DEPARTMENT OF NEUROSCIENCE

Prof. MUDr. Eva Syková, DrSc.
email: sykova@biomed.cas.cz
tel: +420241062204

Laboratory of Diffusion Studies and Imaging Methods

Head: Prof. MUDr. Eva Syková, DrSc.
email: sykova@biomed.cas.cz
tel: +420241062204

Laboratory of Tissue Culture and Stem Cells

Head: RNDr. Pavla Jendelová, PhD.
email: pavla.jendelova@lfmotol.cuni.cz
tel: +420241062619

Scientists:

MVDr. Takashi Amemori, PhD.
RNDr. Kateřina Glogarová, PhD.
Nataliya Kozubenko, PhD.
PharmDr. Šárka Kubinová, PhD., PhD.
Ing. Katarína Likavčanová, PhD.
MUDr. Lucia Urdziková, PhD.
MUDr. Lýdia Vargová, PhD.
Mgr. Ivan Voříšek, PhD.

Ph.D. Students:

Dr. David Arboleda
Mgr. Lesia Dmytrenko
MUDr. Jindřich Fiala
MUDr. Aleš Hejčl
MUDr. Aleš Homola
Mgr. Miroslava Kapcalová
MUDr. Karolína Kroupová
MUDr. Petr Lesný
MUDr. Jiří Šedý
MUDr. Karel Šlais

Technical Assistants:

Dominika Dušková
Hana Hronová

Diffusion properties of the nervous tissue in health and disease

The Laboratory of Diffusion Studies and Imaging Methods studies the changes in the extracellular space that occur during physiological and pathological states. Several animal models of pathological states and diseases attacking the CNS are used, e.g., models of chronic pain, ischemia and ischemic lesions, perinatal and early postnatal anoxia, brain edema, hydrocephalus, multiple sclerosis, Parkinson's disease, Alzheimer's disease, tumors, developmental disorders, changes during aging, and brain and spinal cord injury, as well as models of CNS damage evoked by chemical or physical factors such as neurotoxins and X-irradiation. The research aims are the improvement of therapy and diagnostic methods for CNS diseases and the prevention of CNS damage.

Research at the Laboratory of Diffusion Studies and Imaging Methods focuses on the following main topics:

• The origin, mechanisms and pathophysiological significance of ionic changes in the extracellular space
• Diffusion in the extracellular space: the underlying mechanism of extrasynaptic ("volume") transmission
• Diffusion studies using the real-time TMA⁺ iontophoretic method
• Diffusion studies using diffusion-weighted MR to measure the apparent diffusion coefficient of water
• Extracellular space volume and geometry - factors affecting diffusion in the CNS in health and disease
• Studies using models of pathological states, including transgenic animals
• Magnetic resonance imaging and spectroscopy

Studies at the Laboratory are aimed at understanding the maintenance of ionic and volume homeostasis in the CNS, the extracellular space as a communication channel, the diffusion parameters of the extracellular space, extrasynaptic "volume" transmission and the role of glia in signal transmission, behavior and regeneration.

Regeneration of the CNS using cell therapy and polymer scaffolds

The Laboratory of Tissue Culture and Stem Cells studies the isolation, labeling and application of stem cells in experimental models of brain and spinal cord injury. The implantation of polymer scaffolds into defects in the central nervous system can reduce glial scar formation, bridge the lesion and lead to tissue regeneration within the scaffold. Various cell types, such as mesenchymal stem cells (MSCs) olfactory glial cells, and mouse and human embryonic stem cells, are studied as candidates for supporting neural tissue regeneration. Macroporous polymer hydrogels or nanofiber scaffolds support three-dimensional cell growth under in vitro conditions, and cell-polymer constructs, consisting of a polymer scaffold and cellular elements, are utilized and implanted in order to support regeneration in damaged tissue.

Research at the Laboratory of Tissue Culture and Stem Cells focuses on these main topics:

• Characterization of adult and embryonic stem cells in vitro
• Phenotyping of stem and precursor cells by means of flow cytometry
• Cultivation of human embryonic stem cells
• Regeneration and repair of injured brain and spinal cord using cell therapy and biomaterials
• Macroporous polymer hydrogels designed to bridge spinal cords lesions
• Cell-polymer constructs designed to bridge lesions of the central nervous tissue
• Nanofiber scaffolds for two- and three-dimensional cell cultivation
• The modification of polymer surfaces by bioactive macromolecules
• Labeling of stem cells with superparamagnetic iron-oxide nanoparticles and imaging their migration and fate using MRI

The aim of our cell therapy studies is to replace, repair or improve the biological functioning of defective nervous tissue. This aim can be achieved through the transplantation of cell-polymer constructs or isolated and well-characterized cells into the injured CNS in sufficient numbers and quality so that they can induce the recovery of function.

Fig. 1: Diffusion parameters in a glioblastoma

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Hematoxylin-eosin staining of the human brain cortex and a glioblastoma (WHO grade IV) and representative TMA+ -diffusion curves with the corresponding values of the ECS diffusion parameters α, λ and k´. In comparison with the healthy tissue, the ECS volume fraction (α) is almost doubled and the tortuosity (λ) is significantly increased in a highly malignant glioblastoma.

Fig. 2: Simultaneous measurement of light transmitance, ECS diffusion parameters and changes in extracellular K⁺ concentration

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(A) An example of changes in light transmittance evoked by a 20 min application of 10-4M NMDA. The pseudo-color image reflects the spatial distribution of the percentage of change in light transmittance 5 min after the onset of the application. The time course of light transmittance changes in the area of the dorsal horn is shown below. (B) Typical recordings of TMA+ diffusion curves in agar and in spinal cord gray matter during perfusion with artificial cerebrospinal fluid. (C) Typical recording, using ion-selective microelectrodes, of an increase in [K+]e evoked by 10-4M NMDA application

Fig. 3: Traumatic brain injury

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Coronal rat brain sections stained for GFAP (upper left) and CSPG (upper right) 7 days following a cortical stab wound. The results show a higher level of GFAP expression in the vicinity of the stab wound and a higher level of CSPG expression in the whole cortex of the injured hemisphere. Both the GFAP and CSPG sections are from the same animal. The arrows indicate the site of the stab wound. Lower left: T2-weighted image showing the localization of the wound. Lower right: A pseudocolor image showing a typical ADCW map of an injured rat brain 7 days post-wounding; ADCW was measured along the y-axis (rostrocaudal plane). The scale to the right of the ADCW map shows the relation between the intervals of ADC W values and the colors used for visualization. ADCW at 7 days post-wounding is significantly lower in the entire cortex of the wounded hemisphere compared with control animals, except in the area close to the wound.

Fig. 4: Structural changes in the hippocampal gyrus dentatus region of aged rats

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(A) Astrocytes stained for GFAP in a young rat; note the radial organization of the astrocytic processes between the pyramidal cells (not stained). (B) In an aged rat, the radial organization of the astrocytic processes is lost. (C) Staining for fibronectin in a young adult rat shows densely stained cells, apparently due to perineuronal staining around granular cells. (D) In an aged rat, the fibronectin staining is lost. Scale bar: A and B, 100 μm; C and D, 50 μm.

Fig. 5: TMA⁺ measurements and typical diffusion curves in control and APP23 mice

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(A) A double-barreled TMA+-selective microelectrode (TMA+ ISM) and a micropipette for TMA+ iontophoresis were glued together with dental cement to stabilize the intertip distance at 100–150 μm, and this microelectrode array was introduced into the dorsal brain cortex. (B) Comparison of TMA+ diffusion curves obtained in 17- to 25-month-old transgenic and control mice. The diffusion parameters can be determined from the shape and magnitude of the curves. The ECS was larger, and ADCTMA was lower, in APP23 animals than in controls. (C) ECS volume fraction (α) plotted against the depth of measurements (the zero level corresponds to the brain surface); α was changed in 17- to 25-month-old APP23 mice compared with age-matched controls.

Fig. 6: An APP23 mouse brain stained for amyloid plaques

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(A) A brain slice immunohistochemically stained for β-amyloid, a compound that forms deposits in the brain in Alzheimer’s disease. APP23 mice, an animal model of Alzheimer’s disease, form only subtle deposits in adulthood, and the plaque load is less than 2%. In aged APP23 females the average amyloid plaque load is higher ( 20 ± 2 %) than in males (10 ± 2 %). The plaques are more frequent in the rostral parts of the brain and in the deeper layers of the cortex.  Bottom: adjacent sections stained with thioflavine (B) and Congo red (C).

Fig. 7: Typical apparent diffusion coefficient of water (ADC_W) maps of tenascin
(TN)-R+⁄+ and TN-R–⁄– mice

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ADCW was calculated in five selected areas: motor cortex (M), primary somatosensory cortex (S1), secondary somatosensory cortex (S2), hippocampus (HIP) and thalamus (TH). (A and B) The areas are outlined in the microphotographs of Cresyl violet-stained slices. (C and D) The images show ADCW maps of TN-R+⁄+ and TN-R–⁄– mice; both images are from the same coronal plane as shown in (B). The scale at the bottom of the figure shows the relation between the intervals of ADCW values and the colors used for visualization. Note the lower ADCW throughout the whole slice from the TN-R–⁄– mouse when compared with the TN-R+⁄+ control.

Fig. 8: Mesenchymal stem cells labeled with iron-oxide nanoparticles

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(A) Cells in culture labeled with BrdU (brown nuclei), containing the contrast agent Endorem (Prussian blue staining). (B) Transmission electron microphotograph showing a cluster of iron particles surrounded by a cell membrane, confirming the presence of iron inside the cell. (C-E) Cells labeled with Endorem undergoing cell division, confirming that incorporation of Endorem does not adversely affect cell viability (anti-BrdU and Prussian blue staining). Transplanted nanoparticle-labeled cells can be detected in vivo using MR imaging.

Fig. 9: T2-weighted MR images of a cortical photochemical lesion and MSCs implanted into the contralateral hemisphere or injected intravenously into the femoral vein

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Upper row: The first image (lesion) shows a photochemical lesion 12 hours after a thrombosis evoked by dye/light interaction, prior to any cell implantation. The cell implant labeled with superparamagnetic nanoparticles in the hemisphere contralateral to the lesion is clearly visible as a hypointense area (24h post-implantation). A hypointense signal in the lesion was seen 14 days after grafting (14 d PI), suggesting the migration of implanted cells into the lesion. Lower row: The first image (lesion) shows a photochemical lesion 12 hours after a thrombosis evoked by dye/light interaction, prior to cell implantation. A hypointense signal in the lesion observed 7 days after the intravenous injection of MSCs labeled with nanoparticles (7 days PI) persisited in the lesion for the whole measurement period (7 weeks PI). Inserts shows a higher magnification view of the lesion.

Fig. 10: Spinal cord balloon-induced compression lesion and implantation of stem cells

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A comparison of longitudinal sections of spinal cords with a balloon-induced compression lesion five weeks after the i.v. injection of saline (A), granulocyte-colony stimulating factor to mobilize endogenous bone marrow cells, (B), a freshly prepared mononuclear fraction of bone marrow cells (C) or mesenchymal stem cells (D). Note that the lesion cavity is smaller after cell treatment suggesting a positive effect of  the cells on lesion repair.

Fig. 11: Growth of cells on nanofiber scaffolds

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An electron micrograph of a nanofiber layer made by electrospinning copolymers of hydroxypropylmethacrylamide and ethoxyethylmethacrylate at low magnification (A). These nanofibers can be used as stem cell carriers for tissue regeneration. The growth of olfactory ensheathing cells on a nanofiber scaffold after 24h of cultivation (B). Cells were stained  with CFDA (green). Mesenchymal stem cells on nanofiber layers 24h after seeding (C,D). Cells were stained with phalloidin (red)  and DAPI (blue).

List of current grants:

Name of the grant	Grant agency	Number of the grant	Principal investigator (coinvestigator)	Duration of the grant
Research project: "Molecular, cellular and systems mechanisms of serious diseases of the human organism, their diagnosis, therapy and pharmacotherapy "	AVČR	AV0Z50390512	Syková	2005-10
Research centre: “Centre for cell therapy and tissue repair”.	MŠMT ČR	1M0021620803	Syková	2005-09
Research center: "Centre of neuroscience"	MŠMT ČR	LC554	Syka	2005-09
Autologous transplantation of bone marrow stem cells in patients with spinal cord transection	IGA MZ	NR/8339 - 3	Syková	2005-07
PhD program in neuroscience	GAČR	309/03/H095	Syková	2003-07
Utilization of biocompatible macroporous hydrogels containing cell cultures for therapy of spinal cord injury	IGA MZ	1A8697-5	Syková	2005-09
Utilization of olfactory ensheating glia and bone marrow stromal cells for spinal cord injury repair	GAČR	309/06/1246	Syková (Jendelová)	2006-08
Cellular contrast media and their utilization in MR imaging	GAČR	309/06/1594	Herynek (Jendelová)	2006-08
Combined contrast media for molecular MR imaging	AV ČR	KAN201110651	Lukeš (Syková)	2006-10
Utilization of new synthetized biomaterials in combination with stem cells in the therapy of diseases affecting human tisse derived from the mesoderm: cartilage, bone, ligaments and meniscus	MŠMT ČR	2B06130	Nečas (Syková)	2006-11
Polarized cultures of hepatocytes and mesenchymal cells in nanofiber layers in an experimental bioreactor	GAČR	304/07/1129	Ryska (Syková)	2007-09

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