Microglia survey the brain microenvironment for signals of injury or infection and are essential for the initiation and resolution of pathogen‐ or tissue damage‐induced inflammation. Understanding the mechanism of microglia responses during pathology is hence vital to promote regenerative responses. Here, we analyzed the role of purinergic receptor P2X4 (P2X4R) in microglia/macrophages during autoimmune inflammation. Blockade of P2X4R signaling exacerbated clinical signs in the experimental autoimmune encephalomyelitis (EAE) model and also favored microglia activation to a pro‐inflammatory phenotype and inhibited myelin phagocytosis. Moreover, P2X4R blockade in microglia halted oligodendrocyte differentiation in vitro and remyelination after lysolecithin‐induced demyelination. Conversely, potentiation of P2X4R signaling by the allosteric modulator ivermectin (IVM) favored a switch in microglia to an anti‐inflammatory phenotype, potentiated myelin phagocytosis, promoted the remyelination response, and ameliorated clinical signs of EAE. Our results provide evidence that P2X4Rs modulate microglia/macrophage inflammatory responses and identify IVM as a potential candidate among currently used drugs to promote the repair of myelin damage.
Background: Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy that requires accumulation of boron atoms preferentially in tumour cells. This can be achieved by using nanoparticles as boron carriers and taking advantage of the enhanced permeability and retention (EPR) effect. Here, we present the preparation and characterization of size and shape-tuned gold NPs (AuNPs) stabilised with polyethylene glycol (PEG) and functionalized with the boron-rich anion cobalt bis(dicarbollide), commonly known as COSAN. The resulting NPs were radiolabelled with 124I both at the core and the shell, and were evaluated in vivo in a mouse model of human fibrosarcoma (HT1080 cells) using positron emission tomography (PET). Methods: The thiolated COSAN derivatives for subsequent attachment to the gold surface were synthesized by reaction of COSAN with tetrahydropyran (THP) followed by ring opening using potassium thioacetate (KSAc). Iodination on one of the boron atoms of the cluster was also carried out to enable subsequent radiolabelling of the boron cage. AuNPs grafted with mPEG-SH (5 Kda) and thiolated COSAN were prepared by ligand displacement. Radiolabelling was carried out both at the shell (isotopic exchange) and at the core (anionic absorption) of the NPs using 124I to enable PET imaging. Results: Stable gold nanoparticles simultaneously functionalised with PEG and COSAN (PEG-AuNPs@[4]−) with hydrodynamic diameter of 37.8 ± 0.5 nm, core diameter of 19.2 ± 1.4 nm and ξ-potential of −18.0 ± 0.7 mV were obtained. The presence of the COSAN on the surface of the NPs was confirmed by Raman Spectroscopy and UV-Vis spectrophotometry. PEG-AuNPs@[4]− could be efficiently labelled with 124I both at the core and the shell. Biodistribution studies in a xenograft mouse model of human fibrosarcoma showed major accumulation in liver, lungs and spleen, and poor accumulation in the tumour. The dual labelling approach confirmed the in vivo stability of the PEG-AuNPs@[4]−. Conclusions: PEG stabilized, COSAN-functionalised AuNPs could be synthesized, radiolabelled and evaluated in vivo using PET. The low tumour accumulation in the animal model assayed points to the need of tuning the size and geometry of the gold core for future studies.
The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3′-Co(1,2-C2B9H11)2]−), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.
In vivo positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChRs) is a promising tool for the imaging evaluation of neurologic and neurodegenerative diseases. However, the role of α7 nAChRs after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. In vivo and ex vivo evaluation of α7 nAChRs expression after transient middle cerebral artery occlusion (MCAO) was carried out using PET imaging with [ C]NS14492 and immunohistochemistry (IHC). Pharmacological activation of α7 receptors was evaluated with magnetic resonance imaging (MRI), [ F]DPA-714 PET, IHC, real time polymerase chain reaction (qPCR) and neurofunctional studies. In the ischemic territory, [ C]NS14492 signal and IHC showed an expression increase of α7 receptors in microglia and astrocytes after cerebral ischemia. The role played by α7 receptors on neuroinflammation was supported by the decrease of [ F]DPA-714 binding in ischemic rats treated with the α7 agonist PHA 568487 at day 7 after MCAO. Moreover, compared with non-treated MCAO rats, PHA-treated ischemic rats showed a significant reduction of the cerebral infarct volumes and an improvement of the neurologic outcome. PHA treatment significantly reduced the expression of leukocyte infiltration molecules in MCAO rats and in endothelial cells after in vitro ischemia. Despite that, the activation of α7 nAChR had no influence to the blood brain barrier (BBB) permeability measured by MRI. Taken together, these results suggest that the nicotinic α7 nAChRs play a key role in the inflammatory reaction and the leukocyte recruitment following cerebral ischemia in rats.
BackgroundIn vivo positron-emission tomography (PET) imaging of transporter protein (TSPO) expression is an attractive and indispensable tool for the diagnosis and therapy evaluation of neuroinflammation after cerebral ischemia. Despite several radiotracers have shown an excellent capacity to image neuroinflammation, novel radiotracers such as [18F] VUIIS1008 have shown promising properties to visualize and quantify the in vivo expression of TSPO.MethodsLongitudinal in vivo magnetic resonance (MRI) and PET imaging studies with the novel TSPO radiotracer 2-(5,7-diethyl-2-(4-(2-[18F] fluoroethoxy) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl)-N, N-diethylacetamide ([18F] VUIIS1008), and (N, N-diethyl-2-(2-[4-(2-fluoroethoxy)-phenyl]-5,7-dimethyl-pyrazolo [1,5-a] yrimidin-3-yl)-acetamide ([18F] DPA-714) were carried out before and at days 1, 3, 7, 14, 21, and 28 following the transient middle cerebral artery occlusion (MCAO) in rats.ResultsMRI images showed the extension and evolution of the brain infarction after ischemic stroke in rats. PET imaging with [18F] VUIIS1008 and [18F] DPA714 showed a progressive increase in the ischemic brain hemisphere during the first week, peaking at day 7 and followed by a decline from days 14 to 28 after cerebral ischemia. [18F] DPA714 uptake showed a mild uptake increase compared to [18F] VUIIS1008 in TSPO-rich ischemic brain regions. In vivo [18F] VUIIS1008 binding displacement with VUIIS1008 was more efficient than DPA714. Finally, immunohistochemistry confirmed a high expression of TSPO in microglial cells at day 7 after the MCAO in rats.ConclusionsAltogether, these results suggest that [18F] VUIIS1008 could become a valuable tool for the diagnosis and treatment evaluation of neuroinflammation following ischemic stroke.
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