Eva Syková

Extracellular space structure revealed by diffusion analysis.

The structure of brain extracellular space resembles foam. Diffusing molecules execute random movements that cause their collision with membranes and affect their concentration distribution. By measuring this distribution, the volume fraction (alpha) and the tortuosity (lambda) can be estimated. The volume fraction indicates the relative amount of extracellular space and tortuosity is a measure of hindrance of cellular obstructions. Diffusion measurements with molecules <500 Mr show that alpha approximately 0.2 and lambda approximately 1.6, although some brain regions are anisotropic. Molecules > or =3000 Mr show more hindrance, but molecules of 70000 Mr can move through the extracellular space. During stimulation, and in pathophysiological states, alpha and lambda change, for example in severe ischemia alpha = 0.04 and lambda = 2.2. These data support the feasibility of extrasynaptic or volume transmission in the extracellular space.

Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord.

Nuclear magnetic resonance (MR) imaging provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied, in animals with a cortical photochemical lesion or with a balloon‐induced spinal cord compression lesion, the fate of implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with superparamagnetic iron oxide nanoparticles (Endorem). MSCs were colabeled with bromodeoxyuridine (BrdU), and ESCs were transfected with pEGFP‐C1 (eGFP ESCs). Cells were either grafted intracerebrally into the contralateral hemisphere of the adult rat brain or injected intravenously. In vivo MR imaging was used to track their fate; Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells. During the first week postimplantation, grafted cells migrated to the lesion site and populated the border zone of the lesion. Less than 3% of MSCs differentiated into neurons and none into astrocytes; 5% of eGFP ESCs differentiated into neurons, whereas 70% of eGFP ESCs became astrocytes. The implanted cells were visible on MR images as a hypointense area at the injection site, in the corpus callosum and in the lesion. The hypointense signal persisted for more than 50 days. The presence of GFP‐positive or BrdU‐positive and nanoparticle‐labeled cells was confirmed by histological staining. Our study demonstrates that both grafted MSCs and eGFP ESCs labeled with a contrast agent based on iron oxide nanoparticles migrate into the injured CNS. Iron oxide nanoparticles can therefore be used as a marker for the long‐term noninvasive MR tracking of implanted stem cells.

Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk

Interactions between separate synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits γ-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a physical barrier to diffusion and, in addition, are essential for glutamate uptake, we investigated the physiological contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. Here we show that the reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and volume fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall volume transmission in the brain.

Astroglial networks scale synaptic activity and plasticity

Astrocytes dynamically interact with neurons to regulate synaptic transmission. Although the gap junction proteins connexin 30 (Cx30) and connexin 43 (Cx43) mediate the extensive network organization of astrocytes, their role in synaptic physiology is unknown. Here we show, by inactivating Cx30 and Cx43 genes, that astroglial networks tone down hippocampal synaptic transmission in CA1 pyramidal neurons. Gap junctional networking facilitates extracellular glutamate and potassium removal during synaptic activity through modulation of astroglial clearance rate and extracellular space volume. This regulation limits neuronal excitability, release probability, and insertion of postsynaptic AMPA receptors, silencing synapses. By controlling synaptic strength, connexins play an important role in synaptic plasticity. Altogether, these results establish connexins as critical proteins for extracellular homeostasis, important for the formation of functional synapses.

Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.

Imaging the fate of implanted bone marrow stromal cells labeled with superparamagnetic nanoparticles

Bone marrow stromal cells (MSCs) are pluripotent progenitor cells that have the capacity to migrate toward lesions and induce or facilitate site‐dependent differentiation in response to environmental signals. In animals with a cortical photochemical lesion, the fate of rat MSCs colabeled with magnetic iron‐oxide nanoparticles (Endorem®) and bromodeoxyuridine (BrdU) was studied. MSCs were either grafted intracerebrally into the contralateral hemisphere of adult rat brain or injected intravenously. In vivo MRI was used to track their fate; Prussian blue staining and transmission electron microscopy (TEM) confirmed the presence of iron‐oxide nanoparticles inside the cells. During the first week posttransplantation, the transplanted cells migrated to the lesion site and populated the border zone of the damaged cortical tissue. The implanted cells were visible on MR images as a hypointense area at the injection site and in the lesion. The hypointense signal persisted for more than 50 days. The presence of BrdU‐positive and iron‐containing cells was confirmed by subsequent histological staining. Three to 4 weeks after injection, <3% of MSCs around the lesion expressed the neuronal marker NeuN. Our study demonstrates that a commercially available contrast agent can be used as a marker for the long‐term noninvasive MR tracking of implanted cells. Magn Reson Med 50:767–776, 2003.

Diffusion Barriers Evoked in the Rat Cortex by Reactive Astrogliosis

Changes in extracellular space (ECS) diffusion parameters in astrogliotic tissue around a unilateral cortical stab wound were determined from concentration‐time profiles of tetramethylammonium (TMA+) using TMA+‐selective microelectrodes. Three diffusion parameters—ECS volume fraction α (α = ECS volume/ total tissue volume), tortuosity λ (λ2 = D/ADC; where D is the free and ADC is the apparent diffusion coefficient of TMA+ in the brain), and nonspecific TMA+uptake k′—were determined at 3, 7, 21, and 35 days postwounding (dpw), in the hemispheres ipsilateral and contralateral to the lesion. Following diffusion experiments, tissue sections were immunostained for glial fibrillary acidic protein (GFAP) and chondroitin‐sulphate proteoglycans (CSPG). In the area 300–1000 μm around the wound, α was increased at 3, 7, and 21 dpw by about 20% but returned to control values at 35 dpw; λ was increased at all four intervals, reaching a maximum at 7 dpw. k′ was lower than in the contralateral hemisphere at 7, 21, and 35 dpw. Measurements 1,500–2,000 μm from the wound revealed only an increase in λ at 7 dpw. The time course of changes in ECS diffusion parameters closely correlated with increased staining for GFAP and CSPG. Our results show that astrogliosis significantly changes the diffusion properties of nervous tissue, making it less permissive. Both hypertrophied astrocytic processes and an enhanced formation of some extracellular matrix molecules could affect, through changes in the diffusion of molecules in the ECS, neuron–glia communication, “cross‐talk” between synapses, extrasynaptic transmission, and regenerative processes.  GLIA 28:40–48, 1999.

Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein‐coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

Magnetic Resonance Tracking of Implanted Adult and Embryonic Stem Cells in Injured Brain and Spinal Cord

Abstract: Stem cells are a promising tool for treating brain and spinal cord injury. Magnetic resonance imaging (MRI) provides a noninvasive method to study the fate of transplanted cells in vivo. We studied implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with iron‐oxide nanoparticles (Endorem®) and human CD34+ cells labeled with magnetic MicroBeads (Miltenyi) in rats with a cortical or spinal cord lesion. Cells were grafted intracerebrally, contralaterally to a cortical photochemical lesion, or injected intravenously. During the first week post transplantation, transplanted cells migrated to the lesion. About 3% of MSCs and ESCs differentiated into neurons, while no MSCs, but 75% of ESCs differentiated into astrocytes. Labeled MSCs, ESCs, and CD34+ cells were visible in the lesion on MR images as a hypointensive signal, persisting for more than 50 days. In rats with a balloon‐induced spinal cord compression lesion, intravenously injected MSCs migrated to the lesion, leading to a hypointensive MRI signal. In plantar and Basso‐Beattie‐Bresnehan (BBB) tests, grafted animals scored better than lesioned animals injected with saline solution. Histologic studies confirmed a decrease in lesion size. We also used 3‐D polymer constructs seeded with MSCs to bridge a spinal cord lesion. Our studies demonstrate that grafted adult as well as embryonic stem cells labeled with iron‐oxide nanoparticles migrate into a lesion site in brain as well as in spinal cord.

In vivo tracking of stem cells in brain and spinal cord injury

Cellular magnetic resonance (MR) imaging is a rapidly growing field that aims to visualize and track cells in living organisms. Superparamagnetic iron oxide (SPIO) nanoparticles offer a sufficient signal for T2 weighted MR images. We followed the fate of embryonic stem cells (ESCs) and bone marrow mesenchymal stem cells (MSCs) labeled with iron oxide nanoparticles (Endorem) and human CD34+ cells labeled with magnetic MicroBeads (Miltenyi) in rats with a cortical or spinal cord lesion, models of stroke and spinal cord injury (SCI), respectively. Cells were either grafted intracerebrally, contralaterally to a cortical photochemical lesion, or injected intravenously. During the first post-transplantation week, grafted MSCs or ESCs migrated to the lesion site in the cortex as well as in the spinal cord and were visible in the lesion on MR images as a hypointensive signal, persisting for more than 30 days. In rats with an SCI, we found an increase in functional recovery after the implantation of MSCs or a freshly prepared mononuclear fraction of bone marrow cells (BMCs) or after an injection of granulocyte colony stimulating factor (G-CSF). Morphometric measurements in the center of the lesions showed an increase in white matter volume in cell-treated animals. Prussian blue staining confirmed a large number of iron-positive cells, and the lesions were considerably smaller than in control animals. Additionally, we implanted hydrogels based on poly-hydroxypropylmethacrylamide (HPMA) seeded with nanoparticle-labeled MSCs into hemisected rat spinal cords. Hydrogels seeded with MSCs were visible on MR images as hypointense areas, and subsequent Prussian blue histological staining confirmed positively stained cells within the hydrogels. To obtain better results with cell labeling, new polycation-bound iron oxide superparamagnetic nanoparticles (PC-SPIO) were developed. In comparison with Endorem, PC-SPIO demonstrated a more efficient intracellular uptake into MSCs, with no decrease in cell viability. Our studies demonstrate that magnetic resonance imaging (MRI) of grafted adult as well as ESCs labeled with iron oxide nanoparticles is a useful method for evaluating cellular migration toward a lesion site.