Transient middle cerebral artery occlusion in rats leads to infarction of the lateral part of the striatum and adjacent neocortex, with selective neuronal necrosis in the bordering penumbral zones. Administration of glutamate, cytokine, and leukocyte antagonists have rescued mainly neocortical neurons, indicating differences in the degenerative processes. The aim of this study was, therefore, to describe the microglial/macrophage activation and polymorphonuclear leukocyte recruitment patterns and to correlate these with the ischemia-induced degenerative processes. The analysis showed significant differences in the characteristics and timing of the microglial/macrophage responses between the caudate putamen and neocortical infarct zones, the infarct zones and their associated penumbral zones, as well as between the striatal and the neocortical penumbral zone. Infiltrations with polymorphonuclear leukocytes into the infarct zones were limited and shortlasting and confined to the acutely degenerating striatum and piriform cortex. A delayed, massive infiltration with lipid phagocytes into the caudate putamen infarct markedly contrasted an early recruitment and activation of microglia/macrophages in the adjacent penumbra. Within the neocortex, a later onset of degeneration along the insular-parietal axis was marked by neuronal expression of heat shock protein and a progressive microglial activation with induction of the full repertoire of microglial activation markers, including a widespread microglial major histocompatibility complex (MHC) class II antigen expression. We interpret the present results as delineating two differentially progressing penumbral zones, which are likely to reflect differences in the underlying degenerative processes. Differences in the microglial/macrophage activation pattern attract special attention, as these cells may constitute specific targets for therapeutic intervention.

Oxidative stress and apoptotic cell death have been implicated in the dopaminergic cell loss that characterizes Parkinson's disease. While factors contributing to apoptotic cell death are not well characterized, oxidative stress is known to activate an array of cell signaling molecules that participate in apoptotic cell death mechanisms. We investigated oxidative stress-induced cytotoxicity of hydrogen peroxide (H2O2) in three cell lines, the dopaminergic mesencephalon-derived N27 cell line, the GABAergic striatum-derived M213-20 cell line, and the hippocampal HN2-5 cell line. N27 cells were more sensitive to H2O2-induced cell death than M213-20 and HN2-5 cells. H2O2 induced significantly greater increases in caspase-3 activity in N27 cells than in M213-20 cells. H2O2-induced apoptotic cell death in N27 cells was mediated by caspase-3-dependent proteolytic activation of PKCdelta. Gene expression microarrays were employed to examine the specific transcriptional changes in N27 cells exposed to 100 microM H2O2 for 4 h. Changes in genes encoding pro- or anti-apoptotic proteins included up-regulation of BIK, PAWR, STAT5B, NPAS2, Jun B, MEK4, CCT7, PPP3CC, and PSDM3, while key down-regulated genes included BNIP3, NPTXR, RAGA, STK6, YWHAH, and MAP2K1. Overall, the changes indicate a modulation of transcriptional activity, chaperone activity, kinase activity, and apoptotic activity that appears highly specific, coordinated and relevant to cell survival. Utilizing this in vitro model to identify novel oxidative stress-regulated genes may be useful in unraveling the molecular mechanisms underlying dopaminergic degeneration in Parkinson's disease.

BackgroundElevated levels of an endogenous Na/K‐ATPase inhibitor marinobufagenin accompany salt‐sensitive hypertension and are implicated in cardiac fibrosis. Immunoneutralization of marinobufagenin reduces blood pressure in Dahl salt‐sensitive (Dahl‐S) rats. The effect of the anti‐marinobufagenin monoclonal antibody on blood pressure, left ventricular (LV) and renal remodeling, and gene expression were investigated in hypertensive Dahl‐S rats.Methods and ResultsDahl‐S rats were fed high NaCl (8%, HS; n=14) or low NaCl (0.1%, LS; n=14) diets for 8 weeks. Animals were administered control antibody (LS control antibody, LSC; HS control antibody, HSC; n=7 per group) or anti‐marinobufagenin antibody once on week 7 of diet intervention (n=7 per group). Levels of marinobufagenin, LV, and kidney mRNAs and proteins implicated in profibrotic signaling were assessed. Systolic blood pressure was elevated (211±8 versus 133±3 mm Hg, P<0.01), marinobufagenin increased 2‐fold in plasma (P<0.05) and 5‐fold in urine (P<0.01), LV and kidney weights increased, and levels of LV collagen‐1 rose 3.5‐fold in HSC versus LSC. Anti‐marinobufagenin antibody treatment decreased systolic blood pressure by 24 mm Hg (P<0.01) and reduced organ weights and level of LV collagen‐1 (P<0.01) in hypertensive Dahl salt‐sensitive rats with anti‐marinobufagenin antibody versus HSC. The expression of genes related to transforming growth factor‐β–dependent signaling was upregulated in the left ventricles and kidneys in HSC versus LSC groups and became downregulated following administration of anti‐marinobufagenin antibody to hypertensive Dahl‐S rats. Marinobufagenin also activated transforming growth factor‐β signaling in cultured ventricular myocytes from Dahl‐S rats.ConclusionsImmunoneutralization of heightened marinobufagenin levels in hypertensive Dahl‐S rats resulted in a downregulation of genes implicated in transforming growth factor‐β pathway, which indicates that marinobufagenin is an activator of profibrotic transforming growth factor‐β–dependent signaling in salt‐sensitive hypertension.

Cannabinoid 1 receptor (CB1R) expression is upregulated in the liver with viral hepatitis, cirrhosis, and both alcoholic and non-alcoholic fatty liver disease (FLD), whereas its expression is muted under usual physiological conditions. Inhibiting CB1R has been shown to be beneficial in preserving hepatic function in FLD but it is unclear if inhibiting CB1R during an inflammatory response to an acute hepatic injury, such as toxin-induced injury, would also be beneficial. We found that intrinsic CB1R in hepatocytes regulated liver inflammation-related gene transcription. We tested if nullification of hepatocyte-specific CB1R (hCNR1−/−) in mice protects against concanavalin A (Con A)-induced liver injury. We looked for evidence of liver damage and markers of inflammation in response to Con A by measuring liver enzyme levels and proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, IL-17) in serum collected from hCNR1−/− and control mice. We observed a shift to the right in the dose-response curve for liver injury and inflammation in hCNR1−/− mice. We also found less inflammatory cell infiltration and focal necrosis in livers of hCNR1−/− mice compared to controls, resulting from downregulated apoptotic markers. This anti-apoptotic mechanism results from increased activation of nuclear factor kappa B (NF-κB), especially cAMP-dependent cannabinoid signaling and membrane-bound TNF-α, via downregulated TNF-α receptor 2 (TNFR2) transcription levels. Collectively, these findings provide insight into involvement of CB1R in the pathogenesis of acute liver injury.