Hypoxia is a critical factor for cell death or survival in ischemic stroke, but the pathological consequences of combined ischemia-hypoxia are not fully understood. Here we examine this issue using a modified Levine/Vannucci procedure in adult mice that consists of unilateral common carotid artery occlusion and hypoxia with tightly regulated body temperature. At the cellular level, ischemia-hypoxia produced proinflammatory cytokines and simultaneously activated both prosurvival (eg, synthesis of heat shock 70 protein, phosphorylation of ERK and AKT) and proapoptosis signaling pathways (eg, release of cytochrome c and AIF from mitochondria, cleavage of caspase-9 and -8). However, caspase-3 was not activated, and very few cells completed the apoptosis process. Instead, many damaged neurons showed features of autophagic/lysosomal cell death. At the tissue level, ischemia-hypoxia caused persistent cerebral perfusion deficits even after release of the carotid artery occlusion. These changes were associated with both platelet deposition and fibrin accumulation within the cerebral circulation and would be expected to contribute to infarction. Complementary studies in fibrinogen-deficient mice revealed that the absence of fibrin and/or secondary fibrin-mediated inflammatory processes significantly attenuated brain damage. Together, these results suggest that ischemia-hypoxia is a powerful stimulus for spontaneous coagulation leading to reperfusion deficits and autophagic/lysosomal cell death in brain.

No abstract

The standard method of detecting neurogenesis uses bromodeoxyuridine (BrdU) to label DNA synthesis followed by double labeling with neuronal markers. However, DNA synthesis may occur in events unrelated to neurogenesis including aneuploidy and abortive cell cycle reentry. Hence, it is important to confirm neurogenesis with methods other than BrdU incorporation. To this end, we have generated transgenic nestin-CreER mice that express tamoxifen-inducible Cre recombinase under the control of a nestin enhancer. When crossed with a ubiquitous Enhanced Green Fluorescent Protein (EGFP)-Cre-reporter line, the bitransgenic animals can reveal the nestin-positive progenitors and their progeny with EGFP after tamoxifen induction. This system has many applications including visualization of embryonic neural progenitors, detection of postnatally transformed radial glial cells, and labeling adult neural progenitors in the subventricular zone (SVZ). To examine the contribution of SVZ progenitors to cell replacement after stroke, tamoxifen-induced mice were challenged with focal ischemia or combined ischemia-hypoxia followed by BrdU injection. This analysis revealed only very few EGFP-positive cells outside the SVZ after focal ischemia but robust DNA synthesis by hippocampal neurons without immediate cell death following ischemia-hypoxia. These results suggest that the nestin-CreER system is a useful tool for detecting embryonic and adult neurogensis. They also confirm the existence of nonproliferative DNA synthesis by old neurons after experimental brain injury.

Previously published online a an Autophagy E-publication:

Diffusion tensor imaging (DTI) is a powerful method to visualize white matter, but its use in patients with acute stroke remains limited because of the lack of corresponding histologic information. In this study, we addressed this issue using a hypoxia-ischemia (HI)-induced thrombotic model of stroke in adult mice. At 6, 15, and 24 hours after injury, animals were divided into three groups for (1) in vivo T2-and diffusion-weighted magnetic resonance imaging, followed by histochemistry, (2) ex vivo DTI and electron microscopy, and (3) additional biochemical or immunochemical assays. The temporal changes of diffusion anisotropy and histopathology were compared in the fimbria, internal capsule, and external capsule. We found that HI caused a rapid reduction of axial and radial diffusivities in all three axonal bundles. A large decrease in fractional anisotropy, but not in axial diffusivity per se, was associated with structural breakdown of axons. Furthermore, the decrease in radial diffusivity correlated with swelling of myelin sheaths and compression of the axoplasma. The gray matter of the hippocampus also exhibited a high level of diffusion anisotropy, and its reduction signified dendritic degeneration. Taken together, these results suggest that crossevaluation of multiple DTI parameters may provide a fuller picture of axonal and dendritic injury in acute ischemic stroke.

The immature brains of newborns often respond differently from the brains of adults when exposed to similar insults. Previous studies have indicated that although hypoxia-ischemia (HI) induces persistent thrombosis in adult brains, it only modestly impairs blood perfusion in newborn brains. Here, we used the Vannucci model of HI encephalopathy to study age-related responses to cerebral HI in rat pups. We found that HI triggered fibrin deposition and impaired blood perfusion in both neonatal and adult brains. However, these effects were only transient in neonatal brains (<4 hours) and were accompanied by acute induction of both tissue-type and urinary-type plasminogen activators (tPA and uPA), which was not observed in adult brains subjected to the same insult. Interestingly, activation of the plasminogen system persisted up to 24 hours in neonatal brains, long after the clearance of fibrin-rich thrombi. Furthermore, astrocytes and macrophages outside blood vessels expressed tPA after HI, suggesting the possibility of tPA/plasmin-mediated cytotoxicity. Consistent with this hypothesis, injection of ␣2-antiplasmin into cerebral ventricles markedly ameliorated HI-induced damage to neurofilaments and white matter oligodendrocytes, providing a dose-response reduction of brain injury after 7 days of recovery. Conversely, ventricular injection of tPA increased HI-induced brain damage. Together, these results suggest that tPA/plasmin induction, which may contribute to acute fibrinolysis, is a critical component of extravascular proteolytic damage in immature brains, representing a new therapeutic target for the treatment of HI encephalopathy. (Am J Pathol

e21064 Background: Interleukin-15 (IL-15) is a pro-inflammatory cytokine that stimulates the differentiation and proliferation of T, B, and NK cells. IL-15 is normally tightly bound by its receptor, IL-15Rα. Dendritic cells and monocytes/macrophages primarily express both IL-15 and IL-15-Rα; however, expression has been detected on epithelial cells including those of the lung. In the lung, IL-15 is thought to play a role in the induction of immune responses to infection. Over expression of IL-15 has been shown to induce NK cell activation and cytotoxic T-lymphocyte (CTL) responses leading to tumor regression. Little is known about IL-15 or IL-15Rα expression in lung cancer. Methods: mRNA from 146 primary lung cancers were analyzed by multiplex qPCR for expression of IL-15, IL-15Rα and b-actin (internal control) and compared to expression in normal lung tissue from 45 patients. Of the 146 patients, 50 were stage I (IA=20, IB=30), 49 stage II (IIA=9, IIB=40), 36 stage III (IIIA=18, IIIB=18), and 11 stage IV. Results: Comparing the expression of IL-15 between normal lung and tumor, we found tumors expressed significantly less IL-15 than normal lung (P<0.001). Lung cancers at any given stage, expressed significantly less IL-15 than that seen in normal lung. When comparing stages, stage IV tumors expressed significantly less IL-15 than tumors of stages I (P=0.021), II (P=0.020), or III (P=0.024). There was no difference in expression between stages I, II or III (P>0.05). When examining the expression of IL-15Rα we found no differences between normal lung and tumor. No differences were seen between normal lung and stages I-IIIA; however, significantly less IL-15Rα expression was noted in stages IIIB and IV compared to normal lung. Between stages, stages I and II had significantly more IL-15Rα expression than stage IV tumors. There were no differences between stages I, II and III. Conclusions: Pro-inflammatory cytokines such as IL-15 may be important for the induction of lung immunity. IL-15 expression is down regulated in lung cancers and this down regulation increases with stage. We posit that the down regulation of IL-15 by lung cancers may aid in their evasion of immune responses allowing progression and dissemination of tumor.

577 Background: Oligometastatic CLRM comprises a distinct subset of stage IV colorectal cancer. Liver directed therapies (LDT) including surgery, ablation, radiation, and transarterial chemotherapy/embolization have been shown to improve cancer outcomes in smaller series. We sought to evaluate utilization of LDT and their impact on survival in patients with CLRM in a population-based database. Methods: We analyzed linked SEER-Medicare data. Eligible patients were ≥ 66 years, diagnosed between 1992-2009, carry a code for secondary malignancy of the liver (ICD-9 197.7) and survived ≥ 30 days after diagnosis. LDT (yes vs. no) were defined by ICD-9 and CPT codes of surgery (hepatectomy), ablation (e.g. radiofrequency and cryoablation), radiation (e.g., stereotactic surgery and brachytherapy), and transarterial/embolization (e.g., radioembolization). Treatment and non-treatment groups were matched using a propensity score comprised of a comprehensive set of patient and tumor characteristics including: age, sex, race, marital status, Medicaid status, and tumor histology, grade, and location. Cox Proportional Hazard models were used to compare the impact of the LDT on overall survival among propensity-matched pairs of treated and untreated patients. Results: LDT were performed in 12.7% (n = 1,793) of all 14,150 patients. There were 13.7% of patients over 85 years. Females were 52.1% and 83 % White. Off all, 5.4% had surgery, 3.9% had ablation, 4.6% had radiation, and 1.6% had transarterial chemotherapy/embolization. Unadjusted 5 year overall-survival (OS) was 26.9% for those underdoing LDT vs. 7.5% who did not (HR = 0.44, (CI 0.41,0.46)). Cox modeling demonstrated a survival benefit for each LDT with HR of 0.36 (0.33,0.39) for surgery, 0.35 (0.32,0.39) for ablation, 0.78 (0.72, 0.85) for radiation and 0.42 (0.36,0.49) for transarterial/embolization. Conclusions: While use of LDT for CLRM in this national sample of Medicare patients were low, those who received treatment had markedly improved survival compared to matched patients who did not. Surgery and ablation were the most effective therapies. These findings require evaluation in a prospective clinical study.