Age-specific transcriptional response to stroke.

Increased age is a major risk factor for stroke incidence and post-ischemic mortality. To develop age-adjusted therapeutic interventions, a clear understanding of the complexity of age-related post-ischemic mechanisms is essential. Transient occlusion of the middle cerebral artery--a model that closely resembles human stroke--was used to induce cerebral infarction in mice of 4 different ages (2, 9, 15, 24 months). By using Illumina cDNA microarrays and quantitative PCR we detected a distinct age-dependent response to stroke involving 350 differentially expressed genes. Our analyses also identified 327 differentially expressed genes that responded to stroke in an age-independent manner. These genes are involved in different aspects of the inflammatory and immune response, oxidative stress, cell cycle activation and/or DNA repair, apoptosis, cytoskeleton reorganization and/or astrogliosis, synaptic plasticity and/or neurotransmission, and depressive disorders and/or dopamine-, serotonin-, GABA-signaling. In agreement with our earlier work, aged brains displayed an attenuated inflammatory and immune response (Sieber et al., 2011) and a reduced impairment of post-stroke synaptic plasticity. Our data also revealed a distinct age-related susceptibility for post-ischemic depression, the most common neuropsychiatric consequence of stroke, which has a major influence on functional outcome.

Attenuated inflammatory response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice following stroke.

BACKGROUND: Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear. METHODS AND RESULTS: As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1ß, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed. CONCLUSIONS: Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.

Attenuated inflammatory response in aged mice brains following stroke.

BACKGROUND: Increased age is a major risk factor for stroke incidence, post-ischemic mortality, and severe and long-term disability. Stroke outcome is considerably influenced by post-ischemic mechanisms. We hypothesized that the inflammatory response following an ischemic injury is altered in aged organisms. METHODS AND RESULTS: To that end, we analyzed the expression pattern of pro-inflammatory cytokines (TNF, IL-1α, IL-1ß, IL-6), anti-inflammatory cytokines (IL-10, TGFß1), and chemokines (Mip-1α, MCP-1, RANTES) of adult (2 months) and aged (24 months) mice brains at different reperfusion times (6 h, 12 h, 24 h, 2 d, 7 d) following transient occlusion of the middle cerebral artery. The infarct size was assessed to monitor possible consequences of an altered inflammatory response in aged mice. Our data revealed an increased neuro-inflammation with age. Above all, we found profound age-related alterations in the reaction to stroke. The response of pro-inflammatory cytokines (TNF, and IL-1ß) and the level of chemokines (Mip-1α, and MCP-1) were strongly diminished in the aged post-ischemic brain tissue. IL-6 showed the strongest age-dependent decrease in its post-ischemic expression profile. Anti-inflammatory cytokines (TGFß1, and IL-10) revealed no significant age dependency after ischemia. Aged mice brains tend to develop smaller infarcts. CONCLUSION: The attenuated inflammatory response to stroke in aged animals may contribute to their smaller infarcts. The results presented here highlight the importance of using aged animals to investigate age-associated diseases like stroke, and should be considered as a major prerequisite in the development of age-adjusted therapeutic interventions.

Substantial performance discrepancies among commercially available kits for reverse transcription quantitative polymerase chain reaction: a systematic comparative investigator-driven approach.

Reverse transcription followed by quantitative polymerase chain reaction (rt-qPCR) has become the state-of-the-art tool for quantification of nucleic acids. However, there are still significant problems associated with its sensitivity, reproducibility, and efficiency and the choice of an appropriate rt-qPCR kit. The purpose of this article is to give insights into strategies to optimize and validate the performance of currently available kits for rt-qPCR and to provide up-to-date information about the benefits, potentials, and pitfalls of rt-qPCR assays. A selection of 9 complementary DNA (cDNA) synthesis and 12 qPCR kits were tested using samples obtained from three species (mouse, rat, and human) and three transcripts (Gapdh, Actb, and Hmbs) under highly standardized conditions. Kits with outstanding performance were further analyzed to identify the dynamic range for a reliable quantification of messenger RNA (mRNA). Reverse transcription efficiency varied up to 90-fold depending on the choice of reverse transcriptase, priming strategy, and assay volume. The qPCR kit test revealed variations in mean relative amplification efficiency ranging from 54% to 171%. We conclude that currently available kits for rt-qPCR vary considerably. However, with an appropriate validation strategy and knowledge about capabilities of a particular kit, sensitivity, efficiency, and reliability could be improved significantly.

Transcriptional regulation of ADAMTS13.

The metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-alpha, IL-6, IL-1beta as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.