Digitisation of a modular plug and play 3D printed continuous flow system for chemical synthesis

We describe the development of a digital modular 3D printed continuous flow system to carry out both classical and photochemical synthesis that uses a novel PC based software interface for communication. Using this system, we describe how we were able to both control and monitor reaction conditions at the same time. The system integrates in-line sensors via a simple cassette based system that is analogous to a retro-games console enabling hot-swapping of modules by a user. A PC-interface platform was created to automate both its functional control, including the injection of solvents, and the visualization of sensor-reported data. The utility of the system was demonstrated by performing a series of reactions highlighting the importance that precise control of solvent flow rate and accurate reporting of reaction temperatures can have on standardization and reproducibility and that the system can be easily modified to allow for scale-up synthesis

The local GLP-1 system in the olfactory bulb is required for odor-evoked cephalic phase of insulin release in mice

Objective: The olfactory bulb (OB) codes for sensory information and contributes to the control of energy metabolism by regulating foraging and cephalic phase responses. Mitral cells are the main output neurons of the OB. The glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) system in the OB (GLP-1ᴼᴮ) has been shown to be a major regulator of mitral cell activity but its function in vivo is unclear. Therefore, we investigated the role of GLP-1ᴼᴮ in foraging behavior and odor-evoked Cephalic Phase Insulin Release (CPIR)./ Methods and results: By fluorescent labeling, we confirmed the presence of GLP-1 producing neurons and the expression of GLP-1R in the mouse OB. In response to food odor presentation, we collected blood, quantified plasma insulin by ELISA and showed the existence of an odor-evoked CPIR in lean mice but its absence in obese animals. Expression of shRNA against preproglucagon mRNA in the OB resulted in blunted CPIR in lean mice. Injecting Exendin (9-39), a GLP-1R antagonist, into the OB of lean mice also resulted in decreased CPIR. Since parasympathetic cholinergic input to the pancreas is known to be partly responsible for CPIR, we systemically administered the muscarinic M3 receptor antagonist 4-DAMP which resulted in a reduced odor-evoked CPIR. Finally, local injection of Exendin (9-39) in the OB extinguished olfactory foraging in lean mice whereas the injection of the GLP-1R agonist Exendin-4 rescued the loss of foraging behavior in obese mice./ Conclusions: Our results demonstrate that GLP-1ᴼᴮ controls olfactory foraging and is required for odor-evoked CPIR. We describe a new crucial brain function for GLP-1 and GLP-1R expressed within the brain

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

[Background and aims]: Although the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain. [Materials and methods]: In order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS® Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography. [Results]: Whereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood-brain barrier, when administered either as a free drug or encapsulated into liposomes. [Conclusion]: This study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects.The research leading to these results received funding from the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreements number 201024 and number 202213 (European Stroke Network). Neurovascular Research Laboratory takes part in the Spanish stroke research network INVICTUS (RD12/0014/0005). This study was partially funded by projects FIS 11/0176 on stroke biomarkers research and EC07/90195 on increasing safety and efficacy of simvastatin neuroprotection.Peer Reviewe

Dynamic release of neuronal extracellular vesicles containing miR-21a-5p is induced by hypoxia

Biomarkers; Hypoxia; NeuronBiomarcadores; Hipoxia; NeuronaBiomarcadors; Hipòxia; NeuronaHypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.We thank Benita Löflund and Pasi Laurinmäki (University of Helsinki) for technical assistance in cryoEM. The facilities and expertise of the HiLIFE CryoEM unit at the University of Helsinki, a member of Instruct-ERIC Centre Finland, FINStruct, and Biocenter Finland are gratefully acknowledged. This work was carried out with the support of UEF Cell and Tissue Imaging Unit, University of Eastern Finland, Finland. Moreover, we express our great appreciation to Seppo Ylä-Herttuala and Petri Mäkinen for the access to the NTA facilities (University of Eastern Finland, A.I. Virtanen Institute, Finland). Finally, we would like to extend our thanks to Dora Brites for the facilitation of the N9 cell line (University of Lisbon, Faculty of Farmacy, Portugal) and to Mark Ansel and Eric Wigton (University of California San Francisco, US) for technical help with HITS-clip sequencing. This work was supported by the University of Eastern Finland, Emil Aaltonen Foundation, Paavo Nurmi Foundation, Saastamoinen Foundation, Instrumentarium Science Foundation and Business Finland (Grant number 4399/31/2019). Work with clinical samples was supported by the European Regional Development Fund - Project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451). Work with EVQuant was supported by the IMMPROVE Alpe d'HuZes grant of the Dutch Cancer Society (EMCR2015-8022) and the Daniel den Hoed Foundation grant for Erasmus MC Cancer Treatment Screening Facility. L.R. is supported by a predoctoral fellowship grant (IFI17/00012) and J.M. is the principal investigator of the grant PI18/804 ‘MULTI-BIO-TARGETS: a new strategy for stroke management combining outcome biomarkers and neuroprotection’, both from the Instituto de Salud Carlos III

Benefici de la Simvastatina en el tractament trombolític combinat de l'ictus en models d' isquèmia cerebral en rata

L'ictus és un dels reptes sanitaris més importants al nostre país ja que l'únic tractament disponible és l'administració de trombolítics durant les 4,5 primeres hores i menys d'un 10% dels pacients poden beneficiar-se'n. Publicacions anteriors han demostrat que el tractament de l'ictus amb estatines pot reduir l'extensió del teixit infartat i millorar la funció neurològica, per això proposem fer un estudi experimental usant un model d'isquèmia en rata, que evidenciï si el tractament combinat de Simvastatina i rt-PA incrementa el benefici obtingut únicament amb fàrmacs trombolítics i avaluï la seva seguretat quan s'administra durant la fase aguda (transformacions hemorràgiques i incidència d'infeccions)

Efectes de l'administració de tpa combinat amb dipyridamole sobre cèl·lules endotelials cerebrals humanes sotmeses a deprivació d'oxigen i glucosa

El DP és un agent usat de forma comú en la prevenció secundària de l'ictus per les seves propietats antiplaquetars. Ha estat proposat com a possible teràpia coadjuvant del tractament trombolític en fase aguda de l'ictus isquèmic amb rtPA neuroprotector per les seves propietats neuroprotectores, antiinflamatòries i antioxidants. En el present treball s'analitza la toxicitat de l'administració d'rtPA en cèl·lules endotelials cerebrals humanes sotmeses a isquèmia, així com els seus efectes sobre l'activació de MMPs. Posteriorment es combina l'administració d'rtPA amb Dipyridamole i s'analitzen els efectes en la toxicitat cel·lular i l'expressió d'MMPs

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

Although the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain. In order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS ® Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography. Whereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood-brain barrier, when administered either as a free drug or encapsulated into liposomes. This study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects

Dynamic release of neuronal extracellular vesicles containing miR-21a-5p is induced by hypoxia

© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Hypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.This work was supported by the University of Eastern Finland, Emil Aaltonen Foundation, Paavo Nurmi Foundation, Saastamoinen Foundation, Instrumentarium Science Foundation and Business Finland (Grant number 4399/31/2019). Work with clinical samples was supported by the European Regional Development Fund - Project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451). Work with EVQuant was supported by the IMMPROVE Alpe d'HuZes grant of the Dutch Cancer Society (EMCR2015-8022) and the Daniel den Hoed Foundation grant for Erasmus MC Cancer Treatment Screening Facility. L.R. is supported by a predoctoral fellowship grant (IFI17/00012) and J.M. is the principal investigator of the grant PI18/804 ‘MULTI-BIO-TARGETS: a new strategy for stroke management combining outcome biomarkers and neuroprotection’, both from the Instituto de Salud Carlos III.Peer reviewe

Digitisation of a Modular Plug and Play 3D Printed Continuous Flow System for Chemical Synthesis

We describe the development of a digital modular 3D printed continuous flow system to carry out both classical and photochemical synthesis that uses a novel PC based software interface. Using this system, we describe how we were able to both control and monitor reaction conditions at the same time. The system integrates in-line sensors via a simple cassette based system that is analogous to a games console enabling hot-swopping of modules by a user. To control the system, an PC-interface platform was created to automate the control of the system functionality, as well as the reporting of sensor parameters. The utility of the system was demonstrated by performing a series of reactions highlighting the importance that precise control of solvent flow rate and accurate reporting of reaction temperatures can have on standardization and reproducibility and that the system can be easily modified to allow for scale-up synthesis

Rat middle cerebral artery occlusion is not a suitable model for the study of stroke-induced spontaneous infections.

BACKGROUND: Infections related to stroke-induced immunodepression are an important complication causing a high rate of death in patients. Several experimental studies in mouse stroke models have described this process but it has never been tested in other species such as rats. METHODS: Our study focused on the appearance of secondary systemic and pulmonary infections in ischemic rats, comparing with sham and naive animals. For that purpose, male Wistar rats were subjected to embolic middle cerebral artery occlusion (eMCAO) or to transient MCAO (tMCAO) inserting a nylon filament. Forty-eight hours after ischemia, blood and lung samples were evaluated. RESULTS: In eMCAO set, ischemic rats showed a significant decrease in blood-peripheral lymphocytes (naive = 58.8±18.1%, ischemic = 22.9±16.4%) together with an increase in polymorphonuclears (PMNs) (naive = 29.2±14.7%, ischemic = 71.7±19.5%), while no change in monocytes was observed. The increase in PMNs counts was positively correlated with worse neurological outcome 48 hours after eMCAO (r = 0.55, p = 0.043). However, sham animals showed similar changes in peripheral leukocytes as those seen in ischemic rats (lymphocytes: 40.1±19.7%; PMNs: 51.7±19.2%). Analysis of bacteriological lung growth showed clear differences between naive (0±0 CFU/mL; log10) and both sham (3.9±2.5 CFU/mL; log10) and ischemic (4.3±2.9 CFU/mL; log10) groups. Additionally, naive animals presented non-pathological lung histology, while both sham and ischemic showed congestion, edema or hemorrhage. Concordant results were found in the second set of animals submitted to a tMCAO. CONCLUSIONS: Inflammatory and infection changes in Wistar rats subjected to MCAO models may be attributed not only to the brain ischemic injury but to the surgical aggression and/or anaesthetic stress. Consequently, we suggest that stroke-induced immunodepression in ischemic experimental models should be interpreted with caution in further experimental and translational studies, at least in rat stroke models that entail cervicotomy and cranial trepanation