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Background In moderate acute respiratory distress syndrome (ARDS) several studies support the usage of assisted spontaneous breathing modes. Only limited data, however, focus on the application in systemic sepsis and developing lung injury. The present study examines the effects of immediate initiation of pressure support ventilation (PSV) in a model of sepsis-induced ARDS. Methods 18 anesthetized pigs received a two-staged continuous lipopolysaccharide infusion to induce lung injury. The animals were randomly assigned to PSV or volume controlled (VCV) lung protective ventilation (tidal volume each 6 ml kg-1, n = 2x9) over six hours. Gas exchange parameters, hemodynamics, systemic inflammation, and ventilation distribution by multiple inert gas elimination and electrical impedance tomography were assessed. The post mortem analysis included histopathological scoring, wet to dry ratio, and alveolar protein content. Results Within six hours both groups developed a mild to moderate ARDS with comparable systemic inflammatory response and without signs of improving gas exchange parameters during PSV. The PSV group showed signs of more homogenous ventilation distribution by electrical impedance tomography, but only slightly less hyperinflated lung compartments by multiple inert gas elimination. Post mortem and histopathological assessment yielded no significant intergroup differences. Conclusions In a porcine model of sepsis-induced mild ARDS immediate PSV was not superior to VCV. This contrasts with several experimental studies from non-septic mild to moderate ARDS. The present study therefore assumes that not only severity, but also etiology of lung injury considerably influences the response to early initiation of PSV.

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.

A major challenge in the treatment of brain-injured patients is the decision on indication and timing of prophylactic anticoagulation. In addition, an increasing number of patients suffering from traumatic brain injury (TBI) are on preinjury anticoagulation therapy. Despite clear evidence for an increased risk of venous thromboembolic events and pulmonary embolism in traumatized patients without prophylactic anticoagulation, there is a lack of distinct recommendations and standardized clinical practice guidelines. This review summarizes current research evidence regarding post-traumatic prophylactic anticoagulation and management of patients with prehospital use of anticoagulants.In addition to nonpharmacological techniques like compression stockings, use of low-dose unfractionated heparin or low-molecular-weight heparin is effective in different studies in terms of thromboprophylaxis. If follow-up computed tomography scans and clinical neurological examinations do not show progression within 24 h after initial evaluation, prophylactic anticoagulation does not increase risk for hemorrhage progression and therefore seems to be safe after TBI.Stratification scores for identification of TBI patients with low, moderate, or high risk for spontaneous cerebral bleeding may help to allow early thromboprophylaxis while maintaining a good risk-benefit ratio. So far, these scores require validation by prospective trials. Therefore, current evidence requires control computed tomography scans prior to early pharmacological thromboprophylaxis.

Ischemia, especially pericontusional ischemia, is one of the leading causes of secondary brain damage after traumatic brain injury (TBI). So far efforts to improve cerebral blood flow (CBF) after TBI were not successful because of various reasons. We previously showed that nitric oxide (NO) applied by inhalation after experimental ischemic stroke is transported to the brain and induces vasodilatation in hypoxic brain regions, thus improving regional ischemia, thereby improving brain damage and neurological outcome. As regional ischemia in the traumatic penumbra is a key mechanism determining secondary posttraumatic brain damage, the aim of the current study was to evaluate the effect of NO inhalation after experimental TBI. NO inhalation significantly improved CBF and reduced intracranial pressure after TBI in male C57 Bl/6 mice. Long-term application (24 hours NO inhalation) resulted in reduced lesion volume, reduced brain edema formation and less blood–brain barrier disruption, as well as improved neurological function. No adverse effects, e.g., on cerebral auto-regulation, systemic blood pressure, or oxidative damage were observed. NO inhalation might therefore be a safe and effective treatment option for TBI patients.

After traumatic brain injury (TBI) elderly patients suffer from higher mortality rate and worse functional outcome compared to young patients. However, experimental TBI research is primarily performed in young animals. Aim of the present study was to clarify whether age affects functional outcome, neuroinflammation and secondary brain damage after brain trauma in mice. Young (2 months) and old (21 months) male C57Bl6N mice were anesthetized and subjected to a controlled cortical impact injury (CCI) on the right parietal cortex. Animals of both ages were randomly assigned to 15 min, 24 h, and 72 h survival. At the end of the observation periods, contusion volume, brain water content, neurologic function, cerebral and systemic inflammation (CD3+ T cell migration, inflammatory cytokine expression in brain and lung, blood differential cell count) were determined. Old animals showed worse neurological function 72 h after CCI and a high mortality rate (19.2%) compared to young (0%). This did not correlate with histopathological damage, as contusion volumes were equal in both age groups. Although a more pronounced brain edema formation was detected in old mice 24 hours after TBI, lack of correlation between brain water content and neurological deficit indicated that brain edema formation is not solely responsible for age-dependent differences in neurological outcome. Brains of old naive mice were about 8% smaller compared to young naive brains, suggesting age-related brain atrophy with possible decline in plasticity. Onset of cerebral inflammation started earlier and primarily ipsilateral to damage in old mice, whereas in young mice inflammation was delayed and present in both hemispheres with a characteristic T cell migration pattern. Pulmonary interleukin 1β expression was up-regulated after cerebral injury only in young, not aged mice. The results therefore indicate that old animals are prone to functional deficits and strong ipsilateral cerebral inflammation without major differences in morphological brain damage compared to young.

To investigate the effects of proportional assist ventilation, variable pressure support, and conventional pressure support ventilation on lung function and damage in experimental acute lung injury.: Randomized experimental study.University hospital research facility.: Twenty-four juvenile pigs.Pigs were anesthetized, intubated, and mechanically ventilated. Acute lung injury was induced by saline lung lavage. After resuming of spontaneous breathing, animals were randomly assigned to 6 hrs of assisted ventilation with pressure support ventilation, proportional assist ventilation, or variable pressure support (n = 8 per group). Mean tidal volume was kept at ≈6 mL/kg in all modes.Lung functional parameters, distribution of ventilation by electrical impedance tomography, and breathing patterns were analyzed. Histological lung damage and pulmonary inflammatory response were determined postmortem. Variable -pressure support and proportional assist ventilation improved oxygenation and venous admixture compared with pressure support ventilation. Proportional assist ventilation led to higher esophageal pressure time product than variable pressure support and pressure support ventilation, and redistributed ventilation from central to dorsal lung regions compared to pressure support ventilation. Variable pressure support and proportional assist ventilation yielded higher tidal volume variability than pressure support ventilation. Such pattern was deterministic (self-organized) during proportional assist ventilation and stochastic (random) during variable pressure support. Subject-ventilator synchrony as well as pulmonary inflammatory response and damage did not differ among groups.In a lung lavage model of acute lung injury, both variable pressure support and proportional assist ventilation increased the variability of tidal volume and improved oxygenation and venous admixture, without influencing subject-ventilator synchrony or affecting lung injury compared with pressure support ventilation. However, variable pressure support yielded less inspiratory effort than proportional assist ventilation at comparable mean tidal volumes of 6 mL/kg.

Rationale: Stroke is the third most common cause of death in industrialized countries. The main therapeutic target is the ischemic penumbra, potentially salvageable brain tissue that dies within the first few hours after blood flow cessation. Hence, strategies to keep the penumbra alive until reperfusion occurs are needed. Objective: To study the effect of inhaled nitric oxide on cerebral vessels and cerebral perfusion under physiological conditions and in different models of cerebral ischemia. Methods and Results: This experimental study demonstrates that inhaled nitric oxide (applied in 30% oxygen/70% air mixture) leads to the formation of nitric oxide carriers in blood that distribute throughout the body. This was ascertained by in vivo microscopy in adult mice. Although under normal conditions inhaled nitric oxide does not affect cerebral blood flow, after experimental cerebral ischemia induced by transient middle cerebral artery occlusion it selectively dilates arterioles in the ischemic penumbra, thereby increasing collateral blood flow and significantly reducing ischemic brain damage. This translates into significantly improved neurological outcome. These findings were validated in independent laboratories using two different mouse models of cerebral ischemia and in a clinically relevant large animal model of stroke. Conclusions: Inhaled nitric oxide thus may provide a completely novel strategy to improve penumbral blood flow and neuronal survival in stroke or other ischemic conditions.

BACKGROUND: Autologous blood transfusion (ABT), for example, by means of cell saver equipment, is used to reduce the need for allogenic blood transfusion in patients with high perioperative blood loss. This study investigated the effect of blood/extracorporal surface interaction during withdrawal and retransfusion of shed autologous blood on cerebral inflammation in rats. Rats subjected to hypotension with cerebral ischemia served as positive controls. METHODS: Eighty-eight male Sprague-Dawley rats were anesthetized with sevoflurane, instrumented, and randomly assigned to the following groups: sham-operation (SHAM), autologous blood withdrawal/transfusion only (ABT), or bilateral carotid artery occlusion and autologous blood withdrawal/transfusion (BCAO/ABT). Inflammatory gene expression was investigated with real-time RT-polymerase chain reaction at 6, 12, and 24 hours after SHAM, ABT, or BCAO/ABT in brain hippocampal tissue. Naive rats were investigated as reference. RESULTS: ABT alone had no impact on hippocampal inflammatory gene expression, whereas after BCAO/ABT tumor necrosis factor-alpha (10.7 fold at 24 h), interleukin-1β (2.1 fold at 6 h), interleukin-6 (35.7 fold at 24 h), COX-2 (9.3 fold at 6 h), and inducible nitric oxide synthase (3.4 fold at 24 h) increased compared with SHAM. CONCLUSIONS: ABT by itself did not provoke an inflammatory reaction in the healthy brain. However, in combination with cerebral ischemia the induction of a broad spectrum of inflammatory parameters indicates an inflammatory reaction of the hippocampus beginning after 6 hours and being most pronounced after 24 hours. Therefore, this study shows that cerebral inflammation is not induced by ABT after contact with extracorporal surfaces in rats.

Background: Cerebral edema is an important risk factor for death and poor outcome following subarachnoid hemorrhage (SAH). However, underlying mechanisms are still poorly understood. Matrix metalloproteinase (MMP)-9 is held responsible for the degradation of microvascular basal lamina proteins leading to blood-brain barrier dysfunction and, thus, formation of vasogenic cerebral edema. The current study was conducted to clarify the role of MMP-9 for the development of cerebral edema and for functional outcome after SAH. Methods: SAH was induced in FVB/N wild-type (WT) or MMP-9 knockout (MMP-9–/–) mice by endovascular puncture. Intracranial pressure (ICP), regional cerebral blood flow (rCBF), and mean arterial blood pressure (MABP) were continuously monitored up to 30 min after SAH. Mortality was quantified for 7 days after SAH. In an additional series neurological function and body weight were assessed for 3 days after SAH. Subsequently, ICP and brain water content were quantified. Results: Acute ICP, rCBF, and MABP did not differ between WT and MMP-9–/– mice, while 7 days’ mortality was lower in MMP-9–/– mice (p = 0.03; 20 vs. 60%). MMP-9–/– mice also exhibited better neurological recovery, less brain edema formation, and lower chronic ICP. Conclusions: The results of the current study suggest that MMP-9 contributes to the development of early brain damage after SAH by promoting cerebral edema formation. Hence, MMP- 9 may represent a novel molecular target for the treatment of SAH.

Existing murine models of global cerebral ischemia are technically challenging thereby hampering the use of genetically engineered mice to study cardiac arrest-induced brain damage. We therefore investigated, if disconnecting the cerebral circulation from vertebral collateral blood flow by proximal occlusion of the basilar artery together with temporary bilateral common carotid artery occlusion (BCCAo) may be a more feasible approach. C57/Bl6 mice were anesthetized and the basilar artery was occluded through a ventral approach. Ten days later BCCAo was performed for 8-14min. Increasing durations of ischemia resulted in enhanced neuronal cell death in cortex, striatum, and hippocampus (22-63%) and increased neurological dysfunction and mortality (0-36%). Following 10min of BCCAo, the duration of global ischemia with the most favorable mortality/neuronal cell death ratio, hippocampal damage started 6h after the insult while cortical and striatal damage was delayed by at least 24h. No further loss of neuronal cells was observed later than 3 days. The proposed two-step approach resulted in complete cerebral ischemia and caused neuronal damage with high reproducibility and small variability. In combination with transgenic and knock-out mice this technically feasible model may help to extend our knowledge on the pathophysiology of cardiac arrest-induced brain damage.

The role of the endothelial contractile apparatus in the process of brain edema formation after brain trauma is not characterized. Phosphorylation of myosin light chains by myosin light chain kinases (MLCK) activates endothelial contractile elements and results in a rearrangement of the cytoskeleton. This may enhance post-traumatic blood-brain barrier dysfunction. In order to investigate the role of the MLCK on brain edema formation and blood-brain barrier permeability after brain injury, mice were anesthetized and subjected to a controlled cortical impact (CCI). MLCK expression is significantly up-regulated after CCI with a maximum 12 h post-injury. Specific inhibition of MLCK by ML-7 resulted in a reduction of phosphorylation of myosin light chains and improved blood-brain-barrier integrity. Accordingly, ML-7 attenuated post-traumatic brain edema formation and intracranial hypertension 24 h after CCI. Prevention of brain edema formation did not translate into improved neurological outcome or reduced brain lesion. In conclusion, the results confirm that the endothelial contractile apparatus is activated by CCI and opens the endothelial barrier leading to vasogenic brain edema formation. Lack of neurological and histological improvement suggests that specific targeting of vasogenic brain edema at the endothelial level is not sufficient to limit secondary brain damage and has, therefore, to be combined with other potential neuroprotective strategies.

Quantitative measurements of gene expression require correction for tissue sample size, RNA quantity, and reverse transcription efficiency. This can be achieved by normalization with control genes. The study was designed to identify candidates not altered after brain trauma. Male C57Bl/6 mice were anesthetized with isoflurane, and a pneumatic brain trauma was induced by controlled cortical impact (CCI) on the right parietal cortex. Brains were removed at 15 min, and 3, 6, 12 and 24 h after CCI and from naive animals (n = 6 each). Absolute copies of six control genes (beta-2-microglobin [B2M], cyclophilin A, beta-actin, hypoxanthine ribosyltransferase [HPRT], porphobilinogen deaminase [PBGD], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and one example target gene (iNOS) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR; Lightcycler) in the traumatic focus and contralateral tissue. Control gene expression was stable until 12 h after CCI. At 24 h after CCI expression of B2M, cyclophilin A and HPRT remained stable in the contusion, while expression of beta-actin, GAPDH, and PBGD increased. Due to variations between animals (+/-85%), increases in beta-actin (+64%) and GAPDH (+59%) did not reach the level of significance. In non-contused tissue, expression of all genes dropped 24 h after CCI (range, -17% to -61%). Due to low variations between animals and stable expression after CCI, B2M and cyclophilin A seem to be suitable to serve as single normalizer. Normalization of the example target gene iNOS resulted in varying relative expression extending from onefold (PBDG) to 10-fold (HPRT). The results suggest that the knowledge of the temporal profile of control genes is essential to properly interpret results of mRNA quantification.

Vasogenic brain edema is one of the major determinants for mortality following subarachnoid hemorrhage (SAH). Although the formation of vasogenic brain edema occurs on the microvascular level by opening of endothelial tight junctions and disruption of the basal lamina, microvascular changes following experimental SAH are poorly characterized. The aim of the present study was therefore to investigate the time course of blood-brain barrier (BBB) dysfunction and basal lamina damage following SAH as a basis for the better understanding of the pathophysiology of SAH. SAH was induced in Sprague-Dawley rats by an endovascular filament. Animals were sacrificed 6, 24, 48, and 72 h thereafter (n=9 per group). Microvascular basal lamina damage was quantified by collagen type IV immunostaining. Western blotting was used to quantify collagen IV protein content and bovine serum albumin (BSA) extravasation as a measure for basal lamina damage and blood-brain barrier disruption, respectively. BSA Western blot revealed significant (p

PURPOSE OF REVIEW This article presents an overview of the most recent and important strategies to reduce secondary brain damage. RECENT FINDING There is currently no magic bullet available to protect the brain after neuronal injury. This is related to the complex pathophysiology of cerebral ischemia, which makes it unlikely that a single pharmacological intervention results in sustained neuroprotection. Analyses of clinical studies reveal that acute physiologic derangements (e.g. fever, hypertension and hypotension, hypoxemia, hypercapnia, hyperglycemia) are the most important predictors of unfavorable outcome after brain injury and have to be treated. The effectiveness of anesthetic agents to extend the ischemic tolerance of neurons has been demonstrated in experimental settings, but such benefits have not been demonstrated in humans. The effectiveness of osmodiuretics to decrease elevated intracranial pressure, a factor with relevance to outcome, has been demonstrated. Infusion of magnesium in patients with subarachnoidal hemorrhage can reduce the occurrence of delayed ischemia caused by cerebrovascular spasm. The prophylactic administration of glucocorticoids should be avoided. While the positive effects of chronic administration of statins to reduce the incidence of stroke has been demonstrated in several clinical studies, the protective effect of acute administration of statins after a cerebral insult has do be defined. SUMMARY Control of physiological variables, avoidance of hyperthermia, intensive control of plasma glucose concentrations, use of anesthetic agents and osmodiuretics to control intracranial hypertension and the possible prophylactic administration of magnesium in patients at risk of vasospasm and of statins in patients with cerebrovascular risk factors are currently the most important strategies to reduce neuronal injury.

Despite the constantly increasing use of genetically engineered mice in biomedical research, control of crucial physiological parameters such as blood pressure and arterial blood gases is difficult to achieve in temporarily anesthetized mice due to lack of techniques for reversible arterial cannulation. Here we report that arterial blood pressure and blood gases can be measured reliably in anaesthetized and artificially ventilated mice using non-invasive technology. C57Bl6 mice were anaesthetized by i.p. injection of midazolam, fentanyl, and medetomidin, intubated, and ventilated for 3h. End tidal pCO2 was monitored by micro-capnometry. Arterial blood pressure was measured non-invasively using a tail cuff. Non-invasive blood pressure (NIBP) correlated strongly with the invasive arterial blood pressure measured at the external carotid artery (r = 0.99, P < 0.001) and end tidal pCO2 values correlated very well with arterial blood pCO2 (r = 0.93, P < 0.001). The current results demonstrate that it is possible to reliably measure and control the most relevant physiological parameters in anesthetized mice. Thereby the current study may help to reduce animal numbers and perform mice experiments under more defined and controlled physiological conditions in the future.

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema.

It is unclear whether a single, brief, 15-minute episode of background anesthesia already modulates delayed secondary processes after experimental brain injury. Therefore, this study was designed to characterize three anesthesia protocols for their effect on molecular and histological study endpoints. Mice were randomly separated into groups that received sevoflurane (sevo), isoflurane (iso) or an intraperitoneal anesthetic combination (midazolam, fentanyl and medetomidine; comb) prior to traumatic brain injury (controlled cortical impact, CCI; 8 m/s, 1 mm impact depth, 3 mm diameter). Twenty-four hours after insult, histological brain damage, neurological function (via neurological severity score), cerebral inflammation (via real-time RT-PCR for IL6, COX-2, iNOS) and microglia (via immunohistochemical staining for Iba1) were determined. Fifteen minutes after CCI, the brain contusion volume did not differ between the anesthetic regimens (sevo = 17.9±5.5 mm(3); iso = 20.5±3.7 mm(3); comb = 19.5±4.6 mm(3)). Within 24 hours after injury, lesion size increased in all groups (sevo = 45.3±9.0 mm(3); iso = 31.5±4.0 mm(3); comb = 44.2±6.2 mm(3)). Sevo and comb anesthesia resulted in a significantly larger contusion compared to iso, which was in line with the significantly better neurological function with iso (sevo = 4.6±1.3 pts.; iso = 3.9±0.8 pts.; comb = 5.1±1.6 pts.). The expression of inflammatory marker genes was not significantly different at 15 minutes and 24 hours after CCI. In contrast, significantly more Iba1-positive cells were present in the pericontusional region after sevo compared to comb anesthesia (sevo = 181±48/mm(3); iso = 150±36/mm(3); comb = 113±40/mm(3)). A brief episode of anesthesia, which is sufficient for surgical preparations of mice for procedures such as delivering traumatic brain injury, already has a significant impact on the extent of secondary brain damage.

Evolution of brain edema after subarachnoid hemorrhage (SAH) in rats Serge C Thal, Sonja Sporer, Nikolaus Plesnila, Karsten Schoeller, Robert Schmid-Elsaesser and Stefan Zausinger 1. Department of Anaesthesiology, Johannes Gutenberg University, Mainz, Germany 2. Institute for Surgical Research, Ludwig Maximilians University, Munich, Germany 3. Department of Neurosurgery, Ludwig Maximilians University, Munich, Germany

Mechanisms of early cerebral edema following SAH are poorly understood. Previously, we demonstrated a peak of edema evolution at 24 hours after experimental SAH in rats. Furthermore, brain water content correlated with neurological deficits of animals. In focal cerebral ischemia, alteration of the microvascular basal lamina, a constituent of the blood-brain barrier, and its relevance for development of cerebral edema and hemorrhagic transformation has previously been demonstrated. The present study was designed to demonstrate microvascular basal lamina damage in the context of blood-brain barrier dysfunction as a potential target for therapeutical interventions to reduce vasogenic brain edema following SAH.