Tissue acidosis associated with ischemic stroke to guide nimodipine delivery

Background: Ischemic stroke is a leading cause of death and disability worldwide. Yet, the effective therapy of focal cerebral ischemia has been an unresolved challenge. We propose here that ischemic tissue acidosis, a sensitive metabolic indicator of injury progression in cerebral ischemia, can be harnessed for the targeted delivery of neuroprotective agents. Ischemic tissue acidosis, which represents the accumulation of lactic acid in malperfused brain tissue is significantly exacerbated by the recurrence of SD events. Deepening acidosis itself activates specific ion channels to cause neurotoxic cellular Ca2+ accumulation and cytotoxic edema. These processes are thought to contribute to the loss of the ischemic penumbra. Importantly, acidosis in the ischemic penumbra may also be used to guide therapeutic intervention. Nimodipine, an L-type VGCC antagonist dilates cerebral arterioles, but its systemic administration may cause potential side effects (mainly hypotension). We have constructed chitosan nanoparticles as drug carriers, which release nimodipine in response to decreasing pH typical of cerebral ischemia. Here we have set out to evaluate this nanomedical approach to deliver nimodipine selectively to acidic ischemic brain tissue. Methods: Two sets of experiments are presented in this thesis. In Experimental Project I, nimodipine was applied in solution (100 μM), then global forebrain ischemia was induced in half of the animals by bilateral common carotid artery occlusion under isoflurane anesthesia. Functional hyperemia in the somatosensory cortex was created by mechanical stimulation of the contralateral whisker pad under α‐chloralose anesthesia. SD events were elicited subsequently by 1 M KCl. LFP and CBF in the parietal somatosensory cortex were monitored by electrophysiology and LDF. In Experimental Project II, nimodipine was associated with pH-sensitive nanoparticles in suspension. After washing the nanoparticle suspension with or without nimodipine (100 μM) on the exposed brain surface of anesthetized rats, both common carotid arteries were occluded to create forebrain ischemia. SDs were elicited by 1 M KCl to deepen the ischemic insult. LFP, CBF and tissue pH were recorded from the cerebral cortex. Microglia activation and neuronal survival were evaluated in brain sections by immunocytochemistry. Results: Nimodipine in solution attenuated evoked potentials and SD. In addition to the elevation of baseline CBF, nimodipine augmented hyperemia in response to both somatosensory stimulation and SD, the drug effect was particularly discernable under ischemia. Ischemia-induced tissue acidosis initiated nimodipine release from nanoparticles, confirmed by the significant elevation of baseline CBF. Nimodipine shortened the duration of both SD itself, and the associated tissue acidosis, moreover it enhanced the SD-related hyperemia. Chitosan nanoparticles did not activate microglia. Conclusions: The administered nanoparticles release nimodipine in acidic tissue environment, which reliably delineates sites at risk of injury. The data support the concept that tissue acidosis linked to cerebral ischemia can be employed as a trigger for targeted drug delivery. Nimodipine-mediated vasodilation and SD inhibition can be achieved by pH-responsive chitosan nanoparticles applied directly to the brain surface. Ultimately, this approach may offer a new way to treat stroke patients with the hope of more effective therapy, and better stroke outcome

Selective and rapid monitoring of dual platelet inhibition by aspirin and P2Y12 antagonists by using multiple electrode aggregometry

<p>Abstract</p> <p>Background</p> <p>Poor platelet inhibition by aspirin or clopidogrel has been associated with adverse outcomes in patients with cardiovascular diseases. A reliable and facile assay to measure platelet inhibition after treatment with aspirin and a P2Y₁₂antagonist is lacking. Multiple electrode aggregometry (MEA), which is being increasingly used in clinical studies, is sensitive to platelet inhibition by aspirin and clopidogrel, but a critical evaluation of MEA monitoring of dual anti-platelet therapy with aspirin and P2Y₁₂antagonists is missing.</p> <p>Design and Methods</p> <p>By performing <it>in vitro </it>and <it>ex vivo </it>experiments, we evaluated in healthy subjects the feasibility of using MEA to monitor platelet inhibition of P2Y₁₂antagonists (clopidogrel <it>in vivo</it>, cangrelor <it>in vitro</it>) and aspirin (100 mg per day <it>in vivo</it>, and 1 mM or 5.4 mM <it>in vitro</it>) alone, and in combination. Statistical analyses were performed by the Mann-Whitney rank sum test, student' t-test, analysis of variance followed by the Holm-Sidak test, where appropriate.</p> <p>Results</p> <p>ADP-induced platelet aggregation in hirudin-anticoagulated blood was inhibited by 99.3 ± 1.4% by <it>in vitro </it>addition of cangrelor (100 nM; p < 0.001) and by 64 ± 35% by oral clopidogrel (600 mg) intake (p < 0.05; values are means ± SD). Pre-incubation of blood with aspirin (1 mM) or oral aspirin intake (100 mg/day for 1 week) inhibited arachidonic acid (AA)-stimulated aggregation >95% and 100 ± 3.2%, respectively (p < 0.01). Aspirin did not influence ADP-induced platelet aggregation, either <it>in vitro </it>or <it>ex vivo</it>. Oral intake of clopidogrel did not significantly reduce AA-induced aggregation, but P2Y₁₂blockade by cangrelor (100 nM) <it>in vitro </it>diminished AA-stimulated aggregation by 53 ± 26% (p < 0.01). A feasibility study in healthy volunteers showed that dual anti-platelet drug intake (aspirin and clopidogrel) could be selectively monitored by MEA.</p> <p>Conclusions</p> <p>Selective platelet inhibition by aspirin and P2Y₁₂antagonists alone and in combination can be rapidly measured by MEA. We suggest that dual anti-platelet therapy with these two types of anti-platelet drugs can be optimized individually by measuring platelet responsiveness to ADP and AA with MEA before and after drug intake.</p

Spreading depolarization remarkably exacerbates ischemia-induced tissue acidosis in the young and aged rat brain

Spreading depolarizations (SDs) occur spontaneously in the cerebral cortex of subarachnoid hemorrhage, stroke or traumatic brain injury patients. Accumulating evidence prove that SDs exacerbate focal ischemic injury by converting zones of the viable but non-functional ischemic penumbra to the core region beyond rescue. Yet the SD-related mechanisms to mediate neurodegeneration remain poorly understood. Here we show in the cerebral cortex of isoflurane-anesthetized, young and old laboratory rats, that SDs propagating under ischemic penumbra-like conditions decrease intra and- extracellular tissue pH transiently to levels, which have been recognized to cause tissue damage. Further, tissue pH after the passage of each spontaneous SD event remains acidic for over 10 minutes. Finally, the recovery from SD-related tissue acidosis is hampered further by age. We propose that accumulating acid load is an effective mechanism for SD to cause delayed cell death in the ischemic nervous tissue, particularly in the aged brain

Bicarbonate Evokes Reciprocal Changes in Intracellular Cyclic di-GMP and Cyclic AMP Levels in Pseudomonas aeruginosa

The formation of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) is one of the most common causes of morbidity and mortality in CF patients. Cyclic di-GMP and cyclic AMP are second messengers regulating the bacterial lifestyle transition in response to environmental signals. We aimed to investigate the effects of extracellular pH and bicarbonate on intracellular c-di-GMP and cAMP levels, and on biofilm formation. P. aeruginosa was inoculated in a brain–heart infusion medium supplemented with 25 and 50 mM NaCl in ambient air (pH adjusted to 7.4 and 7.7 respectively), or with 25 and 50 mM NaHCO3 in 5% CO2 (pH 7.4 and 7.7). After 16 h incubation, c-di-GMP and cAMP were extracted and their concentrations determined. Biofilm formation was investigated using an xCelligence real-time cell analyzer and by crystal violet assay. Our results show that HCO3− exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO3−. The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO3− per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria

Placental galectins regulate innate and adaptive immune responses in pregnancy

IntroductionGalectins are master regulators of maternal immune responses and placentation in pregnancy. Galectin-13 (gal-13) and galectin-14 (gal-14) are expressed solely by the placenta and contribute to maternal-fetal immune tolerance by inducing the apoptosis of activated T lymphocytes and the polarization of neutrophils toward an immune-regulatory phenotype.Furthermore, their decreased placental expression is associated with pregnancy complications, such as preeclampsia and miscarriage. Yet, our knowledge of the immunoregulatory role of placental galectins is incomplete.MethodsThis study aimed to investigate the effects of recombinant gal-13 and gal-14 on cell viability, apoptosis, and cytokine production of peripheral blood mononuclear cells (PBMCs) and the signaling pathways involved.ResultsHerein, we show that gal-13 and gal-14 bind to the surface of non-activated PBMCs (monocytes, natural killer cells, B cells, and T cells) and increase their viability while decreasing the rate of their apoptosis without promoting cell proliferation. We also demonstrate that gal-13 and gal-14 induce the production of interleukin (IL)-8, IL-10, and interferon-gamma cytokines in a concentration-dependent manner in PBMCs. The parallel activation of Erk1/2, p38, and NF-ĸB signaling evidenced by kinase phosphorylation in PBMCs suggests the involvement of these pathways in the regulation of the galectin-affected immune cell functions.DiscussionThese findings provide further evidence on how placenta-specific galectins assist in the establishment and maintenance of a proper immune environment during a healthy pregnancy