Intercellular Conduction Optimizes Arterial Network Function and Conserves Blood Flow Homeostasis During Cerebrovascular Challenges.

OBJECTIVE: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. CONCLUSIONS: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Whey Protein and Its Components Lactalbumin and Lactoferrin Affect Energy Balance and Protect against Stroke Onset and Renal Damage in Salt-Loaded, High-Fat Fed Male Spontaneously Hypertensive Stroke-Prone Rats.

BACKGROUND: Whey protein (WH)-enriched diets are reported to aid in weight loss and to improve cardiovascular health. However, the bioactive components in whey responsible for causing such effects remain unidentified. OBJECTIVE: We determined the effects of whey and its components [α-lactalbumin (LA) and lactoferrin (LF)] on energy balance, glucose tolerance, gut hormones, renal damage, and stroke onset in rats. METHODS: Male spontaneously hypertensive stroke-prone (SHRSP) rats (age 8 wk) were fed isocaloric high-fat (40% kcal) and high-salt (4% wt/wt) diets (n = 8-10/group) and randomized for 8 wk to diets enriched as follows: control (CO): 15% kcal from egg albumin, 45% kcal from carbohydrate; WH: 20%kcal WH isolate + 15% kcal egg albumin; LA: 20% kcal LA  + 15% kcal egg albumin; or LF: 20% kcal lactoferrin + 15% kcal egg albumin. Measurements included energy balance (food intake, energy expenditure, and body composition), stroke-related behaviors, brain imaging, glucose tolerance, metabolic hormones, and tissue markers of renal damage. Data were analyzed by linear mixed models with repeated measures or 1-way ANOVA. RESULTS: Diets enriched with WH, LA, or LF increased survival, with 25% of rats fed these diets exhibiting stroke-associated morbidity, whereas 90% of CO rats were morbid by 8 wk (P < 0.05). The nephritis scores of rats fed WH-, LA-, or LF-enriched diets were 80%, 92%, and 122% lower than those of COs (P = 0.001). The mRNA abundances of renin and osteopontin were 100-600% lower in rats fed WH-, LA-, or LF-enriched diets than in COs (P < 0.05). Urine albumin concentrations and albumin-to-creatinine ratios were 200% lower in rats fed LF-enriched diets than in COs (P < 0.05). Compared with COs, rats fed LF-enriched diets for 2-3 wk had food intake decreased by 29%, body weight decreased by 13-19%, lean mass decreased by 12-19%, and fat mass decreased by 20% (P < 0.001). Relative to COs, rats fed WH and LA had food intake decreased by 10% (P < 0.1), but COs had 12-45% lower weight than rats fed LA- and WH-enriched diets by 3 wk (P < 0.01). Compared with COs, rats fed WH-enriched diets increased energy expenditure by 7%, whereas, rats fed LA-enriched diets had energy expenditure acutely decreased by 7% during the first 4 d, and rats fed LF-enriched diets had energy expenditure decreased by 7-17% throughout the first week ( P < 0.001). Rats fed LA- and LF-enriched diets had blood glucose decreased by 14-19% (P < 0.05) and WH by 9% (P = 0.1), relative to COs. Compared with COs, rats fed LF had GIP decreased by 90% and PYY by 87% (P < 0.05). CONCLUSION: Together, these findings indicate that whey and its components α-lactalbumin and lactoferrin improved energy balance and glycemic control, and protected against the onset of neurological deficits associated with stroke and renal damage in male SHRSP rats.

Magnetic resonance imaging detection of multiple ischemic injury produced in an adult rat model of minor stroke followed by mild transient cerebral ischemia.

OBJECTIVES: To determine whether cumulative brain damage produced adjacent to a minor stroke that is followed by a mild transient ischemia is detectable with MRI and histology, and whether acute or chronic recovery between insults influences this damage. MATERIALS AND METHODS: A minor photothrombotic (PT) stroke was followed acutely (1-2 days) or chronically (7 days) by a mild transient middle cerebral artery occlusion (tMCAO). MRI was performed after each insult, followed by final histology. RESULTS: The initial PT produced small hyperintense T2 and DW infarct lesions and peri-lesion regions of scattered necrosis and modestly increased T2. Following tMCAO, in a slice and a region adjacent to the PT, a region of T2 augmentation was observed when recovery between insults was acute but not chronic. Within the PT slice, a modest region of exacerbated T2 change proximate to the PT was also observed in the chronic group. Corresponding histological changes within regions of augmented T2 included increased vacuolation and cell death. CONCLUSION: Within regions adjacent to an experimental minor stroke, a recurrence of a mild transient cerebral ischemia augmented T2 above increases produced by tMCAO alone, reflecting increased damage in this region. Exacerbation appeared broader with acute versus chronic recovery between insults.

Recurrent mild cerebral ischemia: enhanced brain injury following acute compared to subacute recurrence in the rat.

BACKGROUND: In the current study, a transient cerebral ischemia producing selective cell death was designated a mild ischemic insult. A comparable insult in humans is a transient ischemic attack (TIA) that is associated with functional recovery but can have imaging evidence of minor ischemic damage including cerebral atrophy. A TIA also predicts a high risk for early recurrence of a stroke or TIA and thus multiple ischemic insults are not uncommon. Not well understood is what the effect of differing recovery times between mild ischemic insults has on their pathophysiology. We investigated whether cumulative brain damage would differ if recurrence of a mild ischemic insult occurred at 1 or 3 days after a first insult. RESULTS: A transient episode of middle cerebral artery occlusion via microclip was produced to elicit mild ischemic changes-predominantly scattered necrosis. This was followed 1 or 3 days later by a repeat of the same insult. Brain damage assessed histologically 7 days later was substantially greater in the 1 day recurrent group than the 3 days recurrent group, with areas of damage consisting predominantly of regions of incomplete infarction and pannecrosis in the 1 day group but predominantly regions of selective necrosis and smaller areas of incomplete infarction in the 3 days group (P < 0.05). Enhanced injury was reflected by greater number of cells staining for macrophages/microglia with ED1 and greater alterations in GFAP staining of reactive astrocytes in the 1 day than 3 days recurrent groups. The differential susceptibility to injury did not correspond to higher levels of injurious factors present at the time of the second insult such as BBB disruption or increased cytokines (tumor necrosis factor). Microglial activation, with potential for some beneficial effects, appeared greater at 3 days than 1 day. Also blood analysis demonstrated changes that included an acute increase in granulocytes and decrease in platelets at 1 day compared to 3 days post transient ischemia. CONCLUSIONS: Dynamic changes in multiple inflammatory responses likely contribute to the time dependence of the extent of damage produced by recurrent mild ischemic insults. The time of mild stroke recurrence is crucial with early recurrence producing greater damage than subacute recurrence and this supports urgency for determining and implementing optimal stroke management directly after a TIA.

Magnetic resonance imaging of ischemic injury produced by varying severities of photothrombosis differs in neonatal and adult brain.

Stroke is a major cause of disability in adults and children. Recently, we have developed an adult rat model of minor stroke containing a peri-infarct region with a modest T2 increase and mild ischemic damage. We hypothesized that a neonatal minor stroke with mild peri-ischemic changes could also be produced, but with potential ontogenic differences. Using our minor photothrombosis method, we produced a range of severities of ischemic lesions (mini, minor, moderate and severe) within magnetic resonance imaging (MRI) slices of adult and neonatal rats. In both age groups, the lesion region showed a marked increase in T2 and diffusion-weighted intensity and decrease in apparent diffusion coefficient (ADC), corresponding to a cortical infarct detected using fluorojade and hematoxylin and eosin staining. Perilesional regions showed modest increases in T2 and ADC in adults, but not neonates, and this corresponded to scattered cell death, but not necessarily extravasation of plasma protein, i.e. blood-brain barrier disruption. Mini and minor insults in neonates generally showed homogeneous and rather modest changes in T2 and ADC. MR perfusion maps demonstrated a penumbral area of greater hypoperfusion in adults compared with neonates. Together, the results indicate that, in neonatal cortex, a similar severity of photothrombosis occurs throughout the area of photoactivation, whereas, in adult brain, spontaneous clot lysis and/or partial thrombosis occurs adjacent to permanently occluded vessels. Thus, by comparing differing severities of photothrombotic ischemia in neonates and adults, ontogenic differences were detectable using MRI, with mature brain having a greater penumbral region. Mild ischemic injury and scattered cell death in both neonates and adults could be identified by a modest increase in T2 and decrease in ADC. A better understanding of the effects of development on ischemic responses and associated MRI changes will provide a basis for the improved diagnosis of mild or minor ischemic insults relevant to pediatric and adult stroke.

Magnetization transfer and diffusion imaging of acute axonal damage in the cerebral peduncle following hypoxia-ischemia in neonatal rats.

BACKGROUND: Magnetic resonance imaging (MRI) of axonal degenerative changes in the cerebral peduncle of the corticospinal tract following cerebral hypoxic-ischemic damage might distinguish infants most appropriate for receiving prompt treatment. The optimal MRI sequence for very early diagnosis of axonal degenerative changes is unknown. We hypothesized that magnetization transfer ratio (MTR) imaging would be more sensitive than traditional MRI, e.g., T(2) or diffusion weighted imaging. METHODS: Transient unilateral cerebral hypoxia-ischemia was produced in the neonatal rat followed by MRI of changes in T(2), the apparent diffusion coefficient (ADC) of water, and MTR, with a focus on the parietal cortex (an ischemic damaged region) and the cerebral peduncle (remote within the corticospinal tract). Rats were imaged at 2 h, 1 d, or 1 wk postinsult. RESULTS: In the cerebral peduncle, MTR and T(2) responded similarly, with alterations occurring ipsilaterally at 1 d postinsult. ADC was most sensitive for detecting changes as early as 2 h postinsult, and this corresponded to a reduced staining of axonal filaments ipsilaterally. CONCLUSION: MTR and T(2) imaging have comparable sensitivity for distinguishing early axonal damage in the cerebral peduncle. ADC imaging is highly sensitive for detecting early disruption of corticospinal axons, supporting its potential hyperacute diagnostic use clinically.

Changes in differential functional magnetic resonance signals in the rodent brain elicited by mixed-nutrient or protein-enriched meals.

BACKGROUND & AIMS: The hypothalamus and brain stem have important roles in regulating food intake; the roles of other nonhomeostatic centers in detecting nutrient content of ingested food have been poorly characterized. We used blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) to map brain regions that are responsive to intragastric infusion of isocaloric amounts of a mixed nutrient or protein, and assessed the role of blood glucose in the observed BOLD signal changes. METHODS: Brain images were acquired, using a 9.4 T MRI system, from anesthetized rats during intragastric infusion of saline (n = 7), or 12 kcal of a mixed nutrient (n = 13) or protein (n = 6). Nutrient-induced changes in blood parameters and the effects of intravenous infusion of saline or glucose (n = 5/treatment) on BOLD fMRI signal changes were also evaluated. Intragastric nutrient infusion reduced the BOLD fMRI signal intensity in homeostatic (hypothalamus, nucleus tractus solitarius) and nonhomeostatic (thalamus, hippocampus, caudate putamen, cerebral cortex, cerebellum) centers; these effects were mimicked qualitatively by intravenous glucose. In contrast to a mixed meal, protein load reduced the BOLD fMRI signal in the amygdala. BOLD fMRI signal changes were inversely correlated with circulating concentrations of amylin, insulin, peptide YY, and glucagon-like peptide-1. CONCLUSIONS: The caloric content of a meal is signaled from the gut to the brain and affects activity in homeostatic and non-homeostatic centers; blood glucose concentrations have an important role. The satiety effects of protein are associated with activity changes specifically in the amygdala.

Infrared optical imaging of matrix metalloproteinases (MMPs) up regulation following ischemia reperfusion is ameliorated by hypothermia.

BACKGROUND: We investigated the use of a new MMP activatable probe MMPSense™ 750 FAST (MMPSense750) for in-vivo visualization of early MMP activity in ischemic stroke. Following middle cerebral artery occlusion (MCAO) optical imaging was performed. Near-infrared (NIR) fluorescent images of MMPSense activation were acquired using an Olympus fluorescent microscope, 1.25 x objective, a CCD camera and an appropriate filter cube for detecting the activated probe with peak excitation and emission at 749 and 775 nm, respectively. Images were acquired starting at 2 or 24 hours after reperfusion over the ipsilateral and contralateral cortex before and for 3 hours after, MMPSense750 was injected. RESULTS: Increased intensities ipsilaterally were observed following MMPSense750 injection with ischemic injury but not in sham animals. There were significant ipsilateral and contralateral differences at 15 minutes (P <0.05) in early ischemic reperfusion and at time 0 in 24 hours post ischemia (P <0.05) which persisted at 180 minutes in both these groups (P <0.01), but not following sham surgery. The increase in ipsilateral signal intensity was attenuated by hypothermia. These observations corresponded with a significant increase in the total MMP-9 protein levels, 5 and 24 hours following ischemia reperfusion (P <0.05) and their reduction by hypothermia. CONCLUSIONS: Matrix-metalloproteinase upregulation in ischemia reperfusion can be imaged acutely in-vivo with NIRF using MMPSense750. Hypothermia attenuated both the optical increase in intensity after MMPSense750 and the increase in MMP-9 protein expression supporting the proof of concept that NIRF imaging using MMPSense can be used to assess potential therapeutic strategies for stroke treatment.

Functional MRI response and correlated electrophysiological changes during posterior hypothalamic nucleus deep brain stimulation.

Identification of active networks involved in behavior is central to understanding brain function as an emergent property. Functional magnetic resonance imaging (fMRI) allows the identification of areas with increased or decreased activity, but the cellular correlates to changes in fMRI response is still controversial. Deep brain stimulation of the posterior hypothalamic nucleus (PH) is known to facilitate locomotor behaviors and rescue locomotion in rodent models of parkinsonian akinesia by an unknown mechanism. Here, we performed 9.4 T fMRI during deep brain stimulation of PH in the anesthetized rat as a model system to explore the network substrates for its behavioral consequences. In addition, multi-unit and field potential recordings were made to examine the physiological correlates to changes in fMRI response. The most robust and reliable MR signal increases were observed in the somatosensory and motor cortices, with minor limbic and sparse thalamic activation. Electrophysiological experiments demonstrated that increased fMRI response in the neocortex corresponds to general increases in spiking activity, decreased slow oscillations and increased delta band activity. Forelimb movements evoked by intracortical microstimulation had reduced thresholds and larger representational (motor map) areas during and following PH stimulation. These findings identify the sensorimotor cortices as major contributors for behavioral effects of PH stimulation, and that coincident increase in spiking, synaptic activity and MR signal reflect functional facilitation of neocortical output.

Differential impact of diabetes and hypertension in the brain: adverse effects in white matter.

Humans subjected to diabetes mellitus (DM) and/or hypertension (HTN) develop cognitive decline, cerebral atrophy and white matter abnormalities, but the relative effects of DM and HTN upon myelin and axonal integrity is unknown. We studied models of Type 1 (streptozotocin-induced) and Type 2 DM (ZDF) ± HTN (ZSF-1, SHR) in adult rats using magnetic resonance imaging (MRI) and structural and molecular techniques. Type 1 or 2 DM independently led to loss of myelin associated with changes with MRI T2 and magnetization tensor ratios throughout white matter regions. HTN's effect on myelin loss was minimal. Loss of oligodendroglia and myelin proteins was only identified in either Type 1 or Type 2 DM. Activation of the signal transduction pathways initiated by the receptor for advanced glycation end products (AGEs), RAGE, including upregulation of the signal transducer nuclear factor (NF) κB only occurred with DM. Diabetes is a greater contributor to white matter loss than hypertension in the rat brain, while hypertension only plays a mild additive effect upon neurodegeneration in the presence of diabetes.

Differential impact of diabetes and hypertension in the brain: adverse effects in grey matter.

Diabetes mellitus types 1 and 2 (DM1 and DM2) and/or hypertension (HTN) can contribute to cognitive decline, cerebral atrophy and white matter abnormalities in humans. Adult rat models of streptozotocin-induced DM1 and genetic strains of DM2 and HTN were used to investigate relative contributions of DM and HTN for alterations in cerebral structure and function as well as insulin receptor biology using cognitive testing, magnetic resonance imaging (MRI), and histological and molecular methods. The effects of DM1 or DM2 were generally similar. DM was associated with earlier onset of cognitive impairment than with HTN alone. DM was independently correlated with brain atrophy, whereas HTN had minimal effects on brain volume. The combination of DM and HTN led to identifiable mild hippocampal neuronal loss while either DM or HTN led to synaptic loss. Only DM led to downregulation of the insulin receptor pathways' activation. In contrast, only HTN was associated with vascular luminal reduction and restricted cerebral perfusion on MRI. The impacts of DM and HTN in the brain differ, while their separate contributions can lead to some additive adverse effects within rodent brain grey matter.

Persistent enhancement of functional MRI responsiveness to sensory stimulation following repeated seizures.

PURPOSE: Neural reorganization and interictal behavioral anomalies have been documented in people with epilepsy and in animal seizure models. Alterations in behavior could be due to somatosensory dysfunction. This study was designed to determine whether seizures can lead to changes in somatosensory representations and whether those changes are persistent. METHODS: Twice-daily seizures were elicited by delivering 1 s of electrical stimulation through carbon fiber electrodes implanted in both the corpus callosum and sensorimotor neocortex of young adult male Long-Evans rats until a total of 20 seizures were elicited. Either 1-3 days or 3-5 weeks following the last seizure, functional magnetic resonance imaging (MRI) was used to image the brain during electrical stimulation of each forepaw independently. KEY FINDINGS: Forepaw stimulation in control rats resulted in a focused and contralateral fMRI signal in the somatosensory neocortex. Rats that had repeated seizures had a 151% increase in the number of voxels activated in the contralateral hemisphere 1-3 days after the last seizure and a 166% increase at 3-5 weeks after the last seizure. The number of voxels activated in response to forepaw stimulation was positively correlated with the duration of the longest seizure experienced by each rat. The intensity of the activated voxels was not significantly increased at either time interval from the last seizure. SIGNIFICANCE: The increased area of activation in somatosensory cortex, which is persistent at 3-5 weeks, is consistent with previous observations of larger motor maps following seizures. Seizure-induced changes in the functioning of sensory cortex may also contribute to interictal behavioral anomalies.

Small unilamellar vesicles: a platform technology for molecular imaging of brain tumors.

Molecular imaging enables the non-invasive investigation of cellular and molecular processes. Although there are challenges to overcome, the development of targeted contrast agents to increase the sensitivity of molecular imaging techniques is essential for their clinical translation. In this study, spontaneously forming, small unilamellar vesicles (sULVs) (30 nm diameter) were used as a platform to build a bimodal (i.e., optical and magnetic resonance imaging (MRI)) targeted contrast agent for the molecular imaging of brain tumors. sULVs were loaded with a gadolinium (Gd) chelated lipid (Gd-DPTA-BOA), functionalized with targeting antibodies (anti-EGFR monoclonal and anti-IGFBP7 single domain), and incorporated a near infrared dye (Cy5.5). The resultant sULVs were characterized in vitro using small angle neutron scattering (SANS), phantom MRI and dynamic light scattering (DLS). Antibody targeted and nontargeted Gd loaded sULVs labeled with Cy5.5 were assessed in vivo in a brain tumor model in mice using time domain optical imaging and MRI. The results demonstrated that a spontaneously forming, nanosized ULVs loaded with a high payload of Gd can selectively target and image, using MR and optical imaging, brain tumor vessels when functionalized with anti-IGFBP7 single domain antibodies. The unique features of these targeted sULVs make them promising molecular MRI contrast agents.

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke.

BACKGROUND: The link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Slex) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T1 maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa). RESULTS: MRI confirmed similar infarct sizes and T1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (Kgd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P < 0.03). In the P selectin KO mice, Delta T1 stroke -Delta T1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLeX) group compared to Gd-DTPA, indicative of sLeX mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P < 0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P < 0.001) thereby supporting the observed increase in Kgd permeability in stroke brain of WT compared to KO mice. CONCLUSION: P-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.

Functional brain mapping at 9.4T using a new MRI-compatible electrode chronically implanted in rats.

There is a need for acute and chronic stimulation of the brain within the MRI for studies of epilepsy, as well as deep brain stimulation for movement and behavioral disorders. This work describes the production and characteristics of carbon fiber-based electrodes for acute and chronic stimulation in the brain. Increasing MRI field strengths are making it increasingly difficult to introduce foreign objects without a susceptibility artifact. We describe the production of, and the characteristics of carbon fiber-based electrodes. These are biocompatible and can be implanted for chronic studies. We show the use of these electrodes at 9.4T for studying functional activation. Data are presented showing regional connectivity. Activation not only occurs near the electrode, but at sites distant and often contralateral to the electrode. In addition, there were sites showing strong negative activation to stimulation both with direct stimulation and during a kindling-associated seizure.

X-linked inhibitor of apoptosis protein expression after ischemic injury in the human and rat developing brain.

X-linked inhibitor of apoptosis protein (XIAP) is a potent suppressor of neuronal death. The aim of this study was to investigate the expression of XIAP after ischemia in the human and rat developing brain. Autopsy specimens from 19 children with neuropathologic diagnosis of focal cerebral ischemic infarct were processed immunohistochemically for XIAP expression. XIAP positive cells were compared in pathologically classified acute (1-4 d), subacute (5-30 d), and chronic (months) strokes vs. age-matched controls with normal brain histology. For the animal studies, ischemia was induced in 1-wk-old rats by unilateral carotid artery occlusion and transient hypoxia. XIAP expression was quantified at four time points after ischemia in the infarct core and peri-infarct area. Neuronal XIAP expression was higher in the penumbra of subacute human infarcts compared with controls (p < 0.05). XIAP expression in the peri-infarct of rat pup was highest at 7 d postischemic injury (p < 0.05). The increase in XIAP expression was associated with a reduction in activated caspase-3 in ischemic neonatal rat brain. Our results demonstrate that XIAP expression postischemic injury is delayed in both species and may continue for several days. Therefore, potentiation of XIAP expression may be neuroprotective in the developing brain.

Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.

Insulin deficiency in type I diabetes may lead to cognitive impairment, cerebral atrophy and white matter abnormalities. We studied the impact of a novel delivery system using intranasal insulin (I-I) in a mouse model of type I diabetes (streptozotocin-induced) for direct targeting of pathological and cognitive deficits while avoiding potential adverse systemic effects. Daily I-I, subcutaneous insulin (S-I) or placebo in separate cohorts of diabetic and non-diabetic CD1 mice were delivered over 8 months of life. Radio-labelled insulin delivery revealed that I-I delivered more rapid and substantial insulin levels within the cerebrum with less systemic insulin detection when compared with S-I. I-I delivery slowed development of cognitive decline within weekly cognitive/behavioural testing, ameliorated monthly magnetic resonance imaging abnormalities, prevented quantitative morphological abnormalities in cerebrum, improved mouse mortality and reversed diabetes-mediated declines in mRNA and protein for phosphoinositide 3-kinase (PI3K)/Akt and for protein levels of the transcription factors cyclic AMP response element binding protein (CREB) and glycogen synthase kinase 3beta (GSK-3beta) within different cerebral regions. Although the murine diabetic brain was not subject to cellular loss, a diabetes-mediated loss of protein and mRNA for the synaptic elements synaptophysin and choline acetyltransferase was prevented with I-I delivery. As a mechanism of delivery, I-I accesses the brain readily and slows the development of diabetes-induced brain changes as compared to S-I delivery. This therapy and delivery mode, available in humans, may be of clinical utility for the prevention of pathological changes in the diabetic human brain.

Transient hypertension concurrent with forepaw stimulation enhances functional MRI responsiveness in infarct and peri-infarct regions.

Although functional magnetic resonance imaging (fMRI) is gaining use as a tool to assess cerebral recovery following various insults, the effects of potential confounders such as hypertension are poorly defined. We hypothesized that after stroke, transient hypertension during an fMRI study could produce a detected activation unrelated to neuronal activity within the infarct. Thus, the effect of norepinephrine induced increases in blood pressure (BP) on the fMRI response to forepaw stimulation were investigated in controls or 1 week after transient middle cerebral artery occlusion in rats. Images were smoothed spatially and voxels correlating to either forepaw stimulation or the change in BP time courses were analyzed. Transient hypertension increased the signal intensity and numbers of voxels correlating to the BP time courses within and adjacent to the ischemic infarct and these exceeded the response in the contralateral hemisphere or in controls. With left paw stimulation at normotension, there was a loss of activation in right sensory-motor cortex -- a region with necrosis and disruption of cerebral vessels. As BP increased left paw stimulation also resulted in the detection of activation in the infarcted sensory-motor cortex and peri-infarct regions. Thus, BP changes synchronous with tasks in fMRI studies can result in MR signal changes consistent with a loss of cerebral blood flow (CBF) autoregulation rather than neuronal activation in necrotic brain. After stroke, the use of stressful tasks associated with BP changes in fMRI studies should be limited or the BP change should be considered as a potential source of MR signal changes.

Blood-oxygen-level-dependent magnetic resonance signal and cerebral oxygenation responses to brain activation are enhanced by concurrent transient hypertension in rats.

Neuronal activation results in increases in blood-oxygen-level-dependent (BOLD) signal increases in magnetic resonance images, increases in cerebral blood flow (CBF), and changes in tissue oxygenation. We hypothesized that transient hypertension concurrent with neuronal activation would interfere with the normal physiological responses to neuronal activation potentially leading to additive responses. Anesthetized rats were prepared for functional magnetic resonance imaging studies in which increases in BOLD signal were measured in response to: (1) electrical forepaw stimulation, (2) different graded levels of transient hypertension produced with norepinephrine, and both 1 and 2. In other experiments with a similar protocol, changes in CBF and cortical oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb) were measured using Laser Doppler Flowmetry and near-infrared (IR) spectroscopy. BOLD signal within the sensory-motor cortex increased during forepaw stimulation. These matched increases in CBF and oxyHb and decreases in deoxyHb. During moderate or severe transient hypertension, there was a blood pressure-dependent increase in BOLD signal, CBF, and oxyHb; and a decrease in deoxyHb. When transient hypertension and forepaw stimulation were combined, the responses of oxyHb, deoxyHb, or BOLD signal were generally a summation of each response. In contrast, the CBF response to forepaw stimulation was relatively unaffected by transient hypertension. We conclude that during stimulation with concurrent hypertension, the normal changes in tissue oxygenation that accompany neuronal activation are enhanced by the increases produced by hypertension despite an excellent autoregulation of CBF. The latter could reflect highly transient decreases in oxygen consumption or likely a redistribution of flow through more nonexchange vessels.

Combined damage produced by multiple mild cerebral insults assessed using MRI in neonatal rats.

PURPOSE: To determine whether damage to neonatal brain is exacerbated with multiple mild cerebral insults as detected with MRI and corroborated using histology. MATERIALS AND METHODS: The combined brain injury produced by multiple procedures was compared in neonatal rats having: Sham surgery at P5, Sham surgery at P5 plus a diffuse mild transient unilateral cerebral hypoxia ischemia (HI) at P7, HI alone, and a minor photothrombotic (PT) stroke at P5 followed by HI. MRI after the ischemic insults was followed by final histology. RESULTS: PT produced lesions with increased T2 and decreased apparent diffusion coefficient for water (ADC) but no significant effects of a second HI. However, near the PT lesion/parietal cortex there were patchy areas of enhanced T2 and decreased ADC in 6/9, 3/8 and 0/8 animals in the PT+HI, Sham+HI and HI groups, respectively (P<0.05). Patches corresponded histologically to increased vacuolation and cell death and were more pronounced in the PT+HI and Sham+PT groups than the HI group. CONCLUSION: The extent of damage produced by a minor neonatal stroke followed by a diffuse HI two days later results in heterogeneous enhancement of T2, ADC and histological injury near the lesion. Surgical procedures including mechanical head manipulation followed by HI also produced some enhanced heterogeneity of hypoxic-ischemic injury affirming the need for sham controls.