Christina H. Liu

Microemboli may link spreading depression, migraine aura, and patent foramen ovale

Patent foramen ovale and pulmonary arteriovenous shunts are associated with serious complications such as cerebral emboli, stroke, and migraine with aura. The pathophysiological mechanisms that link these conditions are unknown. We aimed to establish a mechanism linking microembolization to migraine aura in an experimental animal model.

Migraine Mutations Increase Stroke Vulnerability by Facilitating Ischemic Depolarizations

Background— Migraine is an independent risk factor for stroke. Mechanisms underlying this association are unclear. Familial hemiplegic migraine (FHM), a migraine subtype that also carries an increased stroke risk, is a useful model for common migraine phenotypes because of shared aura and headache features, trigger factors, and underlying glutamatergic mechanisms. Methods and Results— Here, we show that FHM type 1 (FHM1) mutations in CaV2.1 voltage-gated Ca2+ channels render the brain more vulnerable to ischemic stroke. Compared with wild-type mice, 2 FHM1 mutant mouse strains developed earlier onset of anoxic depolarization and more frequent peri-infarct depolarizations associated with rapid expansion of infarct core on diffusion-weighted magnetic resonance imaging and larger perfusion deficits on laser speckle flowmetry. Cerebral blood flow required for tissue survival was higher in the mutants, leading to infarction with milder ischemia. As a result, mutants developed larger infarcts and worse neurological outcomes after stroke, which were selectively attenuated by a glutamate receptor antagonist. Conclusions— We propose that enhanced susceptibility to ischemic depolarizations akin to spreading depression predisposes migraineurs to infarction during mild ischemic events, thereby increasing the stroke risk.

Exogenous contrast agent improves sensitivity of gradient-echo functional magnetic resonance imaging at 9.4 T.

Relative to common clinical magnetic field strengths, higher fields benefit functional brain imaging both by providing additional signal for high‐resolution applications and by improving the sensitivity of endogenous contrast due to the blood oxygen level dependent (BOLD) mechanism, which has limited detection power at low magnetic fields relative to the use of exogenous contrast agent. This study evaluates the utility of iron oxide contrast agent for gradient echo functional MRI at 9.4 T in rodents using cocaine and methylphenidate as stimuli. Relative to the BOLD method, the use of high iron doses and short echo times provided a roughly twofold global increase in functional sensitivity, while also suppressing large vessel signal and reducing susceptibility artifacts. Furthermore, MRI measurements of the functional percentage change in cerebral blood volume (CBV) showed excellent agreement with results obtained at much lower magnetic field strengths, demonstrating that MRI estimates of this quantity are roughly independent of magnetic field when appropriate techniques are employed. The derived field dependencies for relative sensitivity and MRI estimates of the percentage change in CBV suggest that the benefits provided by exogenous agents will persist even at much higher magnetic fields than 9.4 T. Magn Reson Med 52:1272–1281, 2004.

Inhaled Nitric Oxide Improves Outcomes After Successful Cardiopulmonary Resuscitation in Mice

Background— Sudden cardiac arrest (CA) is a leading cause of death worldwide. Breathing nitric oxide (NO) reduces ischemia/reperfusion injury in animal models and in patients. The objective of this study was to learn whether inhaled NO improves outcomes after CA and cardiopulmonary resuscitation (CPR). Methods and Results— Adult male mice were subjected to potassium-induced CA for 7.5 minutes whereupon CPR was performed with chest compression and mechanical ventilation. One hour after CPR, mice were extubated and breathed air alone or air supplemented with 40 ppm NO for 23 hours. Mice that were subjected to CA/CPR and breathed air exhibited a poor 10-day survival rate (4 of 13), depressed neurological and left ventricular function, and increased caspase-3 activation and inflammatory cytokine induction in the brain. Magnetic resonance imaging revealed brain regions with marked water diffusion abnormality 24 hours after CA/CPR in mice that breathed air. Breathing air supplemented with NO for 23 hours starting 1 hour after CPR attenuated neurological and left ventricular dysfunction 4 days after CA/CPR and markedly improved 10-day survival rate (11 of 13; P=0.003 versus mice breathing air). The protective effects of inhaled NO on the outcome after CA/CPR were associated with reduced water diffusion abnormality, caspase-3 activation, and cytokine induction in the brain and increased serum nitrate/nitrite levels. Deficiency of the &agr;1 subunit of soluble guanylate cyclase, a primary target of NO, abrogated the ability of inhaled NO to improve outcomes after CA/CPR. Conclusions— These results suggest that NO inhalation after CA and successful CPR improves outcome via soluble guanylate cyclase–dependent mechanisms.

Imaging Cerebral Gene Transcripts in Live Animals

To circumvent the limitations of using postmortem brain in molecular assays, we used avidin–biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15–20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and β-actin mRNA (SPION-cfos and SPION-βactin, respectively) (14–22 nm). The Stern–Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 × 106/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 μg of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T2* or R2* maps (R2* = 1/T2*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T2* signal in the right cortex and striatum; retention of SPION-cfos and SPION-βactin positively correlated with c-fos and β-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.

Direct CSF injection of MnCl2 for dynamic manganese-enhanced MRI

MnCl2 was injected intrathecally through the cisterna magna in rats, allowing infusion of divalent manganese ions (Mn++) into the CSF space and thence into the brain, without breaking the blood–brain barrier (BBB). Mn++ uptake and washout dynamics in the brain were measured by serial T1‐weighted MRI and EPI T1 and T2 mapping for up to 3 weeks after injection. Observations within the first 6 hr after injection demonstrated anterograde and bilateral distribution of the Mn++ within the CSF space, from the olfactory bulb and frontal cortex to the brain stem. Enhancement increased in most brain areas up to 4 days after injection, and then slowly decreased. Relaxation maps at each time point demonstrated higher concentrations of Mn in basal ganglia. Residual concentrations were still observable after 3 weeks in all brain regions. With the use of MnCl2 calibration phantoms, the maximum Mn concentration in the brain was estimated to be approximately 27 ± 16 μM, corresponding to changes in relaxation rates of 0.49 ± 0.30 s−1 for R1 and 3.9 ± 2.4 s−1 for R2. For comparison, an intrathecal GdDTPA injection was performed. This injection showed different distribution dynamics: it remained chiefly within the CSF spaces, and was largely washed out after 1 day. This method shows promise as a means of supplying Mn++ uniformly to the whole brain for a variety of chronic functional activation studies. Magn Reson Med 51:978–987, 2004.

MR contrast probes that trace gene transcripts for cerebral ischemia in live animals

The aim of this research was to validate transcription magnetic resonance (MR) imaging (MRI) for gene transcript targeting in acute neurological disorders in live subjects. We delivered three MR probe variants with superparamagnetic iron oxide nanopar‐ticles (SPION’ a T2 susceptibility agent) linked to a phosphorothioate‐modified oligodeoxynucleotide (sODN) complementary to c‐fos mRNA (SPION‐cfos) or β‐actin mRNA (SPION‐β‐actin) and to sODN with random sequence (SPION‐Ran). Each probe (1 μgFe in 2 μl) was delivered via intracerebroventricular infusion to the left cerebral ventricle of male C57Black6 mice. We demonstrated SPION retention’ measured as decreased T2* signal or increased R2* value (R2* = 1/ T2*). Animals that received the SPION‐β‐actin probe exhibited the highest R2* values’ followed (in descending order) by SPION‐cfos and SPION‐Ran. SPION‐cfos retention was localiZed in brain regions where SPION‐cfos was present and where hybrids of SPION‐cfos and its target c‐fos mRNA were detected by in situ reverse transcription PCR. In animals that experienced cerebral ischemia, SPION‐cfos retention was significantly increased in locations where c‐fos mRNA increased in response to the ischemic insult;these elevations were not observed for SPION‐β‐actin and SPION‐Ran. This study should enable MR detection of mRNA alteration in disease models of the central nervous system.—Liu C. H., Huang, S., Cui, J., Kim, Y. R., Farrar, C. T., Moskowitz M. A., Rosen B. R., Liu P. K. MR contrast probes that trace gene transcripts for cerebral ischemia in live animals. FASEB J. 21, 3004–3015 (2007)

Three-Dimensional High-Resolution Diffusion Tensor Imaging and Tractography of the Developing Rabbit Brain

Diffusion tensor imaging (DTI) is sensitive to structural ordering in brain tissue particularly in the white matter tracts. Diffusion anisotropy changes with disease and also with neural development. We used high-resolution DTI of fixed rabbit brains to study developmental changes in regional diffusion anisotropy and white matter fiber tract development. Imaging was performed on a 4.7-tesla Bruker Biospec Avance scanner using custom-built solenoid coils and DTI was performed at various postnatal ages. Trace apparent diffusion coefficient, fractional diffusion anisotropy maps and fiber tracts were generated and compared across the ages. The brain was highly anisotropic at birth and white matter anisotropy increased with age. Regional DTI tractography of the internal capsule showed refinement in regional tract architecture with maturation. Interestingly, brains with congenital deficiencies of the callosal commissure showed selectively strikingly different fiber architecture compared to age-matched brains. There was also some evidence of subcortical to cortical fiber connectivity. DTI tractography of the anterior and posterior limbs of the internal capsule showed reproducibly coherent fiber tracts corresponding to known corticospinal and corticobulbar tract anatomy. There was some minor interanimal tract variability, but there was remarkable similarity between the tracts in all animals. Therefore, ex vivo DTI tractography is a potentially powerful tool for neuroscience investigations and may also reveal effects (such as fiber tract pruning during development) which may be important targets for in vivo human studies.

Noninvasive delivery of gene targeting probes to live brains for transcription MRI

We aimed to test the feasibility of detecting gliosis in living brains when the blood‐brain barrier (BBB) is disrupted. We designed a novel magnetic resonance (MR) probe that contains superparamag‐netic iron oxide nanoparticles (SPION, a T2 susceptibility contrast agent) linked to a short DNA sequence complementary to the cerebral mRNA of glial fibrillary acidic protein (GFAP) found in glia and astrocytes. As a control, we also used a sequence complementary to the mRNA of β‐actin. Our objectives are to demonstrate that this new probe, SPION‐gfap, could be delivered to the brain when administered by eyedrop solution to the conjunctival sac. We induced BBB leakage by puncture wound, global cerebral ischemia, and cortical spreading depression in C57BL6 mice;1 day after probe delivery we acquired T2* MR images and R2* (R2* = 1/T2*) maps using a transcription MRI technique in live mice. We found that the SPION‐gfap probe reported foci with elevated signal in subtraction R2* maps and that these foci matched areas identified as having extensive glial network (gliosis) in postmortem immunohistochemistry. Similarly, animals adminis‐tered the control probe exhibited foci of R2* elevation that matched β‐actin‐expressing endothelia in the vascular wall. We conclude that our modular MR probe, delivered in an eyedrop solution, effectively reports gliosis associated with acute neurological disorders in living animals. As BBB leakage is often observed in acute neurological disorders, this study also served to validate noninvasive delivery of MR probes to the brains of live animals after acute neurological disorders. Liu C. H., You, Z., Ren JQ., Kim, Y. R., Eikermann‐Haerter, K., Liu P. K. Noninvasive delivery of gene targeting probes to live brains for transcription MRI. FASEB J. 22, 1193–1203 (2008)

Manipulation of tissue contrast using contrast agents for enhanced MR microscopy in ex vivo mouse brain.

Detailed 3D mouse brain images may promote better understanding of phenotypical differences between normal and transgenic/mutant mouse models. Previously, a number of magnetic resonance microscopy (MRM) studies have successfully established brain atlases, revealing genotypic traits of several commonly used mouse strains. In such studies, MR contrast agents, mainly gadolinium (Gd) based, were often used to reduce acquisition time and improve signal-to-noise ratio (SNR). In this paper, we intended to extend the utility of contrast agents for MRM applications. Using Gd-DTPA and MnCl(2), we exploited the potential use of MR contrast agents to manipulate image contrast by drawing upon the multiple relaxation mechanisms and tissue-dependent staining properties characteristic of each contrast agent. We quantified r(1) and r(2) of Gd-DTPA and MnCl(2) in both aqueous solution and brain tissue and demonstrated the presence of divergent relaxation mechanisms between solution and tissue for each contrast agent. Further analyses using nuclear magnetic resonance dispersion (NMRD) of Mn(2+) in ex vivo tissue strongly suggested macromolecule binding of Mn(2+), leading to increased T(1) relaxation. Moreover, inductively coupled plasma (ICP) mass spectroscopy revealed that MnCl(2) had higher tissue affinity than Gd-DTPA. As a result, multiple regions of the brain stained by the two agents exhibited different image contrasts. Our results show that differential MRM staining can be achieved using multiple MR contrast agents, revealing detailed cytoarchitecture, and may ultimately offer a window for investigating new techniques by which to understand biophysical MR relaxation mechanisms and perhaps to visualize tissue anomalies even at the molecular level.