Midori A. Yenari

Correlation of perfusion- and diffusion-weighted MRI with NIHSS score in acute (<6.5 hour) ischemic stroke

Background: Diffusion-weighted (DWI) and perfusion-weighted (PWI) MRI are powerful new techniques for the assessment of acute cerebral ischemia. However, quantitative data comparing the severity of clinical neurologic deficit with the results of DWI or PWI in the earliest phases of stroke are scarce. Such information is vital if MRI is potentially to be used as an objective adjunctive measure of stroke severity and outcome. Objective: The authors compared initial DWI and PWI lesion volumes with subsequent 24-hour neurologic deficit as determined by National Institutes of Health Stroke Scale (NIHSS) score in acute stroke patients. Initial DWI and PWI volumes were also compared with T2W MRI lesion volume at 1 week to assess the accuracy of these MRI techniques for the detection of acute cerebral ischemia. Methods: Patients with stroke underwent MRI scanning within 6.5 hours of symptom onset. Lesion volumes on DWI and PWI were measured and compared with 24-hour NIHSS score. Initial DWI and PWI volumes were also compared with T2W lesion size at 1 week. Results: There was a high correlation between 24-hour NIHSS score and lesion volume as determined by PWI (r = 0.96, p < 0.001) or DWI(r = 0.67, p = 0.03). A similar high correlation was seen between T2W stroke size at 7 days and initial DWI and PWI lesion size(r = 0.99, p < 0.00001). Conclusions: Both DWI and PWI are highly correlated with severity of neurologic deficity by 24-hour NIHSS score. These findings may have substantial implications for the use of MRI scanning in the assessment and management of acute stroke patients.

Serial MRI After Transient Focal Cerebral Ischemia in Rats: Dynamics of Tissue Injury, Blood-Brain Barrier Damage, and Edema Formation

BACKGROUND AND PURPOSE With the advent of thrombolytic therapy for acute stroke, reperfusion-associated mechanisms of tissue injury have assumed greater importance. In this experimental study, we used several MRI techniques to monitor the dynamics of secondary ischemic damage, blood-brain barrier (BBB) disturbances, and the development of vasogenic edema during the reperfusion phase after focal cerebral ischemia in rats. METHODS Nineteen Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion of 30 minutes, 60 minutes, or 2.5 hours with the suture occlusion model. MRI, including diffusion-weighted imaging (DWI), T2-weighted imaging, perfusion-weighted imaging, and T1-weighted imaging, was performed 5 to 15 minutes before reperfusion, as well as 0.5, 1.5, and 2.5 hours and 1, 2, and 7 days after withdrawal of the suture. Final infarct size was determined histologically at 7 days. RESULTS In the 30-minute ischemia group (and partially also after 60 minutes), DWI abnormalities reversed transiently during the early reperfusion period but recurred after 1 day, probably due to secondary ischemic damage. After 2.5 hours of ischemia, DWI abnormalities no longer reversed, and signal intensity on both DWI and T2-weighted images increased rapidly in the previously ischemic region due to BBB damage (enhancement on postcontrast T1-weighted images) and edema formation. Early BBB damage during reperfusion was found to be predictive of relatively pronounced edema at subacute time points and was probably related to the increased mortality rates in this experimental group (3 of 7). CONCLUSIONS Reperfusion after short periods of ischemia (30 to 60 minutes) appears to be mainly complicated by secondary ischemic damage as shown by the delayed recurrence of the DWI lesions, whereas BBB damage associated with vasogenic edema becomes a dominant factor with longer occlusion times (2.5 hours).

Optimal Depth and Duration of Mild Hypothermia in a Focal Model of Transient Cerebral Ischemia Effects on Neurologic Outcome, Infarct Size, Apoptosis, and Inflammation

BACKGROUND AND PURPOSE Mild hypothermia is possibly the single most effective method of cerebroprotection developed to date. However, many questions regarding mild hypothermia remain to be addressed before its potential implementation in the treatment of human stroke. Here we report the results of 2 studies designed to determine the optimal depth and duration of mild hypothermia in focal stroke and its effects on infarct size, neurological outcome, programmed cell death, and inflammation. METHODS Rats underwent a 2-hour occlusion of the left middle cerebral artery. In the first study (I) animals were kept (intraischemically) at either 37 degreesC (n=8), 33 degreesC (n=8), or 30 degreesC (n=8). Study II consisted of 4 groups: (1) controls (37 degreesC, n=10), (2) 30 minutes of hypothermia started at ischemic onset (33 degreesC, n=9), (3)1 hour (33 degreesC, n=8), and (4) 2 hours (33 degreesC, n=8). Brain temperature was measured by a thermocouple probe placed in the contralateral cortex. After suture removal, all animals were rewarmed and reperfused for 22 hours (I) or 70 hours (II). RESULTS Mild hypothermia to 33 degreesC or 30 degreesC was neuroprotective (17+/-7% and 27+/-6%, respectively) relative to controls (53+/-8%, P<0.02), but 33 degreesC was better tolerated and recovery from anesthesia was faster. The neurological score of hypothermic animals was significantly better than that of controls (I & II) at both 24 and 72 hours postischemia except for the 30-minute group (II), which showed no improvement. In Study II, 2 hours of hypothermia reduced injury by 59%, 1 hour reduced injury by 84% whereas 30 minutes did not reduce injury. Normalized for infarct size, 2 hours of mild hypothermia decreased neutrophil accumulation by 57% whereas both 1 hour and 30 minutes had no effect. At 72 hours, 1 and 2 hours of mild hypothermia decreased transferase dUTP nick-end labeling (TUNEL) staining by 78% and 99%, respectively, and 30 minutes of hypothermia had no effect. CONCLUSIONS Intraischemic mild hypothermia must be maintained for 1 to 2 hours to obtain optimal neuroprotection against ischemic cell death due to necrosis and apoptosis.

Neuroprotective mechanisms of hypothermia in brain ischaemia

Cooling can reduce primary injury and prevent secondary injury to the brain after insults in certain clinical settings and in animal models of brain insult. The mechanisms that underlie the protective effects of cooling — also known as therapeutic hypothermia — are slowly beginning to be understood. Hypothermia influences multiple aspects of brain physiology in the acute, subacute and chronic stages of ischaemia. It affects pathways leading to excitotoxicity, apoptosis, inflammation and free radical production, as well as blood flow, metabolism and blood–brain barrier integrity. Hypothermia may also influence neurogenesis, gliogenesis and angiogenesis after injury. It is likely that no single factor can explain the neuroprotection provided by hypothermia, but understanding its myriad effects may shed light on important neuroprotective mechanisms.

Post-ischemic inflammation: molecular mechanisms and therapeutic implications

Abstract Ischemic stroke is characterized by the disruption of cerebral blood flow (CBF). This reduction of CBF results in energy failure and secondary biochemical disturbances, eliciting a robust in situ inflammatory response. Post-ischemic inflammation is a dynamic process involving a complicated set of interactions among various inflammatory cells and molecules. The resident inflammatory brain cells, microglia, are especially activated in response to ischemic insults, many of which are regulated by nuclear transcription factor, kappa B (NF-kB). As a result, several inflammatory genes are expressed, leading to local generation of various cytokines, which in turn promulgate inflammatory signals. Meanwhile, endothelial cells lining the local cerebral blood vessels are stimulated to produce adhesion molecules, causing the migration of peripheral circulating leukocytes into the compromised brain tissue, an event that amplifies inflammatory signaling cascades. Post-ischemic inflammation appears to serve multiple purposes, depending on its timing and magnitude, as well as the topographic distribution of various inflammatory molecules. Data from experimental manipulations of some inflammatory molecules are yielding insight into therapeutic strategies for ischemic stroke. This review focuses on some recent advances regarding the regulation of inflammatory signaling pathways, the detrimental effects of post-ischemic inflammation and the potential molecular targets for ischemic stroke therapy.

Microglia Potentiate Damage to Blood–Brain Barrier Constituents Improvement by Minocycline In Vivo and In Vitro

Background— Blood–brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models. Methods and Results— Primary astrocytes, ECs, or cocultures were prepared with or without added microglia. Primary ECs were more resistant to oxygen-glucose deprivation/reperfusion than astrocytes. ECs plus astrocytes showed intermediate vulnerability. Microglia added to cocultures nearly doubled cell death. This increase was prevented by minocycline and apocynin. In vivo, minocycline reduced infarct volume and neurological deficits and markedly reduced BBB disruption and hemorrhage in mice after experimental stroke. Conclusions— Inhibition of microglial activation may protect the brain after ischemic stroke by improving BBB viability and integrity. Microglial inhibitors may prove to be an important treatment adjunct to fibrinolysis.

Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity

Bcl‐2 protects against both apoptotic and necrotic death induced by several cerebral insults. We and others have previously demonstrated that defective herpes simplex virus vectors expressing Bcl‐2 protect against various insults in vitro and in vivo, including cerebral ischemia. Because the infarct margin may be a region that is most amenable to treatment, we first determined whether gene transfer to the infarct margin is possible using a focal ischemia model. Since ischemic injury with and without reperfusion may occur by different mechanisms, we also determined whether Bcl‐2 protects against focal cerebral ischemic injury either with or without reperfusion in rats. Bax expression, cytochrome c translocation and activated caspase‐3 expression were also assessed. Viral vectors overexpressing Bcl‐2 were delivered to the infarct margin. Reperfusion resulted in larger infarcts than permanent occlusion. Bcl‐2 overexpression significantly improved neuron survival in both ischemia models. Bcl‐2 overexpression did not alter overall Bax expression, but inhibited cytosolic accumulation of cytochrome c and caspase‐3 activation. Thus, we provide the first evidence that gene transfer to the infarct margin is feasible, that overexpression of Bcl‐2 protects against damage to the infarct margin induced by ischemia with and without reperfusion, and that Bcl‐2 overexpression using gene therapy attenuates apoptosis‐related proteins. This suggests a potential therapeutic strategy for stroke.

A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia.

Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells‐2 (TREM2) promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2‐Fc identified TREM2 ligands (TREM2‐L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2‐L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti‐TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2‐L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti‐TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2‐L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2‐L form a receptor–ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.

The neuroprotective potential of heat shock protein 70 (HSP70)

In response to many metabolic disturbances and injuries, including stroke, neurodegenerative disease, epilepsy and trauma, the cell mounts a stress response with induction of a variety of proteins, most notably the 70-kDa heat shock protein (HSP70). Whether stress proteins are neuroprotective has been hotly debated, as these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury. A few groups have now shown that overproduction of HSP70 leads to protection in several different models of nervous system injury. This review will cover these studies, along with the potential mechanisms by which HSP70 might mediate cellular protection.

Antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection.

Abstract: We and others have previously shown that heat‐shock proteins (HSPs) are involved in protecting the brain from a variety of insults including stroke, epilepsy, and other related insults. While the mechanism of this protection has largely been thought to be due to their chaperone functions (i.e., preventing abnormal protein folding or aggregation), recent work has shown that HSPs may also directly interfere with other cell death pathways such as apoptosis and inflammation. Using models of cerebral ischemic and ischemia‐like injury, we overexpressed the 70‐kDa heat‐shock protein (HSP70) using gene transfer or by studying a transgenic mouse model. HSP70 protected neurons and astrocytes from experimental stroke and stroke‐like insults. HSP70 transgenic mice also had better neurological scores following experimental stroke compared to their wild‐type littermates. Overexpressing HSP70 was associated with less apoptotic cell death and increased expression of the antiapoptotic protein, Bcl‐2. Furthermore, HSP70 suppressed microglial/monocyte activation following experimental stroke. HSP70 overexpression also led to the reduction of matrix metalloproteinases. We suggest that HSPs are capable of protecting brain cells from lethal insults through a variety of mechanisms and should be explored as a potential therapy against stroke and other neurodegenerative diseases.