Diana Cash

Tracking Transplanted Stem Cell Migration Using Bifunctional, Contrast Agent-Enhanced, Magnetic Resonance Imaging

The ability to track stem cell transplants in the brain by in vivo neuroimaging will undoubtedly aid our understanding of how these cells mediate functional recovery after neural transplantation. One major challenge for the development and refinement of stem cell transplantation is to map the spatial distribution and rate of migration in situ. Here we report a method for tracking transplanted stem cells in the ischemia-damaged rat hippocampus by magnetic resonance imaging (MRI). Before transplantation, stem cells were labeled in vitro either with a novel bifunctional contrast agent, gadolinium rhodamine dextran (GRID), identifiable by both MRI and fluorescence microscopy, or with PKH26, visible exclusively under fluorescence microscopy. At different time points following engraftment, the brains were evaluated by both histology and ex vivo MR imaging. Transplanted stem cells were identified by MRI only if prelabeled with GRID, whereas fluorescence microscopy detected transplanted cells using either label. The distribution of GRID-labeled stem cells identified by MRI corresponded to those detected using fluorescence microscopy. These results demonstrate that GRID-enhanced MRI can reliably identify transplanted stem cells and their migration in the brain.

Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study

Preferential migration of stem cells toward the site of a lesion is a highly desirable property of stem cells that allows flexibility in the site of graft implantation in the damaged brain. In rats with unilateral stroke damage, neural stem cells transplanted into the contralateral hemisphere migrate across to the lesioned hemisphere and populate the area around the ischaemic infarct. To date, the migration of neural stem cells in the damaged brain has been mainly inferred from snapshot histological images. In this study, we demonstrate that by pre-labelling neural stem cells with the bimodal contrast agent Gadolinium-RhodamIne Dextran [GRID, detectable by both magnetic resonance imaging (MRI) and fluorescent microscopy], the transhemispheric migration of transplanted neural stem cells contralateral to a stroke lesion can be followed in vivo by serial MRI and corroborated by subsequent histological analyses. Our results indicate that neural stem cells migrated from the injection tract mainly along the corpus callosum within 7 days of transplantation and extensively re-populated the peri-lesion area by 14 days following implantation. In contrast, neural stem cells transplanted into sham controls did not show any substantial migration outside of the injection tract, suggesting that the transcallosal migration observed in the stroke-lesioned animals is due to neural stem cells being attracted by the lesion site. In vivo tracking of the migration of neural stem cells responding to damage will greatly enhance our understanding of optimal transplantation strategies as well as how neural stem cells promote functional and anatomical recovery in neurological disorders.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood-brain barrier disruption and neurological deficits in stroke

Disruption of the blood-brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70 min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72 h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4-72 h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke.

Neuroprotective effect of aminoguanidine on transient focal ischaemia in the rat brain

Using serial magnetic resonance imaging we have evaluated the effectiveness of aminoguanidine (AG) as a neuroprotective agent in a rat model of transient middle cerebral artery occlusion (MCAO). Because aminoguanidines neuroprotective properties have primarily been ascribed to its action as iNOS inhibitor, we also performed a biochemical analysis of nitric oxide metabolites and NOS isoforms in our model of ischaemia. Daily injections of AG (100 mg/kg) or saline, were started at 6 h after the occlusion and the effects of this treatment on lesion progression monitored by T(2)-weighted MRI at 6 (pre-treatment scan), 24 and 72 h. Measurements of lesion volumes showed that between 6 and 72 h post-MCAO, lesion growth was slower in AG-treated rats than in control rats. This difference was most pronounced between 24 and 72 h post-MCAO when AG halted the lesion volume expansion observed in control rats. Measurements of plasma NOx (nitrite plus nitrate) at 0, 24, 48 and 72 h after MCAO, showed that NO levels did not differ significantly between the AG- and saline-treated groups at any time-point. Moreover, NOS activity assays revealed that no iNOS activity was present in any of the brains tested and that constitutive neuronal NOS activity was similar across the two hemispheres between both groups. The absence of iNOS protein in the ischaemic and contralateral hemispheres at 48 and 72 h after MCAO (control group only) was confirmed by Western blot analysis. These results suggest that AG treatment reduces the rate of growth of ischaemic lesions, perhaps preserving the functioning of perifocal neurons. Our observations contradict suggestions that high levels of NO generated by iNOS are partially responsible for exacerbating the neuronal damage in the postischaemic phase of MCAO. Although this does not rule out a role for AG as a neuroprotective agent via its ability to inhibit iNOS, these findings indicate that neuroprotective actions of AG may also be mediated via other cellular targets.

Using the BOLD MR signal to differentiate the stereoisomers of ketamine in the rat

RATIONALE Ketamine is a chiral molecule that is reported to model aspects of schizophrenia. OBJECTIVES To investigate the stereospecificity of the isomers of ketamine using pharmacological magnetic resonance imaging (phMRI) in order to further understand ketamines pharmacodynamic actions. METHOD Responses to 25 mg kg-1S(+) isomer, R(-) isomer and racemic ketamine in independent groups of Sprague-Dawley rats were investigated using a prepulse inhibition paradigm, locomotor observations, MRI and 2-deoxyglucose techniques. RESULTS Racemic ketamine and the S(+) isomer were both capable of disrupting sensorimotor gating as measured using prepulse inhibition and produced a longer period of hyperlocomotion comparative to the R(-) isomer. In contrast, large alterations in the BOLD MR signal were observed with R(-) isomer, whereas S(+) isomer and racemate precipitated more localized BOLD signal changes predominantly in cortical, hippocampal and hindbrain regions. Glucose utilization rates in conscious animals are in agreement with previously published data and verify the BOLD responses in the racemic group. However, no significant changes in glucose utilization were observed in the anesthetized cohort. CONCLUSIONS Ketamine and its isomers have stereospecific effects on sensorimotor gating and locomotion that correlate with the enantiomers affinity for the NMDA receptor. It would appear that anesthesia, as required for preclinical MRI procedures, may interact with and potentially attenuate the drugs response. Although analysis of the main effect of isomers in comparison to each other or the racemate offers an alternative analysis method that should be less susceptible to anesthetic interactions, only the R(-) isomer comparative to the racemate offers significant differences of interest.

Subthalamic nucleus neurones in slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice show irregular, dopamine-reversible firing pattern changes, but without synchronous activity

The loss of dopamine in idiopathic or animal models of Parkinsons disease induces synchronized low-frequency oscillatory burst-firing in subthalamic nucleus neurones. We sought to establish whether these firing patterns observed in vivo were preserved in slices taken from dopamine-depleted animals, thus establishing a role for the isolated subthalamic-globus pallidus complex in generating the pathological activity. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed significant reductions of over 90% in levels of dopamine as measured in striatum by high pressure liquid chromatography. Likewise, significant reductions in tyrosine hydroxylase immunostaining within the striatum (>90%) and tyrosine hydroxylase positive cell numbers (65%) in substantia nigra were observed. Compared with slices from intact mice, neurones in slices from MPTP-lesioned mice fired significantly more slowly (mean rate of 4.2 Hz, cf. 7.2 Hz in control) and more irregularly (mean coefficient of variation of inter-spike interval of 94.4%, cf. 37.9% in control). Application of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) and the GABA(A) receptor antagonist picrotoxin caused no change in firing pattern. Bath application of dopamine significantly increased cell firing rate and regularized the pattern of activity in cells from slices from both MPTP-treated and control animals. Although the absolute change was more modest in control slices, the maximum dopamine effect in the two groups was comparable. Indeed, when taking into account the basal firing rate, no differences in the sensitivity to dopamine were observed between these two cohorts. Furthermore, pairs of subthalamic nucleus cells showed no correlated activity in slices from either control (21 pairs) or MPTP-treated animals (20 pairs). These results indicate that the isolated but interconnected subthalamic-globus pallidus network is not itself sufficient to generate the aberrant firing patterns in dopamine-depleted animals. More likely, inputs from other regions, such as the cortex, are needed to generate pathological oscillatory activity.

Gamma shielding materials for MR-compatible PET

As for standard positron emission tomography (PET) scanners, MR-compatible PET scanners will require gamma shielding to suppress the influence of activity outside the PET field of view (FOV). Suitable materials must have very specific properties, including magnetic properties close to those of water, high density, high atomic number, and ideally a low conductivity. In order to identify potential suitable materials, we have selected several heavy-metal-based candidates based on the available data for magnetic and shielding properties. These materials include several nonferromagnetic metals and metal oxides, two scintillating crystals (bismuth germanate and lead tungstate) and two metal/epoxy compounds. The magnetic resonance imaging (MRI) compatibility of these materials was assessed under various conditions, both on a human and a small-animal MRI scanner. In parallel, we assessed the shielding efficiency at 661 keV of the most promising candidates. These experiments showed that there is a range of possibilities for the design of MR-compatible gamma shields. Lead has acceptable magnetic compatibility but can induce significant conductivity-related artefacts. Heavy-metal-based minerals are fully insulating and hot-pressed lead monoxide showed good MR compatibility combined with good shielding properties. Other possibilities include the use of lead based powders and heavy-metal oxide composites.

Delayed intramuscular human neurotrophin-3 improves recovery in adult and elderly rats after stroke

Duricki et al. show that intramuscular delivery of human neurotrophin-3 induces corticospinal plasticity and locomotor recovery in adult and elderly rats 24 hours post-stroke. This time-frame would be clinically feasible for most stroke victims, and the safety and tolerability of neurotrophin-3 in humans have been established for other disorders.

Whole-brain ex-vivo quantitative MRI of the cuprizone mouse model

Myelin is a critical component of the nervous system and a major contributor to contrast in Magnetic Resonance (MR) images. However, the precise contribution of myelination to multiple MR modalities is still under debate. The cuprizone mouse is a well-established model of demyelination that has been used in several MR studies, but these have often imaged only a single slice and analysed a small region of interest in the corpus callosum. We imaged and analyzed the whole brain of the cuprizone mouse ex-vivo using high-resolution quantitative MR methods (multi-component relaxometry, Diffusion Tensor Imaging (DTI) and morphometry) and found changes in multiple regions, including the corpus callosum, cerebellum, thalamus and hippocampus. The presence of inflammation, confirmed with histology, presents difficulties in isolating the sensitivity and specificity of these MR methods to demyelination using this model.

Proteomic analysis of rat plasma following transient focal cerebral ischemia

AIM This study aimed to identify plasma protein changes in a rat model of ischemic stroke using a proteomic approach. MATERIALS & METHODS Four male Sprague-Dawley rats (3-6 months old) were subjected to 90 min of left middle cerebral artery occlusion under anesthesia with 1.5% isoflurane in O(2)/air followed by 24-h reperfusion. Blood samples (~100 µl) were collected at baseline, at the end of 90-min middle cerebral artery occlusion and at 24-h postreperfusion. Brain injuries were assessed by MRI at 24-h postreperfusion. Quantitative comparison of global plasma protein expression was performed using 2D differential in-gel electrophoresis. Differentially expressed protein spots were identified using peptide sequencing tandem mass spectrometry. RESULTS These rats had clear brain infarction in the left hemisphere detected by MRI. Thirty-three protein spots of plasma samples were differentially expressed following focal cerebral ischemia/reperfusion. These protein spots belonged to eight proteins. Six of them (α2-macroglobulin, complement C3, inter-α- trypsin inhibitor heavy chain H3, serum albumin, haptoglobin and transthyretin), which are a class of acute-phase proteins, changed significantly. CONCLUSION This study describes the responses of young rats to focal cerebral ischemia and suggests that future studies should use aged animals to better mimic the clinical ischemic stroke setting.