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.

Diets enriched in whey or casein improve energy balance and prevent morbidity and renal damage in salt-loaded and high-fat-fed spontaneously hypertensive stroke-prone rats.

High-fat diets induce obesity and increase risks of diabetes and cardiovascular and renal disorders. Whey- or casein-enriched diets decrease food intake and weight gain; however, their cardiovascular and renal benefits are unclear. We determined whether whey- and casein-enriched diets improve energy balance and are protective against renal damage and morbidity associated with stroke in an obesogenic and hypertensive experimental setting. We also assessed whether the hypophagic effects of these diets were due to reduced diet preference. In experiment 1, spontaneously hypertensive stroke-prone rats were randomized to (a) control (CON; 14% kcal protein, 33% fat), (b) whey (WHY; 40% protein, 33% fat), (c) casein (CAS; 40% protein, 33% fat) or (d) chow (CHW; 24% protein, 13% fat) for 12 weeks with 1% salt in drinking water for CON, WHY and CAS groups. Our results demonstrated that both WHY and CAS produced short-term hypophagia, moderately increased energy expenditure and decreased respiratory quotient, body weight and lean mass, with effects of WHY being more prolonged. Further, only WHY decreased fat mass and blood pressure. Importantly, both WHY and CAS prevented morbidity associated with stroke and decreased indices of renal inflammation (tumor necrosis factor-α, interleukin-6) and damage (osteopontin, renal lesions). In experiment 2, following four initial conditioning trials, the preference for CON, WHY or CAS diet was determined. Both WHY and CAS decreased food intake during conditioning and decreased preference. In conclusion, diets enriched in whey or casein improved energy balance, increased survival and prevented renal damage in salt-loaded and high-fat-fed spontaneously hypertensive stroke-prone rats.

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.

Protection against recurrent stroke with resveratrol: endothelial protection.

Despite increased risk of a recurrent stroke following a minor stroke, information is minimal regarding the interaction between injurious mild cerebral ischemic episodes and the possible treatments which might be effective. The aim of the current study was to investigate recurrent ischemic stroke and whether resveratrol, a nutritive polyphenol with promising cardio- and neuro- protective properties, could ameliorate the associated brain damage. Experiments in adult rats demonstrated that a mild ischemic stroke followed by a second mild cerebral ischemia exacerbated brain damage, and, daily oral resveratrol treatment after the first ischemic insult reduced ischemic cell death with the recurrent insult (P<0.002). Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. The protection observed with resveratrol treatment could not be explained by systemic effects of resveratrol treatment including effects either on blood pressure or body temperature measured telemetrically. Investigation of resveratrol effects on the blood-brain barrier in vivo demonstrated that resveratrol treatment reduced blood-brain barrier disruption and edema following recurrent stroke without affecting regional cerebral blood flow. Investigation of the mechanism in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against an in vitro 'ischemia' resulting in improved viability against oxygen and glucose deprivation (39.6 ± 6.6% and 81.3 ± 9.5% in vehicle and resveratrol treated cells, respectively). An inhibition of nitric oxide synthesis did not prevent the improved cell viability following oxygen glucose deprivation but SIRT-1 inhibition with sirtinol partially blocked the protection (P<0.001) suggesting endothelial protection is to some extent SIRT-1 dependent. Collectively, the results support that oral resveratrol treatment provides a low risk strategy to protect the brain from enhanced damage produced by recurrent stroke which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature.

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.

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.

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.

Mild cerebral hypoxia-ischemia produces a sub-acute transient inflammatory response that is less selective and prolonged after a substantial insult.

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia-ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1-2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1-2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter. Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.

Functional magnetic resonance imaging within the rat spinal cord following peripheral nerve injury.

Functional magnetic resonance imaging (fMRI) was used to detect the effects of graded peripheral nerve injury at the spinal level. Graded peripheral nerve injury in rats was accomplished by transection of nerves entering the spinal cord at the L3 and L4 levels of the spinal cord segments. Electrical stimulation of the hindpaw was used to elicit activity within the spinal cord. The stimulation experimental paradigm consisted of 62 functional images, 5 slices each, with a total of 3 rest and 2 stimulation periods. A 9.4 T MRI system and a quadrature volume rf coil covering the lumbar spinal cord were used for the fMRI study. Sets of fast spin echo images were acquired repeatedly following sham preparatory surgery under control conditions and in rats following sham surgery (pre nerve cut), followed by L3 nerve and then L4 nerve section. In rats with sham surgery, there was a significant activation within the dorsal horn of slices corresponding to L3 and L4 spinal cord segments. Following section of the L3 nerve, there was a reduction in the number of active voxels in the L3 and L4 spinal cord segments. The activation was reduced further by sectioning of the L4 nerve. Thus, following an increasing loss of axonal connections to the spinal cord, there was a decreasing number of active voxels within the spinal cord. The results demonstrate that spinal fMRI in the rat has sufficient sensitivity to detect within the spinal cord the effects of a graded reduction in peripheral connectivity.

White matter damage precedes that in gray matter despite similar magnetic resonance imaging changes following cerebral hypoxia-ischemia in neonatal rats.

We hypothesized that the cerebral injury produced by hypoxia-ischemia (HI) in neonatal rats would differ in white compared with gray matter as detected histologically or with magnetic resonance (MR) imaging methods. Maps of T2 and the apparent diffusion coefficient (ADC) of water were acquired in 1-week-old rats at times prior to cerebral HI (right carotid artery occlusion plus 1.5 h of hypoxia), within the last 5-10 min of HI, and 1 h or 24 h after HI. Near the end of HI, ADC decreased and T2 increased in both cortical gray and subcortical white matter within the cingulum of the HI hemisphere. One hour after HI, ADC partially recovered, but T2 remained increased and then increased further by 24 h post-HI. In contrast to the similar MR responses in white and gray matter, histological evidence for irreversible cell damage occurred in white matter earlier than in gray matter within the HI hemisphere. At 1 h post-HI, rarefied or disrupted nerve fibers and an increase in TUNEL-positive cells were observed within white matter in the cingulum, whereas neurons within the cortical gray matter appeared normal. By 24 h post-HI, damage was apparent in both white and gray matter. Thus, MR imaging detected acute tissue edema following cerebral HI in both gray and white matter but did not distinguish between the early irreversible tissue injury detected histologically in white but not gray matter in this rather severe model of neonatal encephalopathy.

Transient hypoxia-ischemia in rats: changes in diffusion-sensitive MR imaging findings, extracellular space, and Na+-K+ -adenosine triphosphatase and cytochrome oxidase activity.

PURPOSE: To investigate the correlation between diffusion-weighted (DW) magnetic resonance (MR) image changes with alterations in extracellular volume and changes in cytochrome oxidase and Na(+)-K(+)-adenosine triphosphatase (ATPase) activity at various times during and after cerebral hypoxia-ischemia in neonatal and juvenile rats. MATERIALS AND METHODS: One- and 4-week-old rats were randomly assigned to control or transient cerebral hypoxia-ischemia (ie, right carotid artery occlusion plus exposure to 8% oxygen) groups. Hypoxic-ischemic changes compared with normal ipsilateral brain tissue on DW images and the apparent diffusion coefficient of water were measured during and at 1 and 24 hours after hypoxia-ischemia ended. Hypoxic-ischemic changes in extracellular space and ipsilateral versus contralateral differences in Na(+)-K(+)-ATPase and cytochrome oxidase activity were measured. RESULTS: Hyperintensities on DW images obtained during hypoxia-ischemia correlated well (P <.05) with extracellular space reductions, which occurred 15 minutes earlier in the brains of 4-week-old rats than in the brains of 1-week-old rats. Similarly, within 1 hour after hypoxia-ischemia ended, DW image and extracellular space changes normalized. In contrast, Na(+)-K(+)-ATPase and cytochrome oxidase activity decreased in some regions during hypoxia-ischemia and remained reduced 1 hour after the end of hypoxia-ischemia. Twenty-four hours after signal intensity normalization, hyperintense areas reappeared on DW images, and Na(+)-K(+)-ATPase and cytochrome oxidase activity remained decreased. CONCLUSION: Signal intensity alterations with diffusion-sensitive MR imaging during and after transient hypoxia-ischemia are closely associated with a corresponding shrinkage and reexpansion of the extracellular space, irrespective of age. Mechanisms other than Na(+)-K(+)-ATPase changes may induce the early cell volume changes detected with diffusion-sensitive MR imaging.