Khalid A. Hanafy

The role of microglia and the TLR4 pathway in neuronal apoptosis and vasospasm after subarachnoid hemorrhage

BackgroundAlthough microglia and the Toll-like receptor (TLR) pathway have long been thought to play a role in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), thus far only correlations have been made. In this study, we attempted to solidify the relationship between microglia and the TLR pathway using depletion and genetic knockouts, respectively.MethodsSubarachnoid hemorrhage was induced in TLR4−/−, TRIF−/−, MyD88−/− and wild type C57BL/6 mice by injecting 60 μl of autologous blood near the mesencephalon; animals were euthanized 1 to 15 days after SAH for immunohistochemical analysis to detect microglia or apoptotic cells. Lastly, microglial depletion was performed by intracerebroventricular injection of clodronate liposomes.ResultsOn post operative day (POD) 7 (early phase SAH), neuronal apoptosis was largely TLR4-MyD88-dependent and microglial-dependent. By POD 15 (late phase SAH), neuronal apoptosis was characterized by TLR4- toll receptor associated activator of interferon (TRIF)-dependence and microglial-independence. Similarly, vasospasm was also characterized by an early and late phase with MyD88 and TRIF dependence, respectively. Lastly, microglia seem to be both necessary and sufficient to cause vasospasm in both the early and late phases of SAH in our model.ConclusionOur results suggest that SAH pathology could have different phases. These results could explain why therapies tailored to aSAH patients have failed for the most part. Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient’s hospital course could improve outcomes.

Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase-1

Subarachnoid hemorrhage (SAH) carries a 50% mortality rate. The extravasated erythrocytes that surround the brain contain heme, which, when released from damaged red blood cells, functions as a potent danger molecule that induces sterile tissue injury and organ dysfunction. Free heme is metabolized by heme oxygenase (HO), resulting in the generation of carbon monoxide (CO), a bioactive gas with potent immunomodulatory capabilities. Here, using a murine model of SAH, we demonstrated that expression of the inducible HO isoform (HO-1, encoded by Hmox1) in microglia is necessary to attenuate neuronal cell death, vasospasm, impaired cognitive function, and clearance of cerebral blood burden. Initiation of CO inhalation after SAH rescued the absence of microglial HO-1 and reduced injury by enhancing erythrophagocytosis. Evaluation of correlative human data revealed that patients with SAH have markedly higher HO-1 activity in cerebrospinal fluid (CSF) compared with that in patients with unruptured cerebral aneurysms. Furthermore, cisternal hematoma volume correlated with HO-1 activity and cytokine expression in the CSF of these patients. Collectively, we found that microglial HO-1 and the generation of CO are essential for effective elimination of blood and heme after SAH that otherwise leads to neuronal injury and cognitive dysfunction. Administration of CO may have potential as a therapeutic modality in patients with ruptured cerebral aneurysms.

Regulation of remyelination in multiple sclerosis

Multiple sclerosis is a common demyelinating disease that worsens over the course of disease, a significant problem in clinical management. Disability in MS is significantly promoted by poor repair and remyelination of lesions. Both oligodendrocyte recruitment and maturation defects are seen as major causes of poor remyelination in MS. The mechanisms behind impaired remyelination in animal models include involvement of the Notch1, wnt, and hyaluronan/TLR2 pathways. RXR/PPAR signaling has also more recently been identified as an important regulator of remyelination. The local inflammatory milieu also appears to play critical and conflicting roles in promotion and inhibition of remyelination in MS. Understanding the forces regulating remyelination in MS represents an exciting and important initial step towards developing therapeutics targeting chronic disability in MS.

Continuous EEG monitoring: is it ready for prime time?

Purpose of reviewContinuous electroencephalography (cEEG) is being used more frequently in intensive care units to detect epileptic activity and ischemia. This review analyzes clinical applications and limitations of cEEG as a routine neuromonitoring tool. Recent findingscEEG is primarily used to detect nonconvulsive seizures, which are frequent and possibly associated with harm. Cerebral ischemia, such as that from vasospasm after subarachnoid hemorrhage, can be detected earlier by EEG and quantitative EEG (qEEG). Highly skilled technicians and subspecialty-trained physicians are needed to generate good quality EEG and to interpret these data. qEEG allows more efficient interpretation of large amounts of EEG and may trigger prespecified alarms. Currently, there is little high-quality data on cEEG to define indications, cost-saving potential, and impact on outcome. A few studies have demonstrated how cEEG can be integrated into multimodality brain monitoring of severely brain-injured patients. SummarycEEG should be considered as an integral part of multimodality monitoring of the injured brain, particularly in patients at risk for nonconvulsive seizure or ischemia. Automated alarms may help establish cEEG monitoring as an integral part of brain monitoring. All neurological ICUs that routinely care for patients with refractory status epilepticus should have the capability to perform cEEG monitoring. Further research determining the impact on outcome and making EEG monitoring more user friendly may help move this technique out of the subspecialized ICU setting into the general ICU environment. In the future, it may be possible to use specific EEG parameters as endpoints for therapeutic interventions.

Brain interstitial fluid TNF-α after subarachnoid hemorrhage

OBJECTIVE TNF-alpha is an inflammatory cytokine that plays a central role in promoting the cascade of events leading to an inflammatory response. Recent studies have suggested that TNF-alpha may play a key role in the formation and rupture of cerebral aneurysms, and that the underlying cerebral inflammatory response is a major determinate of outcome following subrarachnoid hemorrhage (SAH). METHODS We studied 14 comatose SAH patients who underwent multimodality neuromonitoring with intracranial pressure (ICP) and cerebral microdialysis as part of their clinical care. Continuous physiological variables were time-locked every 8h and recorded at the same point that brain interstitial fluid TNF-alpha was measured in brain microdialysis samples. Significant associations were determined using generalized estimation equations. RESULTS Each patient had a mean of 9 brain tissue TNF-alpha measurements obtained over an average of 72h of monitoring. TNF-alpha levels rose progressively over time. Predictors of elevated brain interstitial TNF-alpha included higher brain interstitial fluid glucose levels (beta=0.066, p<0.02), intraventricular hemorrhage (beta=0.085, p<0.021), and aneurysm size >6mm (beta=0.14, p<0.001). There was no relationship between TNF-alpha levels and the burden of cisternal SAH; concurrent measurements of serum glucose, or lactate-pyruvate ratio. INTERPRETATION Brain interstitial TNF-alpha levels are elevated after SAH, and are associated with large aneurysm size, the burden of intraventricular blood, and elevation brain interstitial glucose levels.

Cerebral inflammatory response and predictors of admission clinical grade after aneurysmal subarachnoid hemorrhage

Poor admission clinical grade is the most important determinant of outcome after aneurysmal subarachnoid hemorrhage (aSAH); however, little attention has been focused on independent predictors of poor admission clinical grade. We hypothesized that the cerebral inflammatory response initiated at the time of aneurysm rupture contributes to ultra-early brain injury and poor admission clinical grade. We sought to identify factors known to contribute to cerebral inflammation as well as markers of cerebral dysfunction that were associated with poor admission clinical grade. Between 1997 and 2008, 850 consecutive SAH patients were enrolled in our prospective database. Demographic data, physiological parameters, and location and volume of blood were recorded. After univariate analysis, significant variables were entered into a logistic regression model to identify significant associations with poor admission clinical grade (Hunt-Hess grade 4-5). Independent predictors of poor admission grade included a SAH sum score >15/30 (odds ratio [OR] 2.3, 95% confidence interval [CI] 1.5-3.6), an intraventricular hemorrhage sum score >1/12 (OR 3.1, 95% CI 2.1-4.8), aneurysm size >10mm (OR 1.7, 95% CI 1.1-2.6), body temperature 38.3 degrees C (OR 2.5, 95% CI 1.1-5.4), and hyperglycemia >200mg/dL (OR 2.7, 95% CI 1.6-4.5). In a large, consecutive series of prospectively enrolled patients with SAH, the inflammatory response at the time of aneurysm rupture, as reflected by the volume and location of the hemoglobin burden, hyperthermia, and perturbed glucose metabolism, independently predicts poor admission Hunt-Hess grade. Strategies for mitigating the inflammatory response to aneurysmal rupture in the hyper-acute setting may improve the admission clinical grade, which may in turn improve outcomes.

Carbon Monoxide and the Brain: Time to Rethink the Dogma

Carbon Monoxide (CO), long thought to be a simple environmental pollutant is now known to have a critical role in cellular functions ranging from vasodilation to circadian rhythms. In this review, we will begin with a discussion of the enzyme responsible for CO production: heme oxygenase. Because this review will focus on the effects of CO in the brain, we will transition to CO toxicology and determine if this simple diatomic gas has really earned its nefarious reputation. An in depth analysis of the roles for CO in circadian rhythms and as a gasotransmitter will be provided in the neurological functional role section, followed by its vascular effects derived mainly from interactions with soluble guanylyl cyclase. We will then describe the evidence for COs protective roles through the MAPK pathway, and finally touch upon the potential therapeutic roles for CO in neurological diseases including ischemic stroke, multiple sclerosis, and neuropathic pain.

Relationship between brain interstitial fluid tumor necrosis factor-α and cerebral vasospasm after aneurysmal subarachnoid hemorrhage

Tumor necrosis factor-alpha (TNF-alpha) has a crucial role in the onset of hemolysis-induced vascular injury and cerebral vasoconstriction. We hypothesized that TNF-alpha measured from brain interstitial fluid would correlate with the severity of vasospasm following aneurysmal subarachnoid hemorrhage (aSAH). From a consecutive series of 10 aSAH patients who underwent cerebral microdialysis (MD) and evaluation of vasospasm by CT angiogram (CTA) or digital subtraction angiography (DSA), TNF-alpha levels from MD were measured at 8-hour intervals from aSAH days 4-6 using enzyme-linked immunosorbent assay. An attending neuroradiologist blinded to the study independently evaluated each CTA and DSA and assigned a vasospasm index (VI). Five patients had a VI<2 and 5 patients had a VI>2, where the median VI was 2 (range 0-13). The median log TNF-alpha area under the curve (AUC) was 1.64pg/mL *day (interquartile range 1.48-1.71) for the VI<2 group, and 2.11pg/mL *day (interquartile range 1.95-2.47) for the VI>2 group (p<0.01). Thus, in this small series of poor-grade aSAH patients, the AUC of TNF-alpha levels from aSAH days 4-6 correlates with the severity of radiographic vasospasm. Further analysis in a larger population is warranted based on our preliminary findings.

Antioxidant Strategies in Neurocritical Care

An increase in oxidative stress and overproduction of oxidizing reactive species plays an important role in the pathophysiology of several conditions encountered in the neurocritical care setting including: ischemic and hemorrhagic strokes, traumatic brain injury, acute respiratory distress syndrome, sepsis, and organ failure. The presence of oxidative stress in these conditions is supported by a large body of pre-clinical and clinical studies, and provides a rationale to support a potential therapeutic role for antioxidants. The purpose of this article is to briefly review the basic mechanisms and molecular biology of oxidative stress, summarize its role in critically ill neurological patients, and review available data regarding the potential role of antioxidant strategies in neurocritical care and future directions.

Circadian Rhythm in Stroke - The Influence of Our Internal Cellular Clock on Cerebrovascular Events

The distinct temporal pattern of stroke occurrence in humans has been recognized for decades; yet, the reason underlying the temporal nature of stroke is not completely understood. Several exogenous factors such as seasonal variation, physical activity, diet and sleep/wake cycles can influence stroke occurrence. Furthermore, it has been increasingly recognized that there are several endogenous physiological functions such as blood pressure, autonomic nervous system activity, and coagulation that show temporal variance and ultimately influence susceptibility to stroke. It was long believed that the neurons within the Suprachiasmatic Nucleus (SCN) controlled all of the body’s circadian rhythm cycles serving as the “master clock”. However, circadian gene expression is inherent to almost every cell in the body, controlling cellular metabolism, and ultimately an organ’s susceptibility to injury. These new insights into the molecular mechanisms regulating circadian rhythmicity might help to explain the phenomenon of circadian variation in stroke occurrence.