Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy.

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

Neuroprotection by dimethyloxalylglycine following permanent and transient focal cerebral ischemia in rats.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1α levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.

Eotaxin and FGF enhance signaling through an extracellular signal-related kinase (ERK)-dependent pathway in the pathogenesis of Eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by the inflammation of the esophagus and the infiltration of eosinophils into the esophagus, leading to symptoms such as dysphagia and stricture formation. Systemic immune indicators like eotaxin and fibroblast growth factor were evaluated for possible synergistic pathological effects. Moreover, blood cells, local tissue, and plasma from EoE and control subjects were studied to determine if the localized disease was associated with a systemic effect that correlated with presence of EoE disease. METHOD: Real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMC), immunohistochemistry from local esophageal biopsies, fluid assays on plasma, and fluorescence-activated cell sorting on peripheral blood cells from subjects were used to study the systemic immune indicators in newly diagnosed EoE (n = 35), treated EoE (n = 9), Gastroesophageal reflux disease (GERD) (n = 8), ulcerative colitis (n = 5), Crohn's disease (n = 5), and healthy controls (n = 8). RESULT: Of the transcripts tested for possible immune indicators, we found extracellular signal-regulated kinase (ERK), Bcl-2, bFGF (basic fibroblast growth factor), and eotaxin levels were highly upregulated in PBMC and associated with disease presence of EoE. Increased FGF detected by immunohistochemistry in esophageal tissues and in PBMC was correlated with low levels of pro-apoptotic factors (Fas, Caspase 8) in PBMC from EoE subjects. Plasma-derived bFGF was shown to be the most elevated and most specific in EoE subjects in comparison to healthy controls and disease control subjects. CONCLUSION: We describe for the first time a possible mechanism by which increased FGF is associated with inhibiting apoptosis in local esophageal tissues of EoE subjects as compared to controls. Eotaxin and FGF signaling pathways share activation through the ERK pathway; together, they could act to increase eosinophil activation and prolong the half-life of eosinophils in local tissues of the esophagus in EoE subjects.

Molecular magnetic resonance imaging of acute vascular cell adhesion molecule-1 expression in a mouse model of cerebral ischemia.

The pathogenesis of stroke is multifactorial, and inflammation is thought to have a critical function in lesion progression at early time points. Detection of inflammatory processes associated with cerebral ischemia would be greatly beneficial in both designing individual therapeutic strategies and monitoring outcome. We have recently developed a new approach to imaging components of the inflammatory response, namely endovascular adhesion molecule expression on the brain endothelium. In this study, we show specific imaging of vascular cell adhesion molecule (VCAM)-1 expression in a mouse model of middle cerebral artery occlusion (MCAO), and a reduction in this inflammatory response, associated with improved behavioral outcome, as a result of preconditioning. The spatial extent of VCAM-1 expression is considerably greater than the detectable lesion using diffusion-weighted imaging (25% versus 3% total brain volume), which is generally taken to reflect the core of the lesion at early time points. Thus, VCAM-1 imaging seems to reveal both core and penumbral regions, and our data implicate VCAM-1 upregulation and associated inflammatory processes in the progression of penumbral tissue to infarction. Our findings indicate that such molecular magnetic resonance imaging (MRI) approaches could be important clinical tools for patient evaluation, acute monitoring of therapy, and design of specific treatment strategies.

Therapeutic hypothermia in experimental models of focal and global cerebral ischemia and intracerebral hemorrhage.

Experimental evidence shows that therapeutic hypothermia (TH) protects the brain from cerebral injury in multiple ways. In different models of focal and global cerebral ischemia, mild-to-moderate hypothermia reduces mortality and neuronal injury and improves neurological outcome. In models of experimental intracerebral hemorrhage (ICH), TH reduces edema formation but does not show consistent benefi cial effects on functional outcome parameters. However, the number of studies of hypothermia on ICH is still limited. TH is most effective when applied before or during the ischemic event, and its neuroprotective properties vary according to species, strains and the model of ischemia used. Intrinsic changes in body and brain temperature frequently occur in experimental models of focal and global cerebral ischemia, and may have infl uenced studies on other neuroprotectants. This might be one explanation for the failure of a large amount of translational clinical neuroprotective trials. Hypothermia is the only neuroprotective therapeutic agent for cerebral ischemia that has successfully managed the transfer from bench to bedside, and it is an approved therapy for patients after cardiac arrest and children with hypoxic-ischemic encephalopathy. However, the implementation of hypothermia in the treatment of stroke patients is still far from routine clinical practice. In this article, the authors describe the development of TH in different models of focal and global cerebral ischemia, point out why hypothermia is so efficient in experimental cerebral ischemia, explain why temperature regulation is essential for further neuroprotective studies and discuss why TH for acute ischemic stroke still remains a promising but controversial therapeutic option.

Improved regional cerebral blood flow is important for the protection seen in a mouse model of late phase ischemic preconditioning.

INTRODUCTION: Ischemic preconditioning (IPC) induces protection to cerebral ischemia. However, it was previously unclear whether this protection resulted from altered susceptibility to ischemia. The current study examines the effects of late phase ischemic preconditioning in a mouse model of middle cerebral artery occlusion (MCAO). Specific examination of the regional cerebral blood flow (rCBF) was conducted. EXPERIMENTAL PROCEDURE: Intra-abdominal radiofrequency probes were implanted in animals and core temperature was regulated. Mice were subjected to MCAO: (1) brief 15 min duration (preconditioning ischemia) and (2) 45 min MCAO (injurious ischemia). Naive (i.e. not preconditioned) animals were compared with preconditioned animals (preconditioning ischemia plus injurious ischemia at 72 h reperfusion). rCBF was measured using laser Doppler flowmetry (LDF) and magnetic resonance cerebral perfusion (MRP) arterial spin labeling. Percentage of brain infarcted was compared between groups. RESULTS: rCBF was significantly improved in the preconditioned cohorts of mice. Naive animals showed flow reductions to 16+/-3.59% (MCAO_45; injurious, unpreconditioned) and 17.1+/-8.6% (MCAO_15; preconditioning ischemia alone) of baseline, while preconditioned animals had flows 33.9+/-13.2% (IPC_45; preconditioned animals with injurious ischemia at 72 h reperfusion) of baseline (p=0.001). Percentage of brain infarcted was 17.2+/-6.2% in naive animals, while it was 5.1+/-4.6% in the preconditioned animals (p=0.003). MRP of the perfusion to the ischemic hemisphere, in a striatal coronal slice of the brain was 26.7+/-5.8% of the contralateral hemisphere in naive animals while preconditioned mice had flows of 38.7+/-6.8% of contralateral (p=0.04). CONCLUSIONS: Improved rCBF is an important factor in the protection of IPC, during injurious MCAO in the mouse. Stringent monitoring of rCBF is required in future studies of IPC.

Early T1- and T2-weighted MRI signatures of transient and permanent middle cerebral artery occlusion in a murine stroke model studied at 9.4T.

Early reperfusion following stroke results in reduced tissue injury. Paradoxically, restoration of blood flow under certain conditions may also cause delayed neuronal damage (reperfusion injury). The interrelationship of changes in T1, T2 and diffusion weighted images of tissue water were studied in mouse models of permanent and transient focal cerebral ischemia. A sham surgery or either permanent or transient (30 min) middle cerebral artery occlusion (MCAO) were induced in 14 mice. Magnetic resonance (MR) images of the brain were acquired including: T2 maps, T1 maps and diffusion weighted spin-echo images to produce apparent diffusion coefficient of water apparent diffusion coefficient (ADC) maps. Images were collected on average 90 min after MCAO in both the transient and permanent ischemia groups. Scans were repeated at 24h post-occlusion in mice with transient ischemia. Permanent MCAO resulted in decreases in ADC and no significant change in T2 acutely following MCAO. There were increases in T1 compared to sham controls within the ischemic region in mice following either transient or permanent MCAO (P<0.001). In contrast to permanent MCAO, there were increases in T2 (P<0.001) in the infarct area present in the reperfusion phase within 90 min of transient MCAO. There was considerable infarct growth at 24h (P<0.001). This study demonstrates that following either type of occlusion there are early increases in T1 suggesting an elevated water content in the stroke lesion, while only following transient MCAO are there early increases in T2, indicative of early vasogenic oedema with breakdown of the blood-brain barrier.

MR molecular imaging of early endothelial activation in focal ischemia.

Focal ischemia followed by reperfusion initiates a harmful P- and E-selectin-mediated recruitment of leukocytes in brain microvasculature. In this study, we tested whether a novel magnetic resonance (MR) contrast agent (Gd-DTPA-sLe(x) A), which is designed to bind to activated endothelium could be detected by MR imaging (MRI) in a focal stroke mouse model. MRIs (9.4T) of the brain were acquired 24 hours after transient middle cerebral artery occlusion. T1 maps were acquired repeatedly before and up to 1.5 hours after the intravenous injection of either Gd-DTPA or Gd-DTPA-sLe(x) A. Analysis of images included a pixel-by-pixel subtraction of T1 maps from the precontrast T1 maps and quantification of T1 within the ischemic area. After injection of Gd-DTPA-sLe(x) A, T1 decreased compared with precontrast levels, and an interhemispheric difference between the pre-post contrast T1 developed within the stroke lesion at a mean time of 52 minutes after injection (p < 0.05). Animals injected with Gd-DTPA did not exhibit changes in T1 signal intensity between regions of the ipsilateral and contralateral hemispheres, indicating that the reductions in T1 observed with Gd-DTPA-sLe(x) A were unrelated to blood-brain barrier breakdown. Fluorescent-labeled sLe(x) A administered intravenously was observed to bind to the endothelium of injured but not control brain. The study suggests that the contrast agent Gd-DTPA-sLe(x) A can be used to visualize early endothelial activation after transient focal ischemia in vivo with MRI.

Temperature-regulated model of focal ischemia in the mouse: a study with histopathological and behavioral outcomes.

BACKGROUND AND PURPOSE: The importance of mouse stroke models has increased with the development of genetically manipulated animals. We hypothesized that immediate postischemia hypothermia may attenuate ischemic brain injury in the mouse. METHODS: Intraabdominal radio frequency probes were implanted in animals and core temperature monitored. Groups included: MCAO-45-REG (45 minutes middle cerebral artery occlusion [MCAO]) temperature-controlled in the postischemic period >34 degrees C for 24 hours; MCAO-45 (45 minutes MCAO) were allowed to self-regulate core temperature during recovery; MCAO-30-REG (30 minutes MCAO), with the same temperature protocol as MCAO-45-REG; MCAO-30 (30 minutes MCAO), with temperature protocol the same as MCAO-45. Behavior and histological score was assessed at 7 days. The qualitative histological score assessed for injury in 18 specified regions. RESULTS: MCAO-45-REG core temperature (mean 34.94 degrees C+/-0.8 degrees C) was significantly different than the self-regulating (MCAO-45, mean 33.1 degrees C+/-2.3 degrees C) for the first 4 hours after anesthesia (P<0.01). There was a trend toward similar differences in temperatures for MCAO-30-REG and MCAO-30 (P=0.08). At 7 days, a greater improvement in behavior score was observed for MCAO-45 and MCAO-30 compared with MCAO-45-REG and MCAO-30-REG (P<0.001). The histological score confirmed reduced injury in unregulated temperature groups (MCAO-45-REG mean 38+/-10 and MCAO-45 30+/-5.1, P<0.05; MCAO-30-REG 41+/-10 and MCAO-30 30+/-9, P<0.05). CONCLUSIONS: Hypothermia is an important confounder of stroke injury in the intraluminal filament mouse model. Future mouse stroke studies must use strict temperature regulation.