Long Lasting Local and Systemic Inflammation after Cerebral Hypoxic ischemia in Newborn Mice

Background: Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice. Methodology/Principal Findings: We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b + and CD11c + antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb + T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb 2 T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls. Conclusions/Significance: Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks afte

Over-expression of DSCR1 protects against post-ischemic neuronal injury

Background and Purpose: The Down syndrome candidate region 1 (DSCR1) gene is located on human chromosome 21 and its protein is over-expressed in brains of Down syndrome individuals. DSCR1 can modulate the activity of calcineurin, a phosphatase abundant in the brain, but its influence on stroke outcome is not clear. We compared stroke outcome in wildtype (WT) and transgenic (DSCR1-TG) mice which over-express isoform 1 of human DSCR1. Methods: Transient cerebral ischemia was produced by occlusion of the middle cerebral artery for 0.5 h. After 23.5 h reperfusion, we assessed neurological impairment, brain infarct and edema volume, leukocyte infiltration and markers of inflammation. Intrinsic resistance to apoptosis following glucose deprivation was also assessed in primary cultures of WT and DSCR1-TG neurons. Results: In contrast to WT, DSCR1-TG mice had an improved neurological deficit score, greater grip strength, attenuated infarct volume and brain swelling, and lacked hippocampal lesions after stroke. Expression of mouse DSCR1-1, but not DSCR1-4, mRNA and protein was increased by ischemia in both WT and DSCR1-TG. Brain calcineurin activity was increased to a similar degree after ischemia in each genotype. DSCR1-TG mice had fewer infiltrating neutrophils and activated microglia compared with WT, in association with an attenuated upregulation of several pro-inflammatory genes. Neurons from DSCR1-TG mice were more resistant than WT neurons to apoptotic cell death following 24 h of glucose deprivation. Conclusions: Over-expression of DSCR1 in mice improves outcome following stroke. Mechanisms underlying this protection may involve calcineurin-independent, anti-inflammatory and anti-apoptotic effects mediated by DSCR1 in neurons

Transcriptomics and proteomics analyses of the PACAP38 influenced ischemic brain in permanent middle cerebral artery occlusion model mice

<p>Abstract</p> <p>Introduction</p> <p>The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with corresponding SHAM control that used 0.9% saline injection.</p> <p>Methods</p> <p>Ischemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expression using DNA microarray chip (4x44K, Agilent) and two-dimensional gel electrophoresis (2-DGE) coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), respectively. Western blotting and immunofluorescent staining were also used to further examine the identified protein factor.</p> <p>Results</p> <p>Our results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral) treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral). Previously known (such as the interleukin family) and novel (<it>Gabra6</it>, <it>Crtam</it>) genes were identified under PACAP influence. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2). The DPYL2, also known as Crmp2, is a marker for the axonal growth and nerve development. Interestingly, PACAP treatment slightly increased its abundance (by 2-DGE and immunostaining) at 6 h but not at 24 h in the ischemic hemisphere, suggesting PACAP activates neuronal defense mechanism early on.</p> <p>Conclusions</p> <p>This study provides a detailed inventory of PACAP influenced gene expressions and protein targets in mice ischemic brain, and suggests new targets for thereaupetic interventions.</p

Stenosis coexists with compromised α1-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a rare disorder caused by a heterozygous deletion of 26-28 contiguous genes that affects the brain and cardiovascular system. Here, we investigated whether WBS affects aortic structure and function in the complete deletion (CD) mouse model harbouring the most common deletion found in WBS patients. Thoracic aortas from 3-4 months-old male CD mice and wild-type littermates were mounted in wire myographs or were processed for histomorphometrical analysis. Nitric oxide synthase (NOS) isoforms and oxidative stress levels were assessed. Ascending aortas from young adult CD mice showed moderate (50%) luminal stenosis, whereas endothelial function and oxidative stress were comparable to wild-type. CD mice showed greater contractions to KCl. However, α1-adrenergic contractions to phenylephrine, but not with a thromboxane analogue, were compromised. Decreased phenylephrine responses were not affected by selective inducible NOS blockade with 1400 W, but were prevented by the non-selective NOS inhibitor L-NAME and the selective neuronal NOS inhibitor SMTC. Consistently, CD mice showed increased neuronal NOS expression in aortas. Overall, aortic stenosis in CD mice coexists with excessive nNOS-derived NO signaling that compromises ascending aorta α1-adrenergic contractions. We suggest that increased neuronal NOS signaling may act as a physiological 'brake' against the detrimental effects of stenosis.This work was supported by Ministerio de Ciencia e Innovación [[SAF2014-56111-R to FJA] and [SAF2016-78508-R (AEI/MINEICO/FEDER, UE) to VC]]; and Generalitat de Catalunya [2017-SGR-645 to FJA]

RETRACTED ARTICLE: Valproic acid attenuates intercellular adhesion molecule-1 and E-selectin through a chemokine ligand 5 dependent mechanism and subarachnoid hemorrhage induced vasospasm in a rat model

Abstract Background Up-regulation of regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) and adhesion molecules is observed in the serum of animals following experimental subarachnoid hemorrhage (SAH). The present study was to examine the effect of valproic acid (VPA) on RANTES and alternation of adhesion molecules in this model. Methods A rodent SAH model was employed. Animals were randomly assigned into six groups. Basilar artery (BA) was harvested for intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule–1 (VCAM-1), and E-selectin evaluation (western blotting) and RANTES (rt-PCR). 1 ng CCL5 recombinant protein intrathecal injection was performed in the VPA + SAH groups. (N = 5). Results Convoluted internal elastic lamina, distorted endothelial wall, and smooth muscle micro-necrosis was prominently observed in the SAH groups, which is absent in the VPA treatment and the healthy controls. Treatment with VPA dose-dependently reduced the ICAM-1, E-selectin and RANTES level, compared with the SAH group (p <0.01). The administration of CCL5 significantly increased CD45(+) glia and ICAM-1 level in the VPA treatment groups. Conclusion VPA exerts its anti-vasospastic effect through the dual effect of inhibiting RANTES expression and reduced adhesion molecules. Besides, VPA also decreased CD45(+) cells transmigrated to the vascular wall. The administration of CCL5 significantly reversed the inhibitory effect of this compound on CD45(+) monocytes, E-selectin, and ICAM-1 level. This study also lends credence to support this compound could attenuate SAH induced adhesion molecules and neuro-inflammation in a CCL5 dependent mechanism