–12 animals per group analyzed three days after MCAO in three or four independent experiments. <i>t</i> test was used to assess statistical significance. (<b>B</b>) Immunohistochemical analysis of absolute numbers of Iba-1 positive brain microglia/macrophages in the ischemic hemisphere of wild type, and <i>Ns^−/−</i> mice 3 days after MCAO. The graphs show means±SD of 3 animals per group. <i>t</i> test was used to assess statistical significance. (<b>C</b>) Immunohistochemical analysis of the activation state (resting, bushy and amoeboid) of Iba-1 positive microglia in the ipsilesional hippocampus and penumbra area and contralesional hippocampus area 3 days following 1 h MCAO. The graphs show means±SD of 3 animals per group and the statistical analysis was assessed using one-way ANOVA with Bonferroni post hoc test (scale bar = 20 µm).</p

Deficiency in neuroserpin does not lead to alterations of the cellular post stroke infiltrate.

<p>(<b>A</b>) Absolute numbers of neutrophils (N), macrophages (Mφ), dendritic cells (DC) and lymphocytes (L) in the CNS-infiltrating cells of <i>wt and Ns^−/−</i> mice. Representative plots are shown for CD45^high and CD45^intermediate cells. Brain macrophages were identified as CD45^high, CD11b⁺, CD11c⁻ and distinguished from microglia by the higher expression of CD45. Dendritic cells were identified as CD45^high, CD11b⁺, CD11c⁺, neutrophils as CD45^high, CD11b⁺, Ly6G⁺ and lymphocytes as CD45^high, CD11b⁻, CD11c⁻. The graphs show the means±SD of 9 animals per group analyzed three days after MCAO in three or four independent experiments. One-way ANOVA with Bonferroni post-hoc test was used to assess statistical significance. (<b>B</b>) Immunohistochemical staining of T cells (CD3), neutrophils (Ly6G) and astrocytes (GFAP) in wt and <i>Ns^−/−</i> mice three days after MCAO (scale bar = 50 µm).</p

Deficiency in neuroserpin is detrimental in stroke.

<p>(<b>A</b>) TTC staining for evaluation of infarct volume at day three (left panel) and (<b>B</b>) neurological scores at days one and three (right panel) of wt and <i>Ns^−/−</i> mice after MCAO. Data are represented as means±SD of 10 wt and eight <i>Ns^−/−</i> animals. <i>t</i> test was used to assess statistical significance for infarct sizes and Mann-Whitney U test for neurological scores. (<b>C</b>) Survival rate of wt (n = 13) and <i>Ns^−/−</i> mice (n = 13). Survival was analyzed by the <i>χ</i>² test (survival rate). (<b>D</b>) In all mice subjected to MCAO, regional cerebral blood flow (rCBF) was measured with Laser Doppler. The decrease in rCBF of approximately 90% was similar between <i>Ns^−/−</i> and wt mice. Ten minutes after reperfusion rCBF was reconstituted to at least 60% of baseline levels and was unaltered between <i>Ns^−/−</i> and wt animals. (<b>E</b>) Accumulation of fibrin(ogen) in the infarcted and in the contralesional hemispheres of wt (n = 3) and <i>Ns^−/−</i> mice (n = 3). Fibrin(ogen) formation was analyzed by immunoblotting following fixation with 4% PFA (upper panel) or w/o 4% PFA-fixation (lower panel) using a rabbit polyclonal fibrin/fibrinogen-specific antibody 24 h following ischemia. Asterisks indicate additional bands representing fibrin degradation. <i>t</i> test was used to assess statistical significance.</p

Activation of microglia is increased in the absence of neuroserpin.

<p>(<b>A</b>) Frequency of TNF-α positive brain microglia in wt and <i>Ns^−/−</i> mice analyzed by flow cytometry three days after stroke. Representative dot plots show CD11b⁺ CD45^intermediate-gated microglia. The graphs show means±SD of 9–12 animals per group analyzed three days after MCAO in three or four independent experiments. <i>t</i> test was used to assess statistical significance. (<b>B</b>) Immunohistochemical analysis of TNF-αexpression in ischemic hemispheres 3 days following 1 h MCAO showing an increased TNF-α immunoreactivity in ischemic tissue from <i>Ns^−/−</i> mice compared to wt mice (scale bar = 50 µm).</p

Magnetic Particle Imaging for Real-Time Perfusion Imaging in Acute Stroke

The fast and accurate assessment of cerebral perfusion is fundamental for the diagnosis and successful treatment of stroke patients. Magnetic particle imaging (MPI) is a new radiation-free tomographic imaging method with a superior temporal resolution, compared to other conventional imaging methods. In addition, MPI scanners can be built as prehospital mobile devices, which require less complex infrastructure than computed tomography (CT) and magnetic resonance imaging (MRI). With these advantages, MPI could accelerate the stroke diagnosis and treatment, thereby improving outcomes. Our objective was to investigate the capabilities of MPI to detect perfusion deficits in a murine model of ischemic stroke. Cerebral ischemia was induced by inserting of a microfilament in the internal carotid artery in C57BL/6 mice, thereby blocking the blood flow into the medial cerebral artery. After the injection of a contrast agent (superparamagnetic iron oxide nanoparticles) specifically tailored for MPI, cerebral perfusion and vascular anatomy were assessed by the MPI scanner within seconds. To validate and compare our MPI data, we performed perfusion imaging with a small animal MRI scanner. MPI detected the perfusion deficits in the ischemic brain, which were comparable to those with MRI but in real-time. For the first time, we showed that MPI could be used as a diagnostic tool for relevant diseases <i>in vivo</i>, such as an ischemic stroke. Due to its shorter image acquisition times and increased temporal resolution compared to that of MRI or CT, we expect that MPI offers the potential to improve stroke imaging and treatment

CTC grade of SAE vs relatedness to DBS therapy.

<p>CTC grade of SAE vs relatedness to DBS therapy.</p

DBS-related neurological and psychiatric adverse events.

<p>DBS-related neurological and psychiatric adverse events.</p

Sum of AEs defined by same severity, reversibility, and attribution to DBS therapy.

<p>Green, reversible; orange, non reversible; grey, unknown. The actual number of AEs is presented. The dotted area indicates AEs that were <i>severe</i> or worse and at least <i>possibly</i> related to DBS therapy and, thus, regarded the most critical. N.B. The number of affected patients may be less than the number indicated because individual patients may have suffered from more than one AE of respective groups (e.g. impairment of gait and speech rated as <i>mild</i>, <i>probably</i> related and <i>non-reversible</i>).</p

Reporting of adverse events in prospective multicentric DBS studies for movement disorders.

<p>Reporting of adverse events in prospective multicentric DBS studies for movement disorders.</p