AT2 activation does not influence brain damage in the early phase after experimental traumatic brain injury in male mice

Antagonism of the angiotensin II type 1 receptor (AT1) improves neurological function and reduces brain damage after experimental traumatic brain injury (TBI), which may be partly a result of enhanced indirect angiotensin II type 2 receptor (AT2) stimulation. AT2 stimulation was demonstrated to be neuroprotective via anti-inflammatory, vasodilatory, and neuroregenerative mechanisms in experimental cerebral pathology models. We recently demonstrated an upregulation of AT2 after TBI suggesting a protective mechanism. The present study investigated the effect of post-traumatic (5 days after TBI) AT2 activation via high and low doses of a selective AT2 agonist, compound 21 (C21), compared to vehicle-treated controls. No differences in the extent of the TBI-induced lesions were found between both doses of C21 and the controls. We then tested AT2-knockdown animals for secondary brain damage after experimental TBI. Lesion volume and neurological outcomes in AT2-deficient mice were similar to those in wild-type control mice at both 24 h and 5 days post-trauma. Thus, in contrast to AT1 antagonism, AT2 modulation does not influence the initial pathophysiological mechanisms of TBI in the first 5 days after the insult, indicating that AT2 plays only a minor role in the early phase following trauma-induced brain damage

Der Einfluss des Angiotensin-II-Typ-2-Agonisten Compound 21 auf den neuronalen Schaden bei der Maus nach traumatischer Hirnläsion mittels Controlled Cortical Impact

Die Beeinflussung des sekundären Hirnschadens nach SHT ist Hauptansatzpunkt der klinischen Therapie. Gleichzeitig sind die zugrunde liegenden Mechanismen zum Großteil unklar und Gegenstand aktueller Forschung. In verschiedenen Studien scheint sich das Renin-Angiotensin-Aldosteron-System als Teil der Pathophysiologie bei der Entstehung des sekundären Hirnschadens und der neuronalen Inflammation nach SHT herauszukristallisieren. Der Fokus richtet sich mitunter auf den AT2- Rezeptor, dessen protektive Wirkung in verschiedensten Ischämiemodellen und Geweben gezeigt werden konnte.rnIn der vorliegenden Promotionsarbeit wurde erstmalig die Wirkung einer AT2- Stimulation auf histologische, inflammatorische und neuromotorische Parameter nach CCI untersucht. Eine neuroprotektive Wirkung der AT2-Stimulation konnte dabei nicht nachgewiesen werden. Es kam weder zu einer Beeinflussung histologischer noch neuromotorischer Parameter, allerdings scheint der AT2-Agonist C21 bei einer Dosierung von 0,03 mg/kg KG zu einer Stimulation der inflammatorischen Reaktion zu führen

Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results

<div><p>Real-time reverse transcription polymerase chain reaction (PCR) is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.</p></div

Schematic presentation of the study design.

<p><b>A)</b> RNA of brain tissue from six naïve animals was extracted and pooled to create an identical dilution series. cDNA synthesis was then performed with each of the four kits. <b>B)</b> RNA of brain tissue from 6 naïve animals and 6 animals suffering traumatic brain injury was extracted. Each sample was reverse-transcribed, using all four kits, again ensuring the exact same starting material for each kit.</p

Influence of kit selection on linearity and efficacy of cDNA synthesis.

<p>The figure depicts absolute cDNA copy numbers after reverse transcription of one mRNA dilution series (pooled from mRNA of six naïve animals) with four different cDNA synthesis kits and RT-qPCR for the housekeeping genes ß2M and PPIA and the low-copy, regulated gene iNOS. All four kits show a linear correlation between the mRNA concentration and the cDNA output for ß2M and PPIA (Spearman's rank-order correlation coefficient; r²>0.99), except at a ß2M mRNA concentration of 1μg. Only kit 3 shows a strong linear correlation between iNOS cDNA and mRNA (r²>0.99; c). The mRNA/cDNA ratio varies significantly between the individual kits and the genes, although the individual kits show a certain consistency regarding the single gene dilution series (d-f). All plots show mean ± S.D.</p

Description of cDNA synthesis kits.

<p>Description of cDNA synthesis kits.</p

Influence of kit selection on absolute copy numbers.

<p>This figure demonstrates absolute cDNA copy numbers from naïve (n = 5) vs. trauma (n = 5) samples for PPIA (high-copy, non-regulated gene), iNOS and IL-1β (both low-copy, regulated genes). An equal amount of each sample was reverse-transcribed, using all four kits. A significantly higher cDNA expression was present in trauma vs. naïve samples for PPIA (kit 2, p = 0.002; kit 3, p = 0.014; and kit 4, p = 0.027), iNOS and IL-1β (all kits, p<0.05). Absolute and relative results vary significantly between the kits used (a-c). In accordance with our previous findings, the mRNA/cDNA ratio varies substantially between the kits (d) (although identical starting material was used) and between genes (although reverse-transcribed by the same kit under equal experimental conditions). All bar charts show mean ± S.D.</p

Impact of kit selection on expression data in an experimental paradigm of brain injury.

<p>To demonstrate whether discrepancies in the efficacy of cDNA synthesis between the four kits influence the results after normalization, the regulated genes iNOS and IL-1β were normalized against the non-regulated PPIA. Although equal amounts of mRNA were used, even after normalization, results for iNOS and IL-1β vary significantly (a, b). After calculating the percentage of normalized trauma cDNA from normalized naïve cDNA for each kit, no significant difference between the kits could be shown neither for iNOS nor for IL-1β (c, d) (p = n.s.). All bar charts show mean ± S.D.</p

Effect of gelatin-polysuccinat on cerebral oxygenation and microcirculation in a porcine haemorrhagic shock model

Abstract Background During early treatment of haemorrhagic shock maintenance of cerebral and end-organ oxygen supply by fluid resuscitation is mandatory. Gelatin-polysuccinat (GP) recently regained attention despite a still unclear risk profile and widely unknown effects on cerebral and peripheral microcirculation. This study investigates the effects of GP versus balanced electrolyte solution (BEL) with focus on cerebral regional oxygen saturation and peripheral microcirculation in a porcine haemorrhagic shock model. Methods After Animal Care Committee approval haemorrhagic shock was induced by arterial blood withdrawal in 27 anaesthetized pigs. Consequently, the animals received rapid fluid resuscitation by either GP or BEL to replace the removed amount of blood, or remained untreated (n = 3 × 9). Over two hours cerebral regional oxygen saturation by near-infrared spectroscopy and peripheral buccal microcirculation by combined white-light spectrometry and laser-Doppler flowmetry were recorded. Secondary parameters included extended haemodynamics, spirometry, haematological and blood gas parameters. Results Both fluid resuscitation regimes sufficiently stabilized the macro- and microcirculation in haemorrhagic shock with a more pronounced effect following GP infusion. GP administration led to a persisting, critical impairment of cerebral regional oxygen saturation through considerable haemodilution. Survival rates were 100% in both fluid resuscitation groups, but only 33% in the untreated control. Conclusion Equal amounts of GP and BEL sufficiently stabilize systemic circulation and microcirculatory perfusion. Forced fluid resuscitation by GP should be applied with caution to prevent haemodilution-induced impairment of cerebral oxygen delivery

Influence of rosuvastatin treatment on cerebral inflammation and nitro-oxidative stress in experimental lung injury in pigs

Background!#!Many patients with acute respiratory distress syndrome (ARDS) suffer from cognitive impairment after hospital discharge. Different mechanisms have been implicated as potential causes for this impairment, inter alia cerebral inflammation. A class of drugs with antioxidant and anti-inflammatory properties are β-HMG-CoA-reductase inhibitors ('statins'). We hypothesized that treatment with rosuvastatin attenuates cerebral cytokine mRNA expression and nitro-oxidative stress in an animal model of acute lung injury.!##!Methods!#!After approval of the institutional and state animal care committee, we performed this prospective randomized controlled animal study in accordance with the international guidelines for the care and use of laboratory animals. Thirty-two healthy male pigs were randomized to one of four groups: lung injury by central venous injection of oleic acid (n = 8), statin treatment before and directly after lung injury (n = 8), statin treatment after lung injury (n = 8), or ventilation-only controls (n = 8). About 18 h after lung injury and standardized treatment, the animals were euthanised, and the brains and lungs were collected for further examinations. We determined histologic lung injury and cerebral and pulmonal cytokine and 3-nitrotyrosine production.!##!Results!#!We found a significant increase in hippocampal IL-6 mRNA after lung injury (p &amp;lt; 0.05). Treatment with rosuvastatin before and after induction of lung injury led to a significant reduction of hippocampal IL-6 mRNA (p &amp;lt; 0.05). Cerebral 3-nitrotyrosine was significantly higher in lung-injured animals compared with all other groups (p &amp;lt; 0.05 vs. animals treated with rosuvastatin after lung injury induction; p &amp;lt; 0.001 vs. all other groups). 3-Nitrotyrosine was also increased in the lungs of the lung-injured pigs compared to all other groups (p &amp;lt; 0.05 each).!##!Conclusions!#!Our findings highlight cerebral cytokine production and nitro-oxidative stress within the first day after induction of lung injury. The treatment with rosuvastatin reduced IL-6 mRNA and 3-nitrotyrosine concentration in the brains of the animals. In earlier trials, statin treatment did not reduce mortality in ARDS patients but seemed to improve quality of life in ARDS survivors. Whether this is attributable to better cognitive function because of reduced nitro-oxidative stress and inflammation remains to be elucidated