Marina Dobrivojević

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.

Involvement of bradykinin in brain edema development after ischemic stroke

Stroke is the third leading cause of death in the Western world. Ischemic stroke is characterized by a rapid loss of brain function due to disturbance in the blood supply to a part of the brain. Due to fixed intracranial space, any increase in intracranial fluid volume, or progressive brain edema formation, contributes to further deterioration of the already impaired brain function. Bradykinin increases blood–brain barrier permeability and raises intracranial capillary blood pressure by arterial dilatation and venous constriction leading to brain edema formation. The aim of this paper is to summarize the recent research in the field of bradykinin function (structure, synthesis, signaling pathways, mechanism of action) followed by characterization of different types of brain edema development related to ischemic brain injury, together with the involvement of bradykinin in edema formation. Since there is currently no causal treatment addressing brain edema after ischemic stroke, specific bradykinin receptor antagonists are proposed as a possible new therapeutic approach.

Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells.

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 μM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

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

Summary 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.

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.

Urodilatin reverses the detrimental influence of bradykinin in acute ischemic stroke

Occlusion of cerebral arteries leads to ischemic stroke accompanied by subsequent brain edema. Bradykinin (BK) is involved in the formation of cerebral edema, and natriuretic peptides (NPs) potentially have beneficial effects on brain edema formation via a still unknown mechanism. The aim of this study was clarifying the mechanisms of action of NPs on BK signaling, and their interactive effects after ischemic brain injury. We used a mouse model for stroke, the middle cerebral artery (MCA) occlusion. Brain lesion and edema were measured by microcomputerized tomography volumetric measurements. To determine the effects of NPs on the BK signaling pathway in the MCAs we measured changes in vessel diameter and membrane potentials in endothelial cells. To determine the effects of NPs on BK signaling pathway in isolated astrocytes and neurons, membrane potentials and intercellular Ca2+ concentrations were measured. Urodilatin inhibited and when applied together with BK, reduced the formation of the ischemic lesion via activation of G-Protein-Signaling Protein Type 4 at the cellular (atrocities, neurons) and blood vessel (endothelial cells and isolated MCA) level as well as in in vivo experiments. The results of this study show the existence of a natural antagonist of BK in the brain, and the possible use of NPs in the treatment of stroke.

Natriuretic peptides in brain physiology

Natriuretic peptides (NPs) regulate salt and water homeostasis by inducing natriuresis and diuresis in the kidney. These actions in addition to those via the heart and vascular system play important roles in the regulation of blood pressure. In the central nervous system NPs play a significant role in neuronal development, synaptic transmission and neuroprotection. Currently, six different human NPs have been described: atrial natriuretic peptide (ANP), urodilatin (URO, renal natriuretic peptide), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as guanylin and uroguanylin. ANP, URO and BNP activate the natriuretic peptide receptor A (NPR-A or guanylate cyclase A (GC-A)) while CNP activates natriuretic peptide receptor B (NPR-B or guanylate cyclase B (GC-B)). Guanylin and uroguanylin are known to activate guanylate cyclase C (GC-C). The receptors GC-A, GC-B, and GC-C are widely expressed in the human body. Currently, GC-B and CNP seems to have the highest expression in central nervous system compared to other NPs and their receptors. All known NPs generate intracellular cyclic GMP (cGMP) by activating their specific guanylate cyclase receptors. Subsequently, cGMP is able to activate protein kinase I or II (PKG I or II) and/or directly regulate transmembrane proteins such as ion channels, transporters and pumps. NPs also bind to the natriuretic peptide receptor C (also called clearance receptor NPR-C) which is a major pathway for the degradation of NPs and has no guanylate cyclase activity. In this review we will focus on new insights regarding the physiological effects of NPs in the brain, especially specific areas of their signaling pathways in neurons and glial cells.

The effect of natriuretic peptides and bradykinin on development of brain oedema after ischemic stroke

Background Ischemic stroke is characterized by a rapid loss of brain function due to disturbance in blood supply to a part of the brain. Due to fixed intracranial space, any increase in intracranial fluid volume, or progressive brain oedema formation, contributes to further deterioration of the already impaired brain function. Bradykinin (BK), which levels increase during ischemic stroke, promotes blood– brain barrier permeability and raises intracranial capillary blood pressure, leading to brain oedema formation. Furthermore, BK induces glutamate release from neurons and astrocytes via activation of BK receptor type 2. suggesting involvement of BK in glutamate neurotoxicity. It has been recently shown that humans without functional natriuretic peptides (NPs) suffer from massive stokes [1,2]. NPs can reduce brain oedema and have a neuroprotective role in acute ischemic stroke as well as during recovery after stroke. Although mechanisms are still not clear, it appears that NPs enhance angiogenesis, neurogenesis and oligodenrogenesis [3,4]. One of the possible beneficiary effects of NPs during the stroke could be an inhibition of BK pathological function. Materials and methods Aim of our study is to determine beneficial effects of the NPs in stroke development in murine model (middle cerebral artery occlusion – MCAO). The symptoms of the stroke are determined by behavioural studies. The sizes of the lesion and brain oedema are established by μCT. Furthermore, we determined the effects of NPs on the BK signalling pathway in primary culture of neurons and astrocytes using whole cell patch clamp experiments to measure membrane potential and measurements of intracellular Ca 2+ concentration.

Interaction between bradykinin and urodilatin - a possible mechanism of clinical relevance

Background Bradykinin (BK) plays a significant role in pathophysiology of different diseases from angioedema to brain stroke and heart attack by inducing vasodilatation and increasing capillary permeability. We investigate the potential effects of natriuretic peptides on BK signaling by measuring membrane potential (Vm) of HEK293 cells using the whole cell patch clamp technique. HEK293cells are an excellent model since they display the natriuretic peptide receptor type A as well as both BK receptors (BR1 and BR2).