Imaging the Effects of ß-Hydroxybutyrate on Peri-Infarct Neurovascular Function and Metabolism.

Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body ß-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Attenuation of functional hyperemia to visual stimulation in mild Alzheimer's disease and its sensitivity to cholinesterase inhibition.

Despite the growing recognition of the significance of cerebrovascular impairment in the etiology and progression of Alzheimer's disease (AD), the early stage brain vascular dysfunction and its sensitivity to pharmacological interventions is still not fully characterized. Due to the early and aggressive treatment of probable AD with cholinesterase inhibitors (ChEI), which in and of themselves have direct effects on brain vasculature, the vast majority of hemodynamic measurements in early AD subjects reported hitherto have consequently been made only after the start of treatment, complicating the disentanglement of disease- vs. treatment-related effects on the cerebral vasculature. To address this gap, we used pseudo continuous arterial spin labeling MRI to measure resting perfusion and visual stimulation elicited changes in cerebral blood flow (CBF) and blood oxygenation dependent (BOLD) fMRI signal in a cohort of mild AD patients immediately prior to, 6months post, and 12months post commencement of open label cholinesterase inhibitor treatment. Although patients exhibited no gray matter atrophy prior to treatment and their resting perfusion was not distinguishable from that in age, education and gender-matched controls, the patients' visual stimulation-elicited changes in BOLD fMRI and blood flow were decreased by 10±4% (BOLD) and 23±2% (CBF), relative to those in controls. Induction of cholinesterase inhibition treatment was associated with a further, 7±2% reduction in patients' CBF response to visual stimulation, but it stabilized, at this new lower level, over the follow-up period. Likewise, MMSE scores remained stable during the treatment; furthermore, higher MMSE scores were associated with higher perfusion responses to visual stimulation. This study represents the initial step in disentangling the effects of AD pathology from those of the first line treatment with cholinesterase inhibitors on cerebral hemodynamics and supports the use of arterial spin labeling MRI for quantitative evaluation of the brain vascular function in mild Alzheimer's disease. The findings provide evidence of a pronounced deficit in the visual cortex hyperemia despite the relative sparing of visual function in early stage AD, its reduction with ChEI treatment induction, and its stabilization in the first year of cholinesterase inhibition treatment. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Neurovascular unit remodelling in the subacute stage of stroke recovery.

Brain plasticity following focal cerebral ischaemia has been observed in both stroke survivors and in preclinical models of stroke. Endogenous neurovascular adaptation is at present incompletely understood yet its potentiation may improve long-term functional outcome. We employed longitudinal MRI, intracranial array electrophysiology, Montoya Staircase testing, and immunofluorescence to examine function of brain vessels, neurons, and glia in addition to forelimb skilled reaching during the subacute stage of ischemic injury progression. Focal ischemic stroke (~100mm3 or ~20% of the total brain volume) was induced in adult Sprague-Dawley rats via direct injection of endothelin-1 (ET-1) into the right sensori-motor cortex, producing sustained impairment in left forelimb reaching ability. Resting perfusion and vascular reactivity to hypercapnia in the peri-lesional cortex were elevated by approximately 60% and 80% respectively seven days following stroke. At the same time, the normal topological pattern of local field potential (LFP) responses to peripheral somatosensory stimulation was abolished and the average power of spontaneous LFP activity attenuated by approximately 50% relative to the contra-lesional cortex, suggesting initial response attenuation within the peri-infarct zone. By 21 days after stroke, perilesional blood flow resolved, but peri-lesional vascular reactivity remained elevated. Concomitantly, the LFP response amplitudes increased with distance from the site of ET-1 injection, suggesting functional remodelling from the core of the lesion to its periphery. This notion was further buttressed by the lateralization of spontaneous neuronal activity: by day 21, the average ipsi-lesional power of spontaneous LFP activity was almost twice that of the contra-lesional cortex. Over the observation period, the peri-lesional cortex exhibited increased vascular density, along with neuronal loss, astrocytic activation, and recruitment and activation of microglia and macrophages, with neuronal loss and inflammation extending beyond the peri-lesional cortex. These findings highlight the complex relationship between neurophysiological state and behaviour and provide evidence of highly dynamic functional changes in the peri-infarct zone weeks following the ischemic insult, suggesting an extended temporal window for therapeutic interventions.

Modulation of the peri-infarct neurogliovascular function by delayed COX-1 inhibition.

PURPOSE: Stroke is the leading cause of adult disability worldwide. The absence of more effective interventions in the chronic stage-that most patients stand to benefit from-reflects uncertainty surrounding mechanisms that govern recovery. The present work investigated the effects of a novel treatment (selective cyclooxygenase-1, COX-1, inhibition) in a model of focal ischemia. MATERIALS AND METHODS: FR122047 (COX-1 inhibitor) was given beginning 7 days following stroke (cortical microinjection of endothelin-1) in 23 adult male rats. Longitudinal continuous-arterial-spin-labeling was performed prior to treatment (7 days), and repeated following treatment (21 days) on a 7T magnetic resonance imaging (MRI) system to estimate resting perfusion and reactivity to hypercapnia. These in vivo measurements were buttressed by immunohistochemistry. RESULTS: Stroke caused an increase in perilesional resting perfusion (peri-/contralesional perfusion ratio of 170 ± 10%) and perfusion responses to hypercapnia (180 ± 10%) at 7 days. At 21 days, placebo-administered rats showed normalized perilesional perfusion (100 ± 20%) but persistent hyperreactivity (190 ± 20%). Treated animals exhibited sustained perilesional hyperperfusion (180 ± 10%). Further, reactivity lateralization did not persist following treatment (peri- vs. contralesional reactivity: P = 0.002 at 7 vs. P = 0.2 at 21 days). Hemodynamic changes were accompanied by neuronal loss, increased endothelial density, and widespread microglial and astrocytic activation. Moreover, relative to controls, treated rats showed increased perilesional neuronal survival (22 ± 1% vs. 14.9 ± 0.8%, P = 0.02) and decreased microglia/macrophage recruitment (17 ± 1% vs. 20 ± 1%, P = 0.05). Finally, perilesional perfusion was correlated with neuronal survival (slope = 0.14 ± 0.05; R2 = 0.7, P = 0.03). CONCLUSION: These findings shed light on the role of COX-1 in chronic ischemic injury and suggest that delayed selective COX-1 inhibition exerts multiple beneficial effects on the neurogliovascular unit. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 4 J. MAGN. RESON. IMAGING 2017;46:505-517.

Magnetic resonance spectroscopy reveals oral Lactobacillus promotion of increases in brain GABA, N-acetyl aspartate and glutamate.

The gut microbiome has been shown to regulate the development and functions of the enteric and central nervous systems. Its involvement in the regulation of behavior has attracted particular attention because of its potential translational importance in clinical disorders, however little is known about the pathways involved. We previously have demonstrated that administration of Lactobacillus rhamnosus (JB-1) to healthy male BALB/c mice, promotes consistent changes in GABA-A and -B receptor sub-types in specific brain regions, accompanied by reductions in anxiety and depression-related behaviors. In the present study, using magnetic resonance spectroscopy (MRS), we quantitatively assessed two clinically validated biomarkers of brain activity and function, glutamate+glutamine (Glx) and total N-acetyl aspartate+N-acetyl aspartyl glutamic acid (tNAA), as well as GABA, the chief brain inhibitory neurotransmitter. Mice received 1×10(9) cfu of JB-1 per day for 4weeks and were subjected to MRS weekly and again 4weeks after cessation of treatment to ascertain temporal changes in these neurometabolites. Baseline concentrations for Glx, tNAA and GABA were equal to 10.4±0.3mM, 8.7±0.1mM, and 1.2±0.1mM, respectively. Delayed increases were first seen for Glx (~10%) and NAA (~37%) at 2weeks which persisted only to the end of treatment. However, Glx was still elevated 4weeks after treatment had ceased. Significantly elevated GABA (~25%) was only seen at 4weeks. These results suggest specific metabolic pathways in our pursuit of mechanisms of action of psychoactive bacteria. They also offer through application of standard clinical neurodiagnostic techniques, translational opportunities to assess biomarkers accompanying behavioral changes induced by alterations in the gut microbiome.

Differences in iron and manganese concentration may confound the measurement of myelin from R1 and R2 relaxation rates in studies of dysmyelination.

A model of dysmyelination, the Long Evans Shaker (les) rat, was used to study the contribution of myelin to MR tissue properties in white matter. A large region of white matter was identified in the deep cerebellum and was used for measurements of the MR relaxation rate constants, R1 = 1/T1 and R2 = 1/T2 , at 7 T. In this study, R1 of the les deep cerebellar white matter was found to be 0.55 ± 0.08 s (-1) and R2 was found to be 15 ± 1 s(-1) , revealing significantly lower R1 and R2 in les white matter relative to wild-type (wt: R1 = 0.69 ± 0.05 s(-1) and R2 = 18 ± 1 s(-1) ). These deviated from the expected ΔR1 and ΔR2 values, given a complete lack of myelin in the les white matter, derived from the literature using values of myelin relaxivity, and we suspect that metals could play a significant role. The absolute concentrations of the paramagnetic transition metals iron (Fe) and manganese (Mn) were measured by a micro-synchrotron radiation X-ray fluorescence (µSRXRF) technique, with significantly greater Fe and Mn in les white matter than in wt (in units of µg [metal]/g [wet weight tissue]: les: Fe concentration,19 ± 1; Mn concentration, 0.71 ± 0.04; wt: Fe concentration,10 ± 1; Mn concentration, 0.47 ± 0.04). These changes in Fe and Mn could explain the deviations in R1 and R2 from the expected values in white matter. Although it was found that the influence of myelin still dominates R1 and R2 in wt rats, there were non-negligible changes in the contribution of the metals to relaxation. Although there are already problems with the estimation of myelin from R1 and R2 changes in disease models with pathology that also affects the relaxation rate constants, this study points to a specific pitfall in the estimation of changes in myelin in diseases or models with disrupted concentrations of paramagnetic transition metals. Copyright © 2016 John Wiley & Sons, Ltd.

An in vivo model of anti-inflammatory activity of subdural dexamethasone following the spinal cord injury.

Current therapies to limit the neural tissue destruction following the spinal cord injury are not effective. Our recent studies indicate that the injury to the white matter of the spinal cord results in a severe inflammatory response where macrophages phagocytize damaged myelin and the fluid-filled cavity of injury extends in size with concurrent and irreversible destruction of the surrounding neural tissue over several months. We previously established that a high dose of 4mg/rat of dexamethasone administered for 1 week via subdural infusion remarkably lowers the numbers of infiltrating macrophages leaving large amounts of un-phagocytized myelin debris and therefore inhibits the severity of inflammation and related tissue destruction. But this dose was potently toxic to the rats. In the present study the lower doses of dexamethasone, 0.125-2.0mg, were administered via the subdural infusion for 2 weeks after an epidural balloon crush of the mid-thoracic spinal cord. The spinal cord cross-sections were analyzed histologically. Levels of dexamethasone used in the current study had no systemic toxic effect and limited phagocytosis of myelin debris by macrophages in the lesion cavity. The subdural infusion with 0.125-2.0mg dexamethasone over 2 week period did not eliminate the inflammatory process indicating the need for a longer period of infusion to do so. However, this treatment has probably lead to inhibition of the tissue destruction by the severe, prolonged inflammatory process.

Cerebral microvascular network geometry changes in response to functional stimulation.

The cortical microvessels are organized in an intricate, hierarchical, three-dimensional network. Superimposed on this anatomical complexity is the highly complicated signaling that drives the focal blood flow adjustments following a rise in the activity of surrounding neurons. The microvascular response to neuronal activation remains incompletely understood. We developed a custom two photon fluorescence microscopy acquisition and analysis to obtain 3D maps of neuronal activation-induced changes in the geometry of the microvascular network of the primary somatosensory cortex of anesthetized rats. An automated, model-based tracking algorithm was employed to reconstruct the 3D microvascular topology and represent it as a graph. The changes in the geometry of this network were then tracked, over time, in the course of electrical stimulation of the contralateral forepaw. Both dilatory and constrictory responses were observed across the network. Early dilatory and late constrictory responses propagated from deeper to more superficial cortical layers while the response of the vertices that showed initial constriction followed by later dilation spread from cortical surface toward increasing cortical depths. Overall, larger caliber adjustments were observed deeper inside the cortex. This work yields the first characterization of the spatiotemporal pattern of geometric changes on the level of the cortical microvascular network as a whole and provides the basis for bottom-up modeling of the hemodynamically-weighted neuroimaging signals.

Optimizing T1-weighted imaging of cortical myelin content at 3.0 T.

With increases in the sensitivity and resolution of anatomical MRI for the brain, methods for mapping the organization of the cerebral cortex by imaging its myelin content have emerged. This identifies major sensory and motor regions and could be used in studies of cortical organization, particularly if patterns of myelination can be visualized over the cortical surface robustly in individual subjects. The imaging problem is difficult, however, because of the relative thinness of the cerebral cortex and the low intracortical tissue contrast. In this paper, we optimize the contrast of T(1)-weighted MRI to help better visualize patterns of myelination. We measure a small but statistically significant difference in T(1) of 171 ± 40 ms between cortical regions with low and high myelin contents in the human cortex at 3T, and then perform simulations to choose parameters for an inversion-recovery pulse sequence that utilizes this T(1) difference to increase contrast within the cortex. We show that lengthening the delay between signal acquisition and the next inversion pulse in the sequence increases intracortical contrast more effectively than does image averaging. Using the optimized sequence, we show that major myelinated regions that are relatively thick, such as the primary motor and auditory regions, can be visualized well in individuals at 3T using whole-cortex 3D images made at 1mm isotropic resolution, while thinner regions, such as the primary visual cortex, can be visualized using targeted 3D images made at 0.5mm isotropic resolution. Our findings demonstrate that patterns of myelination can be better visualized in individual subjects when the imaging is optimized to highlight intracortical contrast and can help to pave the way for the creation of matched maps of microanatomy and function in the cortex of living individual humans.

Hemodynamic effects of cholinesterase inhibition in mild Alzheimer's disease.

PURPOSE: To evaluate the spatiotemporal progression of perfusion changes in early stages of Alzheimer's disease (AD), we imaged the perfusion response to pharmacological treatment in a group of mild AD patients and contrasted it to the perfusion of age-, sex-, and education-matched healthy volunteers over the same time interval. MATERIALS AND METHODS: We used pseudo continuous arterial spin labeling (PCASL) MRI for quantitative three-dimensional mapping of perfusion immediately before and 6 months after cholinesterase inhibitor treatment. RESULTS: Before treatment, patients were found hypoperfused relative to their healthy counterparts in the gray matter of lateral temporal lobe, posterior cingulate, and anterior cingulate as well as in the white matter of the posterior cingulate. Most of the cortical regions investigated and the white matter of posterior cingulate and prefrontal regions showed treatment-elicited increases in perfusion, which were not secondary to changes in regional tissue volume nor were they associated with improvement in either MMSE or ADAS-Cog scores, although lack of deterioration suggested a cognitive benefit. CONCLUSION: This study provides a hemodynamic profile of mild AD and sheds light on the perfusion changes related to prolonged cholinesterase inhibition in this early disease stage.

Human aortic endothelial cell labeling with positive contrast gadolinium oxide nanoparticles for cellular magnetic resonance imaging at 7 Tesla.

Positive T1 contrast using gadolinium (Gd) contrast agents can potentially improve detection of labeled cells on magnetic resonance imaging (MRI). Recently, gadolinium oxide (Gd2O3) nanoparticles have shown promise as a sensitive T1 agent for cell labeling at clinical field strengths compared to conventional Gd chelates. The objective of this study was to investigate Gado CELLTrack, a commercially available Gd2O3 nanoparticle, for cell labeling and MRI at 7 T. Relaxivity measurements yielded r1  =  4.7 s⁻¹ mM⁻¹ and r2/r1  =  6.2. Human aortic endothelial cells were labeled with Gd2O3 at various concentrations and underwent MRI from 1 to 7 days postlabeling. The magnetic resonance relaxation times T1 and T2 of labeled cell pellets were measured. Cellular contrast agent uptake was quantified by inductively coupled plasma-atomic emission spectroscopy, which showed very high uptake compared to conventional Gd compounds. MRI demonstrated significant positive T1 contrast and stable labeling on cells. Enhancement was optimal at low Gd concentrations, attained in the 0.02 to 0.1 mM incubation concentration range (corresponding cell uptake was 7.26 to 34.1 pg Gd/cell). Cell viability and proliferation were unaffected at the concentrations tested and up to at least 3 days postlabeling. Gd2O3 is a promising sensitive and stable positive contrast agent for cellular MRI at 7 T.

Efficient sampling of early signal arrival for estimation of perfusion and transit time in whole-brain arterial spin labeling.

Arterial spin labeling can be used to measure both cerebral perfusion and arterial transit time. However, accurate estimation of these parameters requires adequate temporal sampling of the arterial spin labeling difference signal. In whole-brain multislice acquisitions, two factors reduce the accuracy of the parameter estimates: saturation of labeled blood in transit and inadequate sampling of early difference signal in superior slices. Label saturation arises when slices are acquired inferior-to-superior such that slice selection in proximal slices spoils the label for a distal slice. Inadequate sampling arises when the time spent acquiring inferior slices is too long to allow early sampling of the difference signal in superior slices. A novel approach to multislice imaging is proposed to address these two issues. In round-robin arterial spin labeling, slices are acquired in a different order after every pair of control-label acquisitions. Round-robin arterial spin labeling enables the acquisitions of all slices across the same range of postlabel delays in a descending superior-to-inferior order. This eliminates the temporal sampling problem and greatly reduces label saturation. Arterial transit time estimates obtained for the whole brain with round-robin arterial spin labeling show better agreement with a single-slice acquisition than do conventional multislice acquisitions.

Dipolar chemical shift correlation spectroscopy for homonuclear carbon distance measurements in proteins in the solid state: application to structure determination and refinement.

High-resolution solid-state NMR spectroscopy has become a promising tool for protein structure determination. Here, we describe a new dipolar-chemical shift correlation experiment for the measurement of homonuclear 13C-13C distances in uniformly 13C,15N-labeled proteins and demonstrate its suitability for protein structure determination and refinement. The experiments were carried out on the beta1 immunoglobulin binding domain of protein G (GB1). Both intraresidue and interresidue distances between carbonyl atoms and atoms in the aliphatic side chains were collected using a three-dimensional chemical shift correlation spectroscopy experiment that uses homogeneously broadened rotational resonance recoupling for carbon mixing. A steady-state approximation for the polarization transfer function was employed in data analysis, and a total of 100 intramolecular distances were determined, all in the range 2.5-5.5 A. An additional 41 dipolar contacts were detected, but the corresponding distances could not be accurately quantified. Additional distance and torsional restraints were derived from the proton-driven spin diffusion measurements and from the chemical shift analysis, respectively. Using all these restraints, it was possible to refine the structure of GB1 to a root-mean square deviation of 0.8 A. The approach is of general applicability for peptides and small proteins and can be easily incorporated into a structure determination and refinement protocol.

Reduced Cerebrovascular Reactivity and Increased Resting Cerebral Perfusion in Rats Exposed to a Cafeteria Diet.

To better understand the effects of a diet high in fat, sugar, and sodium on cerebrovascular function, Sprague Dawley rats were chronically exposed to a Cafeteria diet. Resting cerebral perfusion and cerebrovascular reactivity was quantified using continuous arterial spin labeling (CASL) magnetic resonance imaging (MRI). In addition, structural changes to the cerebrovasculature and susceptibility to ischemic lesion were examined. Compared to control animals fed standard chow (SD), Cafeteria diet (CAF) rats exhibited increased resting brain perfusion in the hippocampus and reduced cerebrovascular reactivity in response to 10% inspired CO2 challenges in both the hippocampus and the neocortex. CAF rats switched to chow for one month (SWT) exhibited improved resting perfusion in the hippocampus as well as improved cerebrovascular reactivity in the neocortex. However, the diet switch did not correct cerebrovascular reactivity in the hippocampus. These changes were not accompanied by alterations in the structural integrity of the cerebral microvasculature, examined using rat endothelial cell antigen-1 (RECA-1) and immunoglobulin G (IgG) immunostaining. Also, the extent of tissue damage induced by endothelin-1 injection into sensorimotor cortex was not affected by the Cafeteria diet. These results demonstrate that short-term consumption of an ultra-processed diet reduces cerebrovascular reactivity. This effect persists after dietary normalization despite recovery of peripheral symptomatology.

(13)C-(13)C distance measurements in U-(13)C, (15)N-labeled peptides using rotational resonance width experiment with a homogeneously broadened matching condition.

In this publication, we introduce a version of the rotational resonance width experiment with a homogeneously broadened matching condition. The increase in the bandwidth is achieved by the reduction of the proton decoupling power during mixing, which results in the reduction of zero-quantum relaxation, and broadens the rotational resonance condition. We show that one can achieve recoupling of the carbonyl-aliphatic side chain dipolar interactions band selectively, while avoiding the recoupling of strongly interacting C'-Calpha and C'-Cbeta spin pairs. The attenuation of the multi-spin effects in the presence of short zero-quantum relaxation enables a two-spin approximation to be employed for the analysis of the experimental data. The systematic error introduced by this approximation is estimated by comparing the results with a three-spin simulation. The experiment is demonstrated in [U-(13)C,(15)N]N-acetyl-L-Val-L-Leu dipeptide, where 11 distances, ranging from 2.5 to 6 A, were measured.

A physiological characterization of the Cafeteria diet model of metabolic syndrome in the rat.

Many promising findings from pre-clinical research have failed to translate to the clinic due to their inability to incorporate human disease co-morbidity. A variety of rodent diets and feeding durations are currently used in models of human metabolic syndrome, obesity and diabetes. One model, the Cafeteria (CAF) diet, makes use of grocery store-purchased food items that more closely approximate the human ultra-processed diet than commercial high-fat or high-sugar rodent diets. The present study describes the development of metabolic syndrome in rats fed a CAF diet as well as the recovery of metabolic syndrome following a healthy "lifestyle" change. In addition, we explored the effects of CAF diet on spatial learning and memory and on neuroinflammation. Three-week old male Sprague-Dawley rats were fed a CAF diet for three months that consisted of 16 highly palatable human food items along with standard chow and a 12% sucrose solution to mimic soda consumption. Thereafter, a sub-group of CAF diet rats was switched to a chow diet (SWT) for one month. Both CAF and SWT groups were compared to control rats maintained on a standard chow diet (SD). Prior to the diet switch, CAF and SWT animals developed features akin to metabolic syndrome. Both groups of rats displayed significant abdominal obesity with increased visceral adiposity, hyperinsulinemia, glucose intolerance and dyslipidemia with elevated serum triglyceride levels and reduced HDL cholesterol. Switching to a chow diet for one month completely reversed these features in SWT animals. Although acquisition of the Barnes maze was not affected by the CAF diet, these animals exhibited greater hippocampal neuroinflammation compared to both SD and SWT rats as assessed by Iba1 staining. These results demonstrate that the CAF diet is very effective in creating metabolic syndrome with hippocampal inflammation in rats over a relatively short time span. This model may be of great heuristic importance in determining potential reversibility of metabolic and cerebrovascular pathologies across the lifespan and as a co-morbid factor in other disease models such as stroke.

The effects of delayed reduction of tonic inhibition on ischemic lesion and sensorimotor function.

To aid in development of chronic stage treatments for sensorimotor deficits induced by ischemic stroke, we investigated the effects of GABA antagonism on brain structure and fine skilled reaching in a rat model of focal ischemia induced via cortical microinjections of endothelin-1 (ET-1). Beginning 7 days after stroke, animals were administered a gamma-aminobutyric acid (GABAA) inverse agonist, L-655,708, at a dose low enough to afford α5-GABAA receptor specificity. A week after stroke, the ischemic lesion comprised a small hypointense necrotic core (6±1 mm(3)) surrounded by a large (62±11 mm(3)) hyperintense perilesional region; the skilled reaching ability on the Montoya staircase test was decreased to 34%±2% of the animals' prestroke performance level. On L-655,708 treatment, animals showed a progressive decrease in total stroke volume (13±4 mm(3) per week), with no change in animals receiving placebo. Concomitantly, treated animals' skilled reaching progressively improved by 9%±1% per week, so that after 2 weeks of treatment, these animals performed at 65%±6% of their baseline ability, which was 25%±11% better than animals given placebo. These data indicate beneficial effects of delayed, sustained low-dose GABAA antagonism on neuroanatomic injury and skilled reaching in the chronic stage of stroke recovery in an ET-1 rat model of focal ischemia.

Prolonged Subdural Infusion of Kynurenic Acid Is Associated with Dose-Dependent Myelin Damage in the Rat Spinal Cord.

BACKGROUND: Kynurenic acid (KYNA) is the end stage metabolite of tryptophan produced mainly by astrocytes in the central nervous system (CNS). It has neuroprotective activities but can be elevated in the neuropsychiatric disorders. Toxic effects of KYNA in the CNS are unknown. The aim of this study was to assess the effect of the subdural KYNA infusion on the spinal cord in adult rats. METHODS: A total of 42 healthy adult rats were randomly assigned into six groups and were infused for 7 days with PBS (control) or 0.0002 pmol/min, 0.01 nmol/min, 0.1 nmol/min, 1 nmol/min, and 10 nmol/min of KYNA per 7 days. The effect of KYNA on spinal cord was determined using histological and electron microscopy examination. Myelin oligodendrocyte glycoprotein (MOG) was measured in the blood serum to assess a degree of myelin damage. RESULT: In all rats continuous long-lasting subdural KYNA infusion was associated with myelin damage and myelin loss that was increasingly widespread in a dose-depended fashion in peripheral, sub-pial areas. Damage to myelin sheaths was uniquely related to the separation of lamellae at the intraperiod line. The damaged myelin sheaths and areas with complete loss of myelin were associated with limited loss of scattered axons while vast majority of axons in affected areas were morphologically intact. The myelin loss-causing effect of KYNA occurred with no necrosis of oligodendrocytes, with locally severe astrogliosis and no cellular inflammatory response. Additionally, subdural KYNA infusion increased blood MOG concentration. Moreover, the rats infused with the highest doses of KYNA (1 and 10 nmol/min) demonstrated adverse neurological signs including weakness and quadriplegia. CONCLUSIONS: We suggest, that subdural infusion of high dose of KYNA can be used as an experimental tool for the study of mechanisms of myelin damage and regeneration. On the other hand, the administration of low, physiologically relevant doses of KYNA may help to discover the role of KYNA in control of physiological myelination process.

Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C,15N-labeled peptides and proteins.

In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly (13)C,(15)N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i-2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.