Johanna Närväinen

Proliferating resident microglia after focal cerebral ischaemia in mice.

Cerebral ischaemia usually results in the rapid death of neurons within the immediate territory of the affected artery. Neuronal loss is accompanied by a sequence of events, including brain oedema, blood-brain barrier (BBB) breakdown, and neuroinflammation, all of which contribute to further neuronal death. Although the role of macrophages and mononuclear phagocytes in the expansion of ischaemic injury has been widely studied, the relative contribution of these cells, either of exogenous or intrinsic central nervous system (CNS) origin is still not entirely clear. The purpose of this study, therefore, was to use different durations of transient middle cerebral artery occlusion (tMCAo) in the mouse to investigate fully post-occlusion BBB permeability and cellular changes in the brain during the 72 h post-MCAo period. This was achieved using in vivo magnetic resonance imaging (MRI) and cell labelling techniques. Our results show that BBB breakdown and formation of the primary ischaemic damage after tMCAo is not associated with significant infiltration of neutrophils, although more are observed with longer periods of MCAo. In addition, we observe very few infiltrating exogenous macrophages over a 72 h period after 30 or 60 mins of occlusion, instead a profound increase in proliferating resident microglia cells was observed. Interestingly, the more severe injury associated with 60 mins of MCAo leads to a markedly reduced proliferation of resident microglial cells, suggesting that these cells may play a protective function, possibly through phagocytosis of infiltrating neutrophils. These data further support possible beneficial actions of microglial cells in the injured brain.

Synthesis and spectroscopic properties of a prototype single molecule dual imaging agent comprising a heterobimetallic rhenium-gadolinium complex.

A luminescent d−f hybrid containing a [Re(bpy)(Ar)(CO)3]+ chromophore and a gadolinium ion bound in a DO3A-derived binding pocket has been prepared. This complex combines long-lived luminescence from the 3MLCT state of the transition metal chromophore with high relaxivity, making it suitable for MRI imaging and luminescence imaging. The analogous complex containing ytterbium exhibits sensitized emission from both the ytterbium center and long-lived emission from the 3MLCT state.

Antiangiogenic Gene Therapy With Soluble VEGFR-1, -2, and -3 Reduces the Growth of Solid Human Ovarian Carcinoma in Mice

We studied antiangiogenic and antilymphangiogenic effects of sVEGFR-1 (sFlt-1), sVEGFR-2 (sFlk-1/KDR), and sVEGFR-3 (sFlt-4) gene transfers and their combinations in intraperitoneal ovarian cancer xenograft mice (Balb/c-Anu, n = 55). Gene therapy was initiated when the presence of sizable tumors was confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously via tail vein as follows: AdLacZ as a control (group I), AdsFlt-1 (group II), AdsKDR (group III), AdsFlt-4 (group IV) and two combination groups of AdsFlt-1 and AdsFlt-4 (group V) and AdsFlt-1, AdsKDR, and AdsFlt-4 (group VI). Antitumor effectiveness was assessed by sequential MRI, immunohistochemistry, microvessel density, overall tumor growth, and survival time. In combination group VI, intraperitoneal tumors were significantly smaller than in the control group at the end of the follow-up (P < 0.001). Furthermore, in group VI the microvessel density (microvessels/mm(2)) in tumor tissue and the total area of tumors covered by microvessels were significantly smaller than in the controls. One mouse in group V was cured. The combined antiangiogenic gene therapy with soluble VEGFRs reduced tumor growth, tumor vascularity, and ascites formation in ovarian cancer xenografts. The results suggest that the combined antiangiogenic gene therapy is a potential approach for the treatment of ovarian cancer patients.

Z-spectroscopy with Alternating-Phase Irradiation

Magnetization transfer (MT) MRI and Z-spectroscopy are tools to study both water-macromolecule interactions and pH-sensitive exchange dynamics between water and the protons of mobile chemical groups within these macromolecules. Both rely on saturation of frequencies offset from water and observation of the on-resonance water signal. In this work, an RF saturation method called Z-spectroscopy with Alternating-Phase Irradiation (ZAPI) is introduced. Based on the T(2)-selectivity of the irradiation pulse, ZAPI can be used to separate the different contributions to a Z-spectrum, as well as to study the T(2) distribution of the macromolecules contributing to the MT signal. ZAPI can be run at resonance for water and with low power, thus minimizing problems with specific absorption rate (SAR) limits in clinical applications. In this paper, physical and practical aspects of ZAPI are discussed and the sequence is applied in vitro to sample systems and in vivo to rat head to demonstrate the method.

Comparison of seven different anesthesia protocols for nicotine pharmacologic magnetic resonance imaging in rat.

Pharmacologic MRI (phMRI) is a non-invasive in vivo imaging method, which can evaluate the drug effects on the brain and provide complementary information to ex vivo techniques. The preclinical phMRI studies usually require anesthesia to reduce the motion and stress of the animals. The anesthesia, however, is a crucial part of the experimental design, as it may modulate the neural drug-induced (de)activation and hemodynamic coupling. Therefore, the aim of the present study was to address this methodologic question by performing phMRI experiments with five anesthetics (α-chloralose, isoflurane, medetomidine, thiobutabarbital, and urethane) and seven anesthesia protocols. Nicotine, a widely studied psychostimulant, was administered to rats while measuring blood oxygenation level-dependent (BOLD) signals. Notably different responses were observed depending on the anesthetic used. The highest responses were measured in urethane-anesthetized rats whereas the responses were hardly noticeable in α-chloralose group. As urethane is not commonly used in phMRI, hemodynamic coupling under urethane anesthesia was investigated with functional cerebral blood flow (CBF) and volume-weighted (CBVw) imaging, and simultaneous electrophysiologic and BOLD measurements. The BOLD, CBF, and CBVw measurements in response to nicotine were highly correlated (R(2) ≥ 0.70, p<0.001). BOLD values correlated well (R(2)=0.43, p<10(-6)) with local field potential (LFP) spectral power (13-70Hz) during pharmacologic stimulation. These findings indicate that urethane anesthesia combined with BOLD contrast provides a robust protocol for nicotine phMRI studies. As urethane has mild effects to individual receptor systems, and coupling between electrophysiologic activity and hemodynamic response is maintained, this anesthetic may also be suitable for other phMRI studies.

Short and Long-Term Effects of hVEGF-A165 in Cre-Activated Transgenic Mice

We have generated a transgenic mouse where hVEGF-A165 expression has been silenced with loxP-STOP fragment, and we used this model to study the effects of hVEGF-A165 over-expression in mice after systemic adenovirus mediated Cre-gene transfer. Unlike previous conventional transgenic models, this model leads to the expression of hVEGF-A165 in only a low number of cells in the target tissues in adult mice. Levels of hVEGF-A165 expression were moderate and morphological changes were found mainly in the liver, showing typical signs of active angiogenesis. Most mice were healthy without any major consequences up to 18 months after the activation of hVEGF-A165 expression. However, one mouse with a high plasma hVEGF-A165 level died spontaneously because of bleeding into abdominal cavity and having liver hemangioma, haemorrhagic paratubarian cystic lesions and spleen peliosis. Also, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in control mice. We conclude that long-term uncontrolled hVEGF-A165 expression in only a limited number of target cells in adult mice can be associated with pathological changes, including possible formation of malignant tumors and uncontrolled bleeding in target tissues. These findings have implications for the design of long-term clinical trials using hVEGF-A165 gene and protein.

Rats Bred for Low Aerobic Capacity Become Promptly Fatigued and Have Slow Metabolic Recovery after Stimulated, Maximal Muscle Contractions

AIM Muscular fatigue is a complex phenomenon affected by muscle fiber type and several metabolic and ionic changes within myocytes. Mitochondria are the main determinants of muscle oxidative capacity which is also one determinant of muscle fatigability. By measuring the concentrations of intracellular stores of high-energy phosphates it is possible to estimate the energy production efficiency and metabolic recovery of the muscle. Low intrinsic aerobic capacity is known to be associated with reduced mitochondrial function. Whether low intrinsic aerobic capacity also results in slower metabolic recovery of skeletal muscle is not known. Here we studied the influence of intrinsic aerobic capacity on in vivo muscle metabolism during maximal, fatiguing electrical stimulation. METHODS Animal subjects were genetically heterogeneous rats selectively bred to differ for non–trained treadmill running endurance, low capacity runners (LCRs) and high capacity runners (HCRs) (n = 15–19). We measured the concentrations of major phosphorus compounds and force parameters in a contracting triceps surae muscle complex using 31P-Magnetic resonance spectroscopy (31P-MRS) combined with muscle force measurement from repeated isometric twitches. RESULTS Our results demonstrated that phosphocreatine re-synthesis after maximal muscle stimulation was significantly slower in LCRs (p<0.05). LCR rats also became promptly fatigued and maintained the intramuscular pH poorly compared to HCRs. Half relaxation time (HRT) of the triceps surae was significantly longer in LCRs throughout the stimulation protocol (p≤0.05) and maximal rate of torque development (MRTD) was significantly lower in LCRs compared to HCRs from 2 min 30 s onwards (p≤0.05). CONCLUSION We observed that LCRs are more sensitive to fatigue and have slower metabolic recovery compared to HCRs after maximal muscle contractions. These new findings are associated with reduced running capacity and with previously found lower mitochondrial content, increased body mass and higher complex disease risk of LCRs.

T2, Carr–Purcell T2 and T1ρ of fat and water as surrogate markers of trabecular bone structure

Magnetic resonance imaging (MRI) techniques have been developed for non-invasive assessment of the structural properties of trabecular bone. These measurements, however, suffer from relatively long acquisition times and low resolution compared to the trabecular size. Spectroscopic measurement of relaxation times could be applied for more detailed and faster assessment of relaxation properties of bone marrow and also provide surrogate information on trabecular structure. In the present study, bovine trabecular bone was investigated with spectroscopic NMR (nuclear magnetic resonance) methods to determine the relationship between structural parameters as measured with micro-CT and T(2), Carr-Purcell T(2) and T(1rho) relaxation times of fat and water. To compare bone with a sample matrix with magnetic susceptibility interfaces, phantoms consisting of glass beads with different diameters in oil or water were used. The behavior of T(2) measured with different sequences and T(1rho) at different magnitudes of spin-lock fields were characterized, and relaxation times were correlated with structural parameters. T(2) and T(1rho) showed significant associations with structural bone parameters. Strongest linear correlations (r = 0.81, p < 0.01) were established between R(1rho) (1/T(1rho)) of fat component and structural model index. For glass beads, the behavior of T(2) and T(1rho) was similar to that of the water compartment of bone marrow. The present results suggest feasibility of spectroscopic NMR measurements to assess trabecular structure. However, further studies are required to determine the sensitivity of this approach to fat content of bone marrow and to lower the field strengths used in clinical devices.

Tuftsin derivatives of FITC, Tb-DOTA or Gd-DOTA as potential macrophage-specific imaging biomarkers

Fluorescein- and terbium-labelled tuftsin (Thr-Lys-Pro-Arg) and pentapeptide (Thr-Lys-Pro-Pro-Arg) were synthesized and their properties were evaluated in vitro by luminescence spectrometry and confocal microscopy as fluorescence probes to target macrophage cells in biological systems. An increase in fluorescence of macrophages incubated with varying concentrations of fluorescein isothiocyanate or Tb-DOTA-tuftsin/pentapeptide conjugates was observed in a concentration-dependent manner. Tb-DOTA-pentapeptide had a greater affinity to macrophages than Tb-DOTA-tuftsin. Lipopolysaccharide (LPS) stimulation strengthened the internalization of peptide conjugates by macrophages through the tuftsin receptor mechanism. Tb-DOTA-tuftsin/pentapeptide conjugates are likely to be a promising optical reagents as probes of the immune response with involvement of macrophage cells in a variety of diseases. Gd-DOTA-tuftsin conjugate was also evaluated as a cell-specific contrast agent in in vitro MRI experiments. In this context, the macrophages labelled by Gd-DOTA-tuftsin were highly magnetic and detectable by MRI, which confirms that this vectorized MRI probe has the potential to image macrophage-mediated inflammation in diseases like brain traumas and stroke. Tuftsin receptor-specific biological-function domain may have a modified in vivo biodistribution profile, bioavailability and pharmacokinetics subsequent to its conjugation to a metal ion-binding backbone.

Ultrasound imaging with bolus delivered contrast agent for the detection of angiogenesis and blood flow irregularities

Highly increased blood flow and vascularity after angiogenic gene therapy have raised concerns of shunting and hemangioma-like blood pool formation that might decrease effective perfusion and ruin the beneficial effects of the therapy. Contrast enhanced ultrasound is a promising noninvasive tool for studying skeletal muscle perfusion. The objectives of the present study were to test bolus and infusion administrations of ultrasound microbubble contrast media in imaging vascular growth in skeletal muscle and assess the functionality of vessels grown with angiogenic gene therapy. Contrast enhanced ultrasound was used to study changes in skeletal muscle perfusion in normal and gene-transduced rabbit hindlimbs 6 days after gene transfer. Adenoviral gene transfer of VEGF (10e(9)-10e(11) viral particles) or β-galactosidase control gene (10e(11) viral particles) was done under anesthesia and induced up to 16-fold increases in relative tissue perfusion. Contrast intensity versus time curves were plotted and analyzed for contrast kinetics. Bolus administration of the contrast media was highly feasible in analyzing skeletal muscle blood flow and its kinetics. Maximal signal intensity of the bolus signal reflected relative changes in both blood flow and volume equally to the infusion method. Flow irregularities were detected after angiogenic gene therapy. In conclusion, bolus delivery of ultrasound contrast agent is highly feasible for the relative analysis of both quantity and quality of blood flow after angiogenic gene therapy. The kinetics of blood flow can and should be studied more extensively in both preclinical and clinical trials of angiogenic gene therapy since there is increasing evidence of flow irregularities in angiogenic vessels.