Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents

Aim To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). Methods Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. Results Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. Conclusion MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema

The effect of natriuretic peptide on the bradykinin signaling pathway after ischemic injury in the mouse brain

Začepljenje moždanih arterija dovodi do moždanog udara te posljedično do moždanog edema koji pridonosi povećanju ishemijske ozljede. Kako je bradikinin izravni uzročnik moždanog edema te natriuretski peptidi smanjuju moždani edem nakon moždanog udara još uvijek nepoznatim mehanizmom, cilj ovog istraživanja bio je razjasniti mogući mehanizam djelovanja natriuretskih peptida na signalni sustav bradikinina te učinak njihovog međudjelovanja na događaje koji slijede nakon ishemijske ozljede mozga. HEK-293 stanice su korištene kao stanični model jer smo pokazali, koristeći RT-PCR, postojanje bradikininskih receptora tipa 1 i 2, gvanilat ciklaze A i B te natriuretskog receptora tipa C. Mjerenjem membranskih potencijala i unutarstanične koncentracije kalcija HEK-293 stanica pokazali smo da natriuretski peptidi, agonisti gvanilat ciklaze-A (atrijski natriuretski peptid, moždani natriuretski peptid, urodilatin), ali ne i gunailat ciklaze-B (C-tip natriuretski peptid) inhibiraju bradikininski signalni put nakon aktivacije njegovog receptora tipa 2. U in vivo pokusima na mišjem modelu moždanog udara pokazali smo da i.v. primjena urodilatina sprječava nastanak ishemijske lezije mozga te posljedičnog moždanog edema koje smo određivali mjerenjem stupnja neurološkog oštećenja, oslikavanjem mozgova mikrokompjuteriziranom tomografijom, serijskim rezovima mozgova histološki bojenim po Nisslu te određivanjem sadržaja vode u mozgu. Rezultati ovog istraživanja po prvi puta pokazuju postojanje prirodnog antagonista bradikininskog receptora tipa 2 te moguću primjenu natriuretskih peptida u liječenju moždanog udara.Occlusion of the cerebral arteries leads to stroke and its consequence brain edema which further increases the brain damage. The aim of this research was clarifying the possible mechanisms of action of natriuretic peptides on bradykinin signaling, and their interactive effects after ischemic brain injury, since bradykinin is involved in the formation of cerebral edema and natriuretic peptides decrease brain edema via still unknown manner. HEK-293 cells were used as a model since using RT-PCR we proved the existence of bradykinin receptors type 1 and 2, guanylyl cyclase A and B, and natriuretic receptor type C. Measuring membrane potentials and intercellular calcium concentrations in HEK-293 cells we showed that natriuretic peptides, guanylyl cyclase A agonists (atrial natriuretic peptide, brain natriuretic peptide, urodilatin), excluding the guanylil cyclase B (C-type natriuretic peptide) inhibited the bradykinin signaling pathway after activation of bradykinin receptor type 2. The in vivo experiments on the animal model showed that urodilatin inhibited the formation of the ischemic lesion and brain edema which was measured by neurological scoring, visualization with microcomputerized tomography, serial Nissl stained sections and brain water content. The results of this research show for the first time the existence of a natural antagonist of the bradykinin type 2 receptor, and the possible use of natriuretic peptides in treatment of stroke

The effect of natriuretic peptide on the bradykinin signaling pathway after ischemic injury in the mouse brain

Začepljenje moždanih arterija dovodi do moždanog udara te posljedično do moždanog edema koji pridonosi povećanju ishemijske ozljede. Kako je bradikinin izravni uzročnik moždanog edema te natriuretski peptidi smanjuju moždani edem nakon moždanog udara još uvijek nepoznatim mehanizmom, cilj ovog istraživanja bio je razjasniti mogući mehanizam djelovanja natriuretskih peptida na signalni sustav bradikinina te učinak njihovog međudjelovanja na događaje koji slijede nakon ishemijske ozljede mozga. HEK-293 stanice su korištene kao stanični model jer smo pokazali, koristeći RT-PCR, postojanje bradikininskih receptora tipa 1 i 2, gvanilat ciklaze A i B te natriuretskog receptora tipa C. Mjerenjem membranskih potencijala i unutarstanične koncentracije kalcija HEK-293 stanica pokazali smo da natriuretski peptidi, agonisti gvanilat ciklaze-A (atrijski natriuretski peptid, moždani natriuretski peptid, urodilatin), ali ne i gunailat ciklaze-B (C-tip natriuretski peptid) inhibiraju bradikininski signalni put nakon aktivacije njegovog receptora tipa 2. U in vivo pokusima na mišjem modelu moždanog udara pokazali smo da i.v. primjena urodilatina sprječava nastanak ishemijske lezije mozga te posljedičnog moždanog edema koje smo određivali mjerenjem stupnja neurološkog oštećenja, oslikavanjem mozgova mikrokompjuteriziranom tomografijom, serijskim rezovima mozgova histološki bojenim po Nisslu te određivanjem sadržaja vode u mozgu. Rezultati ovog istraživanja po prvi puta pokazuju postojanje prirodnog antagonista bradikininskog receptora tipa 2 te moguću primjenu natriuretskih peptida u liječenju moždanog udara.Occlusion of the cerebral arteries leads to stroke and its consequence brain edema which further increases the brain damage. The aim of this research was clarifying the possible mechanisms of action of natriuretic peptides on bradykinin signaling, and their interactive effects after ischemic brain injury, since bradykinin is involved in the formation of cerebral edema and natriuretic peptides decrease brain edema via still unknown manner. HEK-293 cells were used as a model since using RT-PCR we proved the existence of bradykinin receptors type 1 and 2, guanylyl cyclase A and B, and natriuretic receptor type C. Measuring membrane potentials and intercellular calcium concentrations in HEK-293 cells we showed that natriuretic peptides, guanylyl cyclase A agonists (atrial natriuretic peptide, brain natriuretic peptide, urodilatin), excluding the guanylil cyclase B (C-type natriuretic peptide) inhibited the bradykinin signaling pathway after activation of bradykinin receptor type 2. The in vivo experiments on the animal model showed that urodilatin inhibited the formation of the ischemic lesion and brain edema which was measured by neurological scoring, visualization with microcomputerized tomography, serial Nissl stained sections and brain water content. The results of this research show for the first time the existence of a natural antagonist of the bradykinin type 2 receptor, and the possible use of natriuretic peptides in treatment of stroke

The effect of natriuretic peptide on the bradykinin signaling pathway after ischemic injury in the mouse brain

Začepljenje moždanih arterija dovodi do moždanog udara te posljedično do moždanog edema koji pridonosi povećanju ishemijske ozljede. Kako je bradikinin izravni uzročnik moždanog edema te natriuretski peptidi smanjuju moždani edem nakon moždanog udara još uvijek nepoznatim mehanizmom, cilj ovog istraživanja bio je razjasniti mogući mehanizam djelovanja natriuretskih peptida na signalni sustav bradikinina te učinak njihovog međudjelovanja na događaje koji slijede nakon ishemijske ozljede mozga. HEK-293 stanice su korištene kao stanični model jer smo pokazali, koristeći RT-PCR, postojanje bradikininskih receptora tipa 1 i 2, gvanilat ciklaze A i B te natriuretskog receptora tipa C. Mjerenjem membranskih potencijala i unutarstanične koncentracije kalcija HEK-293 stanica pokazali smo da natriuretski peptidi, agonisti gvanilat ciklaze-A (atrijski natriuretski peptid, moždani natriuretski peptid, urodilatin), ali ne i gunailat ciklaze-B (C-tip natriuretski peptid) inhibiraju bradikininski signalni put nakon aktivacije njegovog receptora tipa 2. U in vivo pokusima na mišjem modelu moždanog udara pokazali smo da i.v. primjena urodilatina sprječava nastanak ishemijske lezije mozga te posljedičnog moždanog edema koje smo određivali mjerenjem stupnja neurološkog oštećenja, oslikavanjem mozgova mikrokompjuteriziranom tomografijom, serijskim rezovima mozgova histološki bojenim po Nisslu te određivanjem sadržaja vode u mozgu. Rezultati ovog istraživanja po prvi puta pokazuju postojanje prirodnog antagonista bradikininskog receptora tipa 2 te moguću primjenu natriuretskih peptida u liječenju moždanog udara.Occlusion of the cerebral arteries leads to stroke and its consequence brain edema which further increases the brain damage. The aim of this research was clarifying the possible mechanisms of action of natriuretic peptides on bradykinin signaling, and their interactive effects after ischemic brain injury, since bradykinin is involved in the formation of cerebral edema and natriuretic peptides decrease brain edema via still unknown manner. HEK-293 cells were used as a model since using RT-PCR we proved the existence of bradykinin receptors type 1 and 2, guanylyl cyclase A and B, and natriuretic receptor type C. Measuring membrane potentials and intercellular calcium concentrations in HEK-293 cells we showed that natriuretic peptides, guanylyl cyclase A agonists (atrial natriuretic peptide, brain natriuretic peptide, urodilatin), excluding the guanylil cyclase B (C-type natriuretic peptide) inhibited the bradykinin signaling pathway after activation of bradykinin receptor type 2. The in vivo experiments on the animal model showed that urodilatin inhibited the formation of the ischemic lesion and brain edema which was measured by neurological scoring, visualization with microcomputerized tomography, serial Nissl stained sections and brain water content. The results of this research show for the first time the existence of a natural antagonist of the bradykinin type 2 receptor, and the possible use of natriuretic peptides in treatment of stroke

Učinak natriuretskih peptida na signalni put bradikinina nakon ishemijskog oštećenja mišjeg mozga [The effect of natriuretic peptide on the bradykinin signaling pathway after ischemic injury in the mouse brain]

Occlusion of the cerebral arteries leads to stroke and its consequence brain edema which further increases the brain damage. The aim of this research was clarifying the possible mechanisms of action of natriuretic peptides on bradykinin signaling, and their interactive effects after ischemic brain injury, since bradykinin is involved in the formation of cerebral edema and natriuretic peptides decrease brain edema via still unknown manner. HEK-293 cells were used as a model since using RT-PCR we proved the existence of bradykinin receptors type 1 and 2, guanylyl cyclase A and B, and natriuretic receptor type C. Measuring membrane potentials and intercellular calcium concentrations in HEK-293 cells we showed that natriuretic peptides, guanylyl cyclase A agonists (atrial natriuretic peptide, brain natriuretic peptide, urodilatin), excluding the guanylil cyclase B (C-type natriuretic peptide) inhibited the bradykinin signaling pathway after activation of bradykinin receptor type 2. The in vivo experiments on the animal model showed that urodilatin inhibited the formation of the ischemic lesion and brain edema which was measured by neurological scoring, visualization with microcomputerized tomography, serial Nissl stained sections and brain water content. The results of this research show for the first time the existence of a natural antagonist of the bradykinin type 2 receptor, and the possible use of natriuretic peptides in treatment of stroke

Krüppel-like transcription factor 8 (Klf8) is expressed and active in the neurons of the mouse brain

Krüppel-like transcription factor 8 (KLF8) is a transcription factor suggested to be involved in various cellular events, including malignant cell transformation, still its expression in the adult rodent brain remained unknown. To analyze Klf8 in the mouse brain and to identify cell types expressing it, a specific transgenic Klf8(Gt1Gaj) mouse was used. The resulting Klf8 gene-driven β-galactosidase activity was visualized by X-gal histochemical staining of the brain sections. The obtained results were complemented by in situ RNA hybridization and immunohistochemistry. Klf8 was highly expressed throughout the adult mouse brain gray matter including the cerebral cortex, hippocampus, olfactory bulb, hypothalamus, pallidum, and striatum, but not in the cerebellum. Immunofluorescent double-labeling revealed that KLF8-immunoreactive cells were neurons, and the staining was located in their nucleus. This was the first study showing that Klf8 was highly expressed in various regions of the mouse brain and in particular in the neurons, where it was localized in the cell nuclei

Role of uroguanylin's signalling pathway in the development of ischaemic stroke

Stroke is one of the leading causes of mortality and disability worldwide. By affecting bradykinin function, activation of guanylate cyclase (GC)-A has been shown to have a neuroprotective effect after ischaemic stroke, whereas the same has not been confirmed for GC-B; therefore, we aimed to determine the possible role of GC-C and its agonist, uroguanylin (UGN), in the development of stroke. In this study, middle cerebral artery occlusion (MCAO) was performed on wild-type (WT), GC-C KO and UGN KO mice. MR images were acquired before and 24 h after MCAO. On brain slices 48 h after MCAO, the Ca2+ response to UGN stimulation was recorded. Our results showed that the absence of GC-C in GC-C KO mice resulted in the development of smaller ischaemic lesions compared with WT littermates, which is an opposite effect compared with the effects of GC-A agonists on brain lesions. WT and UGN KO animals showed a stronger Ca2+ response upon UGN stimulation in astrocytes of the peri-ischaemic cerebral cortex compared with the same cortical region of the unaffected contralateral hemisphere. This stronger activation was not observed in GC-C KO animals, which may be the reason for smaller lesion development in GC-C KO mice. The reason why GC-C might affect Ca2+ signalling in peri-ischaemic astrocytes is that GC-C is expressed in these cells after MCAO, whereas under normoxic conditions, it is expressed mainly in cortical neurons. Stronger activation of the Ca2+ -dependent signalling pathway could lead to the stronger activation of the Na+ /H+ exchanger, tissue acidification and neuronal death

Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles

Background: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles.Results: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes).Conclusion: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies