Vaccinium bracteatum Leaf Extract Reverses Chronic Restraint Stress-Induced Depression-Like Behavior in Mice: Regulation of Hypothalamic-Pituitary-Adrenal Axis, Serotonin Turnover Systems, and ERK/Akt Phosphorylation.

The leaves of Vaccinium bracteatum Thunb. are a source of traditional herbal medicines found in East Asia. The present study aimed to evaluate the mechanisms underlying the antidepressant-like effects of water extract of V. bracteatum Thunb. leaves (VBLW) in a mouse model of chronic restraint stress (CRS) and to identify the possible molecular in vitro mechanisms of the neuroprotective effects. The CRS-exposed mice were orally administered VBLW (100 and 200 mg/kg) daily for 21 days consecutively. The behavioral effects of VBLW were assessed through the forced swim test (FST) and the open field test (OFT). The levels of serum corticosterone (CORT), corticotropin releasing hormone (CRH), and adrenocorticotropin hormone (ACTH), brain monoamines, such as serotonin, dopamine, and norepinephrine, and serotonin turnover by tryptophan hydroxylase 2 (TPH2), serotonin reuptake (SERT), and monoamine oxidase A (MAO-A) were evaluated, in addition to the extracellular signal-regulated kinases (ERKs)/protein kinase B (Akt) signaling pathway. CRS-exposed mice treated with VBLW (100 and 200 mg/kg) showed significantly reduced immobility time and increased swimming and climbing times in the FST, and increased locomotor activity in the OFT. Moreover, CRS mice treated with VBLW exhibited significantly decreased CORT and ACTH, but enhanced brain monoamine neurotransmitters. In addition, CRS mice treated with VBLW had dramatically decreased protein levels of MAO-A and SERT, but increased TPH2 protein levels in the hippocampus and the PFC. Similarly, VBLW significantly upregulated the ERKs/Akt signaling pathway in the hippocampus and the PFC. Furthermore, VBLW showed neuroprotective effects via increased CREB phosphorylation in CORT-induced cell injury that were mediated through the ERK/Akt/mTOR signaling pathways. These results suggested that the antidepressant-like effects of VBLW might be mediated by the regulation of the HPA axis, glucocorticoids, and serotonin turnover, such as TPH2, SERT, and MAO-A, as well as the concentration of monoamine neurotransmitters, and the activities of ERK and Akt phosphorylation, which were possibly associated with neuroprotective effects.

Molecular MR imaging of fibrosis in a mouse model of pancreatic cancer.

Fibrosis with excessive amounts of type I collagen is a hallmark of many solid tumours, and fibrosis is a promising target in cancer therapy, but tools for its non-invasive quantification are missing. Here we used magnetic resonance imaging with a gadolinium-based probe targeted to type I collagen (EP-3533) to image and quantify fibrosis in pancreatic ductal adenocarcinoma. An orthotopic syngeneic mouse model resulted in tumours with 2.3-fold higher collagen level compared to healthy pancreas. Animals were scanned at 4.7 T before, during and up to 60 min after i.v. injection of EP-3533, or of its non-binding isomer EP-3612. Ex-vivo quantification of gadolinium showed significantly higher uptake of EP-3533 compared to EP-3612 in tumours, but not in surrounding tissue (blood, muscle). Uptake of EP-3533 visualized in T1-weighted MRI correlated well with spatial distribution of collagen determined by second harmonic generation imaging. Differences in the tumour pharmacokinetic profiles of EP-3533 and EP-3612 were utilized to distinguish specific binding to tumour collagen from non-specific uptake. A model-free pharmacokinetic measurement based on area under the curve was identified as a robust imaging biomarker of fibrosis. Collagen-targeted molecular MRI with EP-3533 represents a new tool for non-invasive visualization and quantification of fibrosis in tumour tissue.

Enhanced Thalamic Functional Connectivity with No fMRI Responses to Affected Forelimb Stimulation in Stroke-Recovered Rats.

Neurological recovery after stroke has been extensively investigated to provide better understanding of neurobiological mechanism, therapy, and patient management. Recent advances in neuroimaging techniques, particularly functional MRI (fMRI), have widely contributed to unravel the relationship between the altered neural function and stroke-affected brain areas. As results of previous investigations, the plastic reorganization and/or gradual restoration of the hemodynamic fMRI responses to neural stimuli have been suggested as relevant mechanisms underlying the stroke recovery process. However, divergent study results and modality-dependent outcomes have clouded the proper interpretation of variable fMRI signals. Here, we performed both evoked and resting state fMRI (rs-fMRI) to clarify the link between the fMRI phenotypes and post-stroke functional recovery. The experiments were designed to examine the altered neural activity within the contra-lesional hemisphere and other undamaged brain regions using rat models with large unilateral stroke, which despite the severe injury, exhibited nearly full recovery at ∼6 months after stroke. Surprisingly, both blood oxygenation level-dependent and blood volume-weighted (CBVw) fMRI activities elicited by electrical stimulation of the stroke-affected forelimb were completely absent, failing to reveal the neural origin of the behavioral recovery. In contrast, the functional connectivity maps showed highly robust rs-fMRI activity concentrated in the contra-lesional ventromedial nucleus of thalamus (VM). The negative finding in the stimuli-induced fMRI study using the popular rat middle cerebral artery model denotes weak association between the fMRI hemodynamic responses and neurological improvement. The results strongly caution the indiscreet interpretation of stroke-affected fMRI signals and demonstrate rs-fMRI as a complementary tool for efficiently characterizing stroke recovery.

Layer-specific interhemispheric functional connectivity in the somatosensory cortex of rats: resting state electrophysiology and fMRI studies.

The spontaneous cerebral hemodynamic fluctuations observed during the resting state have been frequently visualized using functional magnetic resonance imaging (rsfMRI). However, the neuronal populations and neuroelectric characteristics underlying the functional connectivity of cerebrohemodynamic activities are poorly understood. We investigated the characteristics of bi-hemispheric functional connectivity via electrophysiology and rsfMRI in the primary sensory cortex of rats anesthetized by α-chloralose. Unlike the evoked responses, the spontaneous electrophysiological activity was concentrated in the infragranular layers and could be classified into subtypes with distinctive current sources and sinks. Both neuroelectric and rsfMRI signals were interhemispherically correlated in a layer-specific manner, suggesting that there are independent neural inputs to infragranular and granular/supragranular layers. The majority of spontaneous electrophysiological activities were bilaterally paired with delays of up to ~50 ms between each pair. The variable interhemispheric delay implies the involvement of indirect, multi-neural pathways. Our findings demonstrated the diverse activity patterns of layer-specific electrophysiological substrates and suggest the recruitment of multiple, non-specific brain regions in construction of interhemispheric functional connectivity.

Reduced microvascular volume and hemispherically deficient vasoreactivity to hypercapnia in acute ischemia: MRI study using permanent middle cerebral artery occlusion rat model.

Vasoreactivity to hypercapnia has been used for assessing cerebrovascular tone and control altered by ischemic stroke. Despite the high prognostic potential, traits of hypercapnia-induced hemodynamic changes have not been fully characterized in relation with baseline vascular states and brain tissue damage. To monitor cerebrovascular responses, T2- and T2*-weighted magnetic resonance imaging (MRI) images were acquired alternatively using spin- and gradient-echo echo plannar imaging (GESE EPI) sequence with 5% CO2 gas inhalation in normal (n=5) and acute stroke rats (n=10). Dynamic relative changes in cerebrovascular volume (CBV), microvascular volume (MVV), and vascular size index (VSI) were assessed from regions of interest (ROIs) delineated by the percent decrease of apparent diffusion coefficient (ADC). The baseline CBV was not affected by middle cerebral artery occlusion (MCAO) whereas the baseline MVV in ischemic areas was significantly lower than that in the rest of the brain and correlated with ADC. Vasoreactivity to hypercapnic challenge was considerably attenuated in the entire ipsilesional hemisphere including normal ADC regions, in which unsolicited, spreading depression-associated increases of CBV and MVV were observed. The lesion-dependent inhomogeneity in baseline MVV indicates the effective perfusion reserve for accurately delineating the true ischemic damage while the cascade of neuronal depolarization is probably responsible for the hemispherically lateralized changes in overall neurovascular physiology.

Response of the primary auditory and non-auditory cortices to acoustic stimulation: a manganese-enhanced MRI study.

Structural and functional features of various cerebral cortices have been extensively explored in neuroscience research. We used manganese-enhanced MRI, a non-invasive method for examining stimulus-dependent activity in the whole brain, to investigate the activity in the layers of primary cortices and sensory, such as auditory and olfactory, pathways under acoustic stimulation. Male Sprague-Dawley rats, either with or without exposure to auditory stimulation, were scanned before and 24-29 hour after systemic MnCl2 injection. Cortex linearization and layer-dependent signal extraction were subsequently performed for detecting layer-specific cortical activity. We found stimulus-dependent activity in the deep layers of the primary auditory cortex and the auditory pathways. The primary sensory and visual cortices also showed the enhanced activity, whereas the olfactory pathways did not. Further, we performed correlation analysis of the signal intensity ratios among different layers of each cortex, and compared the strength of correlations between with and without the auditory stimulation. In the primary auditory cortex, the correlation strength between left and right hemisphere showed a slight but not significant increase with the acoustic simulation, whereas, in the primary sensory and visual cortex, the correlation coefficients were significantly smaller. These results suggest the possibility that even though the primary auditory, sensory, and visual cortices showed enhanced activity to the auditory stimulation, these cortices had different associations for auditory processing in the brain network.

Dynamic monitoring of blood-brain barrier integrity using water exchange index (WEI) during mannitol and CO2 challenges in mouse brain.

The integrity of the blood-brain barrier (BBB) is critical to normal brain function. Traditional techniques for the assessment of BBB disruption rely heavily on the spatiotemporal analysis of extravasating contrast agents. However, such methods based on the leakage of relatively large molecules are not suitable for the detection of subtle BBB impairment or for the performance of repeated measurements in a short time frame. Quantification of the water exchange rate constant (WER) across the BBB using strictly intravascular contrast agents could provide a much more sensitive method for the quantification of the BBB integrity. To estimate WER, we have recently devised a powerful new method using a water exchange index (WEI) biomarker and demonstrated BBB disruption in an acute stroke model. Here, we confirm that WEI is sensitive to even very subtle changes in the integrity of the BBB caused by: (i) systemic hypercapnia and (ii) low doses of a hyperosmolar solution. In addition, we have examined the sensitivity and accuracy of WEI as a biomarker of WER using computer simulation. In particular, the dependence of the WEI-WER relation on changes in vascular blood volume, T1 relaxation of cellular magnetization and transcytolemmal water exchange was explored. Simulated WEI was found to vary linearly with WER for typically encountered exchange rate constants (1-4 Hz), regardless of the blood volume. However, for very high WER (>5 Hz), WEI became progressively more insensitive to increasing WER. The incorporation of transcytolemmal water exchange, using a three-compartment tissue model, helped to extend the linear WEI regime to slightly higher WER, but had no significant effect for most physiologically important WERs (WER < 4 Hz). Variation in cellular T1 had no effect on WEI. Using both theoretical and experimental approaches, our study validates the utility of the WEI biomarker for the monitoring of BBB integrity.

Frequency distribution of causal connectivity in rat sensorimotor network: resting-state fMRI analyses.

Resting-state functional MRI (fMRI) has emerged as an important method for assessing neural networks, enabling extensive connectivity analyses between multiple brain regions. Among the analysis techniques proposed, partial directed coherence (PDC) provides a promising tool to unveil causal connectivity networks in the frequency domain. Using the MRI time series obtained from the rat sensorimotor system, we applied PDC analysis to determine the frequency-dependent causality networks. In particular, we compared in vivo and postmortem conditions to establish the statistical significance of directional PDC values. Our results demonstrate that two distinctive frequency populations drive the causality networks in rat; significant, high-frequency causal connections clustered in the range of 0.2-0.4 Hz, and the frequently documented low-frequency connections <0.15 Hz. Frequency-dependence and directionality of the causal connection are characteristic between sensorimotor regions, implying the functional role of frequency bands to transport specific resting-state signals. In particular, whereas both intra- and interhemispheric causal connections between heterologous sensorimotor regions are robust over all frequency levels, the bilaterally homologous regions are interhemispherically linked mostly via low-frequency components. We also discovered a significant, frequency-independent, unidirectional connection from motor cortex to thalamus, indicating dominant cortical inputs to the thalamus in the absence of external stimuli. Additionally, to address factors underlying the measurement error, we performed signal simulations and revealed that the interactive MRI system noise alone is a likely source of the inaccurate PDC values. This work demonstrates technical basis for the PDC analysis of resting-state fMRI time series and the presence of frequency-dependent causality networks in the sensorimotor system.

Sensitivity of MRI tumor biomarkers to VEGFR inhibitor therapy in an orthotopic mouse glioma model.

MRI biomarkers of tumor edema, vascular permeability, blood volume, and average vessel caliber are increasingly being employed to assess the efficacy of tumor therapies. However, the dependence of these biomarkers on a number of physiological factors can compromise their sensitivity and complicate the assessment of therapeutic efficacy. Here we examine the response of these MRI tumor biomarkers to cediranib, a potent vascular endothelial growth factor receptor (VEGFR) inhibitor, in an orthotopic mouse glioma model. A significant increase in the tumor volume and relative vessel caliber index (rVCI) and a slight decrease in the water apparent diffusion coefficient (ADC) were observed for both control and cediranib treated animals. This contrasts with a clinical study that observed a significant decrease in tumor rVCI, ADC and volume with cediranib therapy. While the lack of a difference between control and cediranib treated animals in these biomarker responses might suggest that cediranib has no therapeutic benefit, cediranib treated mice had a significantly increased survival. The increased survival benefit of cediranib treated animals is consistent with the significant decrease observed for cediranib treated animals in the relative cerebral blood volume (rCBV), relative microvascular blood volume (rMBV), transverse relaxation time (T2), blood vessel permeability (K(trans)), and extravascular-extracellular space (ν(e)). The differential response of pre-clinical and clinical tumors to cediranib therapy, along with the lack of a positive response for some biomarkers, indicates the importance of evaluating the whole spectrum of different tumor biomarkers to properly assess the therapeutic response and identify and interpret the therapy-induced changes in the tumor physiology.

In vivo validation of MRI vessel caliber index measurement methods with intravital optical microscopy in a U87 mouse brain tumor model.

The vessel caliber index (VCI), a magnetic resonance imaging biomarker of the average blood vessel diameter, is increasingly being used as a tool for assessing tumor angiogenesis and response to antiangiogenic therapy. However, although the VCI has been correlated with histological vessel diameters, good quantitative agreement with histology has been lacking. In addition, no VCI validation studies have been performed in vivo where the structural deformations frequently associated with histological tissue preparation are not present. This study employs intravital optical microscopy (IVM) measurements of cerebral blood vessel diameters in a mouse orthotopic glioma model to provide the first such in vivo validation. Two VCI correlation models, both a linear and a 3/2-power dependence on the DeltaR2*/DeltaR2 ratio, were compared with the IVM data. The linear VCI model, determined from steady-state susceptibility contrast (SSC) images, was found to be in excellent quantitative agreement with the intravitally determined VCI for separate tumor size matched groups of mice. In addition, preliminary data indicate that the VCI is independent of whether a dynamic susceptibility contrast or SSC measurement method is used.

Diffusion-weighted magnetic resonance imaging reversal by gene knockdown of matrix metalloproteinase-9 activities in live animal brains.

The involvement of matrix metalloproteinase-9 (MMP-9) activities in the development of abnormal water diffusion in the brain after cardiac arrest is not fully understood. We used magnetic resonance imaging to determine the correlation between MMP-9 activity and the mechanism of abnormal water diffusion after global cerebral ischemia (GCI)-induced brain damage in C57black6 mice. We induced GCI in mice by occluding both carotid arteries for 60 min, then allowing reperfusion. We labeled a short DNA that targets mmp-9 mRNA activity [phosphorothioate-modified oligodeoxynucleotide (sODN)-mmp9] or a control probe without intracellular target (sODN-Ran) with iron-based MR contrast agent [superparamagnetic iron oxide nanoparticle (SPION)-mmp9 or SPION-Ran] or fluorescein isothiocyanate (FITC)-sODN-mmp9 or FITC-sODN-Ran; we then delivered these probes by intracerebroventricular infusion or intraperitoneal injection within 3 h of reperfusion. At low dose (120 pmol/kg) the SPION-mmp9 probe was retained at significant levels in the striatum and cortex of living brains 10 h after GCI. Probe retention was validated by similar elevation of mmp-9 mRNA and antigens in postmortem samples taken from regions that exhibited GCI-induced hyperintensity in diffusion-weighted imaging, and a significant reduction in apparent diffusion coefficient (rADC, p = 0.0006, n = 12). At a higher dose (120 nmol/kg), the FITC-sODN-mmp9 probe revealed significant knockdown of MMP-9 activity, per zymography, and a reversal of striatal rADC (p = 0.004, n = 6). These observations were not duplicated in the control group. We conclude that expression of mmp-9 mRNA is associated with abnormal ADC after GCI.

Functional MRI of delayed chronic lithium treatment in rat focal cerebral ischemia.

BACKGROUND AND PURPOSE: The use of lithium as a neuroprotective agent has been demonstrated using various models in which improvements in infarct size, DNA damage, and neurological function were reported. We further investigated neurohemodynamic aspects of the treatment-associated recovery by assessing the therapeutic efficacy of delayed chronic lithium treatment using functional MRI. METHODS: Ipsilesional functional MRI activations in the somatosensory cortex, acquired 2 weeks after the 90-minute transient middle cerebral artery occlusion, were compared between lithium- and saline-treated rats. Specifically, MRI signal changes based on blood oxygenation level dependence and functional cerebral blood volume responses were examined using electrical stimulation of forelimbs. Additional immunohistochemical assays were performed. RESULTS: The ratio of ipsilesional to contralesional blood oxygenation level dependence response magnitudes significantly improved with lithium treatments. In contrast, the increase of the functional cerebral blood volume response magnitude ratio was not statistically significant. Nonetheless, the lithium treatment induced significant enhancements of total functional MRI activation (defined as a product of activation volume and response magnitude) for both blood oxygenation level dependence and functional cerebral blood volume methods. Increased cerebral blood volume in periinfarct tissues suggests a possible stroke-induced vascular transformation in both saline- and lithium-treated rats; however, other MRI-derived vascular parameters (vascular size index and microvascular volume) and immunohistochemical staining (CD31, glia fibrillary-associated protein, and matrix metalloproteinase-9) may imply that the neoformation of vasculature was differently affected by the lithium treatment. CONCLUSIONS: The delayed chronic lithium treatment enhanced the blood oxygenation level dependence functional MRI response magnitude in the absence of neurological improvement and influenced vascular formation in poststroke animal models.

Transcription MRI: a new view of the living brain.

Altered gene activities are underlying causes of many neurological disorders. The ability to detect, image, and report endogenous gene transcription using magnetic resonance (MR) holds great potential for providing significant clinical benefits. In this review, we present the development of conjugates consisting of gene-targeting short nucleic acids (oligodeoxynucleotides, or sODN) and superparamagnetic iron oxide nanoparticles (SPION, an MR susceptibility T(2) agent) for reporting gene activity using transcription MRI (tMRI). We will discuss 1) the target specificity of sODN, 2) selection of contrast agents for tMRI, 3) the distribution and uptake, 4) sequence specificity, 5) histology of SPION and sODN, 6) data acquisition and quantitative analysis for tMRI, and 7) application of gene transcript-targeting nanoparticles in biology and medicine. We will also discuss methods of validating the correlation between results from conventional assays (in situ hybridization, PCR, histology Prussian blue stain and immunohistochemistry) in postmortem samples and retention of SPION-sODN using tMRI. The application of our novel contrast probe to report and target gene transcripts in the mesolimbic pathways of living mouse brains after amphetamine exposure will be discussed. Because of the targeting ability in the nucleic acid sequence, the concept of tMRI probes with complementary nucleic acid (antisense DNA or short interfering RNA) allows not only tracking, targeting, binding to intracellular mRNA, and manipulating gene action but also tracing cells with specific gene action in living brains. Transcription MRI will lend itself to myriad applications in living organs.

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 superparamagnetic 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 beta-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 administered the control probe exhibited foci of R2* elevation that matched beta-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.

Forebrain ischemia-reperfusion simulating cardiac arrest in mice induces edema and DNA fragmentation in the brain.

Brain injury affects one-third of persons who survive after heart attack, even with restoration of spontaneous circulation by cardiopulmonary resuscitation. We studied brain injury resulting from transient bilateral carotid artery occlusion (BCAO) and reperfusion by simulating heart attack and restoration of circulation, respectively, in live C57Black6 mice. This model is known to induce neuronal death in the hippocampus, striatum, and cortex. We report the appearance of edema after transient BCAO of 60 minutes and 1 day of reperfusion. Hyperintensity in diffusion-weighted magnetic resonance imaging (MRI) was detectable in the striatum, thalamus, and cortex but not in the hippocampus. To determine whether damage to the hippocampus can be detected in live animals, we infused a T(2) susceptibility magnetic resonance contrast agent (superparamagnetic iron oxide nanoparticles [SPIONs]) that was linked to single-stranded deoxyribonucleic acid (DNA) complementary in sequence to c-fos messenger ribonucleic acid (SPION-cfos); we acquired in vivo T(2)*-weighted MRI 3 days later. SPION retention was measured as T(2)* (milliseconds) signal reduction or R(2)* value (s(-1)) elevation. We found that animals treated with 60-minute BCAO and 7-day reperfusion exhibited significantly less SPION retention in the hippocampus and cortex than sham-operated animals. These findings suggest that brain injury induced by cardiac arrest can be detected in live animals.

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 nanoparticles (SPION, a T2 susceptibility agent) linked to a phosphorothioate-modified oligodeoxynucleotide (sODN) complementary to c-fos mRNA (SPION-cfos) or beta-actin mRNA (SPION-beta-actin) and to sODN with random sequence (SPION-Ran). Each probe (1 microg Fe in 2 microl) 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-beta-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-beta-actin and SPION-Ran. This study should enable MR detection of mRNA alteration in disease models of the central nervous system.

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 beta-actin mRNA (SPION-cfos and SPION-beta-actin, respectively) (14-22 nm). The Stern-Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 x 10(6)/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 mug 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 T(2)* or R(2)* maps (R(2)* = 1/T(2)*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T(2)* signal in the right cortex and striatum; retention of SPION-cfos and SPION-beta-actin positively correlated with c-fos and beta-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.

fMRI of delayed albumin treatment during stroke recovery in rats: implication for fast neuronal habituation in recovering brains.

Accumulating experimental and clinical data suggest that albumin may be neuroprotective for stroke. Here, we use functional magnetic resonance imaging (fMRI) to evaluate the therapeutic efficacy of albumin and its effects on the recovery of stimuli-induced cerebral hemodynamics. For this purpose, fMRI activity in the ipsilesional somatosensory (SS) cortex was assessed using a well established rat model of transient 90 min focal ischemia and electrical forelimb stimulation. Rats were treated with either saline or albumin via intracerebroventricular injections at 12 h post-stroke onset. Despite this delayed treatment time, when compared to the saline-treated rats (n=7), there were significant enhancements of the fMRI activation in the albumin-treated rats (n=6) for both blood oxygenation level dependence (BOLD) and functional cerebral blood volume (fCBV) responses. Interestingly, the temporal characteristics of the ipsilesional SS BOLD responses in the albumin-treated rats appeared considerably altered compared to those of contralesional responses while such temporal alterations were not pronounced for the fCBV responses. These characteristic fMRI temporal profiles of the albumin-treated brains may be due to altered neuronal responses rather than altered integrity of neurovascular coupling, which implies an unusually fast habituation of neuronal responses in the lesional SS cortex. The correlation between various MRI-derived structural parameters and the fMRI response magnitude was also characteristic for albumin and control groups. Taken together, these data suggest that restoration of fMRI response magnitudes, temporal profiles, and correlations with structure may reveal the extent and specific traits of albumin treatment associated stroke recovery.

High resolution spatial mapping of nicotine action using pharmacologic magnetic resonance imaging.

Nicotine is one of the most addictive substances known. To better understand the mechanisms of action, we mapped the regional brain response to nicotine administration using pharmacologic magnetic resonance imaging (phMRI) in rats. We measured the regional response of relative cerebral blood volume (rCBV) in rats to a challenge of 0.07 mg/kg (0.43 micromol/kg) of nicotine. The areas of the brain with significant and reproducible changes in the rCBV response were (in descending order of magnitude) infralimbic cortex, hippocampus (subiculum), agranular insular/pyriform cortex, visual cortex, interpeduncular area, nucleus accumbens, cingulate cortex, thalamus, and septum. This pattern of response is consistent with stimulation of both cholinergic and dopaminergic neuronal pathways and is consistent with the known behavioral properties of nicotine. The peak CBV response to nicotine occurred between 9 and 13 min depending upon brain region, and the average full width half-maximum of the rCBV response was 27 min. The high spatial and temporal resolution of the phMRI technique lends itself well to further, more detailed, studies of nicotine dynamics.

Measurements of BOLD/CBV ratio show altered fMRI hemodynamics during stroke recovery in rats.

Brain responses to external stimuli after permanent and transient ischemic insults have been documented using cerebral blood volume weighted (CBVw) functional magnetic resonance imaging (fMRI) in correlation with tissue damage and neurological recovery. Here, we extend our previous studies of stroke recovery in rat models of focal cerebral ischemia by comparing blood oxygen level-dependent (BOLD) and cerebral blood volume (CBV) changes. Responses to forepaw stimulation were measured in normal rats (n=5) and stroke rats subjected to 2 h of middle cerebral artery occlusion (n=6). Functional magnetic resonance imaging was performed 2 weeks after stroke to evaluate the recovery process. After stroke, animals showed variable degrees of fMRI activation in ipsilesional cortex, the extent of which did not correlate with structural damages as measured using apparent diffusion coefficient, fractional anisotropy, blood volume, and vessel size index. While the contralesional cortex showed good overlap between BOLD and CBV-activated regions, the ipsilesional cortex showed low covariance between significantly activated voxels by BOLD and CBVw techniques. In particular, the relative activation during contralateral stimuli in the ipsilesional somatosensory cortex was significantly higher for CBVw responses than BOLD, which might be due to stroke-related alterations in fMRI hemodynamic coupling. Aberrant subcortical activations were also observed. When unaffected forelimbs were stimulated, strong bilateral responses were observed. However, little thalamic responses accompanied stimulation of affected forelimbs despite significant activation in the ipsilesional somatosensory cortex. These results suggest that stroke affects not only local hemodynamics and coupling but also other factors including neural connectivity.