Nathan Yanasak

HIGH MOBILITY GROUP BOX PROTEIN-1 PROMOTES CEREBRAL EDEMA AFTER TRAUMATIC BRAIN INJURY VIA ACTIVATION OF TOLL-LIKE RECEPTOR 4

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Cerebral edema, a life‐threatening medical complication, contributes to elevated intracranial pressure (ICP) and a poor clinical prognosis after TBI. Unfortunately, treatment options to reduce post‐traumatic edema remain suboptimal, due in part, to a dearth of viable therapeutic targets. Herein, we tested the hypothesis that cerebral innate immune responses contribute to edema development after TBI. Our results demonstrate that high‐mobility group box protein 1 (HMGB1) was released from necrotic neurons via a NR2B‐mediated mechanism. HMGB1 was clinically associated with elevated ICP in patients and functionally promoted cerebral edema after TBI in mice. The detrimental effects of HMGB1 were mediated, at least in part, via activation of microglial toll‐like receptor 4 (TLR4) and the subsequent expression of the astrocytic water channel, aquaporin‐4 (AQP4). Genetic or pharmacological (VGX‐1027) TLR4 inhibition attenuated the neuroinflammatory response and limited post‐traumatic edema with a delayed, clinically implementable therapeutic window. Human and rodent tissue culture studies further defined the cellular mechanisms demonstrating neuronal HMGB1 initiates the microglial release of interleukin‐6 (IL‐6) in a TLR4 dependent mechanism. In turn, microglial IL‐6 increased the astrocytic expression of AQP4. Taken together, these data implicate microglia as key mediators of post‐traumatic brain edema and suggest HMGB1‐TLR4 signaling promotes neurovascular dysfunction after TBI. GLIA 2013;62:26–38

Activation of P2X7 Promotes Cerebral Edema and Neurological Injury after Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part to the absence of viable drug targets. In the present study, genetic inhibition (P2X7−/− mice) of the purinergic P2x7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-1β (IL-1β) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, brilliant blue G (BBG), a clinically non-toxic P2X7 inhibitor, inhibited IL-1β expression, limited edemic development, and improved neurobehavioral outcomes after TBI. The beneficial effects of BBG followed either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and attenuated the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.

Demyelination, Astrogliosis, and Accumulation of Ubiquitinated Proteins, Hallmarks of CNS Disease in hsf1-Deficient Mice

The heat shock transcription factors (Hsfs) are responsible for the heat shock response, an evolutionarily conserved process for clearance of damaged and aggregated proteins. In organisms such as Caenorhabditis elegans, which contain a single Hsf, reduction in the level of Hsf is associated with the appearance of age-related phenotypes and increased accumulation of protein aggregates. Mammalian cells express three hsfs (hsf1, hsf2, hsf4) and their role in CNS homeostasis remains unclear. In this study, we examined the effects of deletion of single or multiple hsf genes in the CNS using mutant mice. Our results show that hsf1−/− mice display progressive myelin loss that accompanies severe astrogliosis and this is exacerbated in the absence of either the hsf2 or hsf4 gene. Magnetic resonance imaging and behavioral studies indicate reduction in the white matter tracts of the corpus callosum, and deficiencies in motor activity, respectively, in aged hsf1−/− mice. Concomitantly, hsf1−/− aged CNS exhibit increased activated microglia and apoptotic cells that are mainly positive for GFAP, an astrocyte-specific marker. Studies based on the expression of short-lived ubiquitinated green fluorescent protein (GFPu) in living hsf1−/− cells indicate that they exhibit reduced ability to degrade ubiquitinated proteins, accumulate short-lived GFPu, and accumulate aggregates of the Huntingtons model of GFP containing trinucleotide repeats (Q103-GFP). Likewise, hsf1−/− brain and astrocytes exhibit higher than wild-type levels of ubiquitinated proteins, increased levels of protein oxidation, and increased sensitivity to oxidative stress. These studies indicate a critical role for mammalian hsf genes, but specifically hsf1, in the quality control mechanisms and maintenance of CNS homeostasis during the organisms lifetime.

Temporal and Noninvasive Monitoring of Inflammatory-Cell Infiltration to Myocardial Infarction Sites Using Micrometer-Sized Iron Oxide Particles

Micrometer‐sized iron oxide particles (MPIO) are a more sensitive MRI contrast agent for tracking cell migration compared to ultrasmall iron oxide particles. This study investigated the temporal relationship between inflammation and tissue remodeling due to myocardial infarction (MI) using MPIO‐enhanced MRI. C57Bl/6 mice received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later (n = 7). For controls, two groups underwent either sham‐operated surgery without inducing an MI post‐MPIO injection (n = 7) or MI surgery without MPIO injection (n = 6). The MRIs performed post‐MI showed significant signal attenuation around the MI site for the mice that received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later, compared to the two control groups (P < 0.01). The findings suggested that the prelabeled inflammatory cells mobilized and infiltrated into the MI site. Furthermore, the linear regression of contrast‐to‐noise ratio at the MI site and left ventricular ejection function suggested a positive correlation between the labeled inflammatory cell infiltration and cardiac function attenuation during post‐MI remodeling (r2 = 0.98). In conclusion, this study demonstrated an MRI technique for noninvasively and temporally monitoring inflammatory cell migration into the myocardium while potentially providing additional insight concerning the pathologic progression of a myocardial infarction. Magn Reson Med, 2010.

Monitoring dynamic alterations in calcium homeostasis by T1-weighted and T1-mapping cardiac manganese-enhanced MRI in a murine myocardial infarction model

Manganese has been used as a T1‐weighted MRI contrast agent in a variety of applications. Because manganese ions (Mn2+) enter viable myocardial cells via voltage‐gated Ca2+ channels, manganese‐enhanced MRI is sensitive to the viability and inotropic state of the heart. In spite of the established importance of Ca2+ regulation in the heart both before and after myocardial injury, monitoring strategies to assess Ca2+ homeostasis in affected cardiac tissues are limited. This study implements a T1‐mapping method to obtain quantitative information both dynamically and over a range of MnCl2 infusion doses. To optimize the current Mn2+ infusion protocols, we performed both dose‐dependent and temporal washout studies. A non‐linear relationship between infused MnCl2 solution dose and increase in left ventricular wall relaxation rate (ΔR1) was observed. Control mice also exhibited significant Mn2+ clearance over time, with a decrease in ΔR1 of ∼50% occurring in just 2.5 h. The complicated efflux time dependence possibly suggests multiple efflux mechanisms. With the use of the measured relationship between infused Mn2+ dose, ΔR1, and inductively coupled plasma mass spectrometry data analysis provided a means of estimating the absolute heart Mn concentration in vivo. We show that this technique has the sensitivity to observe or monitor potential alterations in Ca2+ handling in vivo because of the physiological remodeling after myocardial infarction. Left ventricular free wall ΔR1 values were significantly lower (P = 0.005) in the adjacent zone, surrounding the injured myocardial tissue, than in healthy tissue. This inferred reduction in Mn concentration can be used to estimate potentially salvageable myocardium in vivo for future treatment or evaluation of disease progression. Copyright

Age-Dependent Lethality in Novel Transgenic Mouse Models of Central Nervous System Arteriovenous Malformations

Background and Purpose— The lack of an appropriate animal model has been a limitation in studying hemorrhage from arteriovenous malformations (AVMs) in the central nervous system. Methods— Novel mouse central nervous system AVM models were generated by conditionally deleting the activin receptor-like kinase (Alk1; Acvrl1) gene with the SM22-Cre transgene. All mice developed AVMs in their brain and/or spinal cord, and >80% of them showed a paralysis or lethality phenotype due to internal hemorrhages during the first 10 to 15 weeks of life. The mice that survived this early lethal period, however, showed significantly reduced lethality rates even though they carried multiple AVMs. Results— The age-dependent change in hemorrhage rates allowed us to identify molecular factors uniquely upregulated in the rupture-prone AVM lesions. Conclusions— Upregulation of angiopoietin 2 and a few inflammatory genes were identified in the hemorrhage-prone lesions, which may be comparable with human pathology. These models will be an exceptional tool to study pathophysiology of AVM hemorrhage.

Repeated exposure to chlorpyrifos leads to prolonged impairments of axonal transport in the living rodent brain

The toxicity of the class of chemicals known as the organophosphates (OP) is most commonly attributed to the inhibition of the enzyme acetylcholinesterase. However, there is significant evidence that this mechanism may not account for all of the deleterious neurologic and neurobehavioral symptoms of OP exposure, especially those associated with levels that produce no overt signs of acute toxicity. In the study described here we evaluated the effects of the commonly used OP-pesticide, chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) of the optic nerve (ON) projections from the retina to the superior colliculus (SC). T1-weighted MEMRI scans were evaluated at 6 and 24h after intravitreal injection of Mn(2+). As a positive control for axonal transport deficits, initial studies were conducted with the tropolone alkaloid colchicine administered by intravitreal injection. In subsequent studies both single and repeated exposures to CPF were evaluated for effects on axonal transport using MEMRI. As expected, intravitreal injection of colchicine (2.5μg) produced a robust decrease in transport of Mn(2+) along the optic nerve (ON) and to the superior colliculus (SC) (as indicated by the reduced MEMRI contrast). A single subcutaneous (s.c.) injection of CPF (18.0mg/kg) was not associated with significant alterations in the transport of Mn(2+). Conversely, 14-days of repeated s.c. exposure to CPF (18.0mg/kg/day) was associated with decreased transport of Mn(2+) along the ONs and to the SC, an effect that was also present after a 30-day (CPF-free) washout period. These results indicate that repeated exposures to a commonly used pesticide, CPF can result in persistent alterations in axonal transport in the living mammalian brain. Given the fundamental importance of axonal transport to neuronal function, these observations may (at least in part) explain some of the long term neurological deficits that have been observed in humans who have been repeatedly exposed to doses of OPs not associated with acute toxicity.

Assessing manganese efflux using SEA0400 and cardiac T1-mapping manganese-enhanced MRI in a murine model

The sodium–calcium exchanger (NCX) is one of the transporters contributing to the control of intracellular calcium (Ca2+) concentration by normally mediating net Ca2+ efflux. However, the reverse mode of the NCX can cause intracellular Ca2+ concentration overload, which exacerbates the myocardial tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has been shown to therapeutically reduce myocardial injury, no in vivo technique exists to monitor intracellular Ca2+ fluctuations produced by this drug. Cardiac manganese‐enhanced MRI (MEMRI) may indirectly assess Ca2+ efflux by estimating changes in manganese (Mn2+) content in vivo, since Mn2+ has been suggested as a surrogate marker for Ca2+. This study used the MEMRI technique to examine the temporal features of cardiac Mn2+ efflux by implementing a T1‐mapping method and inhibiting the NCX with SEA0400. The change in 1H2O longitudinal relaxation rate, ΔR1, in the left ventricular free wall, was calculated at different time points following infusion of 190 nmol/g manganese chloride (MnCl2) in healthy adult male mice. The results showed 50% MEMRI signal attenuation at 3.4 ± 0.6 h post‐MnCl2 infusion without drug intervention. Furthermore, treatment with 50 ± 0.2 mg/kg of SEA0400 significantly reduced the rate of decrease in ΔR1. At 4.9–5.9 h post‐MnCl2 infusion, the average ΔR1 values for the two groups treated with SEA0400 were 2.46 ± 0.29 and 1.72 ± 0.24 s−1 for 50 and 20 mg/kg doses, respectively, as compared to the value of 1.27 ± 0.28 s−1 for the control group. When this in vivo data were compared to ex vivo absolute manganese content data, the MEMRI T1‐mapping technique was shown to effectively quantify Mn2+ efflux rates in the myocardium. Therefore, combining an NCX inhibitor with MEMRI may be a useful technique for assessing Mn2+ transport mechanisms and rates in vivo, which may reflect changes in Ca2+ transport. Copyright

Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury.

Traumatic brain injury (TBI) induces severe harm and disability in many accident victims and combat‐related activities. The heat‐shock proteins Hsp70/Hsp110 protect cells against death and ischemic damage. In this study, we used mice deficient in Hsp110 or Hsp70 to examine their potential requirement following TBI. Data indicate that loss of Hsp110 or Hsp70 increases brain injury and death of neurons. One of the mechanisms underlying the increased cell death observed in the absence of Hsp110 and Hsp70 following TBI is the increased expression of reactive oxygen species‐induced p53 target genes Pig1, Pig8, and Pig12. To examine whether drugs that increase the levels of Hsp70/Hsp110 can protect cells against TBI, we subjected mice to TBI and administered Celastrol or BGP‐15. In contrast to Hsp110‐ or Hsp70i‐deficient mice that were not protected following TBI and Celastrol treatment, there was a significant improvement of wild‐type mice following administration of these drugs during the first week following TBI. In addition, assessment of neurological injury shows significant improvement in contextual and cued fear conditioning tests and beam balance in wild‐type mice that were treated with Celastrol or BGP‐15 following TBI compared to TBI‐treated mice. These studies indicate a significant role of Hsp70/Hsp110 in neuronal survival following TBI and the beneficial effects of Hsp70/Hsp110 inducers toward reducing the pathological consequences of TBI.

Relationship between blood and myocardium manganese levels during manganese-enhanced MRI (MEMRI) with T1 mapping in rats.

Manganese ions (Mn2+) enter viable myocardial cells via voltage‐gated calcium channels. Because of its shortening of T1 and its relatively long half‐life in cells, Mn2+ can serve as an intracellular molecular contrast agent to study indirect calcium influx into the myocardium. One major concern in using Mn2+ is its sensitivity over a limited range of concentrations employing T1‐weighted images for visualization, which limits its potential in quantitative techniques. Therefore, this study assessed the implementation of a T1 mapping method for cardiac manganese‐enhanced MRI to enable a quantitative estimate of the influx of Mn2+ over a wide range of concentrations in male Sprague‐Dawley rats. This MRI method was used to compare the relationship between T1 changes in the heart as a function of myocardium and blood Mn2+ levels. Results showed a biphasic relationship between ΔR1 and the total Mn2+ infusion dose. Nonlinear relationships were observed between the total Mn2+ infusion dose versus blood levels and left ventricular free wall ΔR1. At low blood levels of Mn2+, there was proportionally less cardiac enhancement seen than at higher levels of blood Mn2+. We hypothesize that Mn2+ blood levels increase as a result of rate‐limiting excretion by the liver and kidneys at these higher Mn2+ doses. Copyright