Cognitive decline in Tg2576 mice shows sex-specific differences and correlates with cerebral amyloid-beta.

Patients suffering from Alzheimer's disease show a sex-dependent decline of cognitive function. The aim of this investigation was to show these differences in an animal model for Alzheimer's disease and to determine whether this effect is correlated to amyloid-beta-induced pathophysiological changes. Therefore, we assessed cognitive performance with the modified hole-board test in female and male Tg2576 and wild type mice at the age of 6, 8, 10, 12, 14, and 16 months and correlated these findings to the total amount of soluble amyloid-beta and insoluble amyloid deposits in the brain. Tg2576 mice perform worse than wild types. Female Tg2576 mice develop an accentuated cognitive impairment (wrong choice total) beginning at the age of 12 months compared to their male littermates. Alterations in the mice's behaviour do not show interference with these deficits. Cognitive impairment is correlated to the amount of soluble amyloid-beta and insoluble amyloid deposits in the brain in a sex-dependent manner.

Intracerebroventricular injection of beta-amyloid in mice is associated with long-term cognitive impairment in the modified hole-board test.

BACKGROUND: The intracerebroventricular injection of beta-amyloid (Aß) in mice allows the investigation of acute effects on cognitive function and cellular pathology. The aim of this investigation was to further characterize the time course of Aß-induced cognitive and behavioural changes and to detect potential molecular mechanisms. METHODS: Cannulas were implanted in the lateral cerebral ventricle. 14days after surgery the mice were injected with Aß1-42 or phosphate buffered saline (PBS). Starting 2, 4 or 8 (PBS only 4) days after injection we evaluated cognitive and behavioural performance using the modified hole board test (mHBT). We determined tumour-necrosis factor alpha (TNF alpha) and caspase 3 by western blotting, on days 10, 12 and 16. Data were analysed using general linear modelling, Kruskall-Wallis and Mann-Whitney-U test. RESULTS: Aß induced a decline in cognitive performance represented as an increased total number of wrong choices during the testing period from day 2-15 (p<0.05). Behavioural parameters were comparable between mice treated with Aß and PBS. There was no difference regarding TNF alpha levels between the groups. Compared to day 16 Caspase 3 levels were increased on day 10 (p=0.004). CONCLUSIONS: Application of Aß in the lateral ventricle of mice is associated with cognitive impairment of declarative memory in the mHBT. There is no interference caused by altered behaviour. Therefore, it represents a valid model for acute Aß-mediated neurotoxic effects. Although the exact mechanisms remain unclear, changes in levels of Caspase 3 suggest apoptosis as an important factor for the development of cognitive dysfunction.

Selenium Pretreatment for Mitigation of Ischemia/Reperfusion Injury in Cardiovascular Surgery: Influence on Acute Organ Damage and Inflammatory Response.

Ischemia/reperfusion injury (IRI) contributes to morbidity and mortality after cardiovascular surgery requiring cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). Multi-organ damage is associated with substantial decreases of blood selenium (Se) levels in patients undergoing cardiac surgery with CPB. We compared the influence of a dietary surplus of Se and pretreatment with ebselen, a mimic of the selenoenzyme glutathione peroxidase, on IRI-induced tissue damage and inflammation. Male Wistar rats were fed either a Se-adequate diet containing 0.3 ppm Se or supplemented with 1 ppm Se (as sodium selenite) for 5 weeks. Two other groups of Se-adequate rats received intraperitoneal injection of ebselen (30 mg/kg) or DMSO (solvent control) before surgery. The animals were connected to a heart-lung-machine and underwent 45 min of global ischemia during circulatory arrest at 16 °C, followed by re-warming and reperfusion. Selenite and ebselen suppressed IRI-induced leukocytosis and the increase in plasma levels of tissue damage markers (AST, ALT, LDH, troponin) during surgery but did not prevent the induction of proinflammatory cytokines (IL-6, TNF-α). Both Se compounds affected phosphorylation and expression of proteins related to stress response and inflammation: Ebselen increased phosphorylation of STAT3 transcription factor in the heart and decreased phosphorylation of ERK1/2 MAP kinases in the lungs. Selenite decreased ERK1/2 phosphorylation and HSP-70 expression in the heart. Pretreatment with selenite or ebselen protected against acute IRI-induced tissue damage during CPB and DHCA. Potential implications of their different actions with regard to molecular stress markers on the recovery after surgery represent promising targets for further investigation.

Electromyographic permutation entropy quantifies diaphragmatic denervation and reinnervation.

Spontaneous reinnervation after diaphragmatic paralysis due to trauma, surgery, tumors and spinal cord injuries is frequently observed. A possible explanation could be collateral reinnervation, since the diaphragm is commonly double-innervated by the (accessory) phrenic nerve. Permutation entropy (PeEn), a complexity measure for time series, may reflect a functional state of neuromuscular transmission by quantifying the complexity of interactions across neural and muscular networks. In an established rat model, electromyographic signals of the diaphragm after phrenicotomy were analyzed using PeEn quantifying denervation and reinnervation. Thirty-three anesthetized rats were unilaterally phrenicotomized. After 1, 3, 9, 27 and 81 days, diaphragmatic electromyographic PeEn was analyzed in vivo from sternal, mid-costal and crural areas of both hemidiaphragms. After euthanasia of the animals, both hemidiaphragms were dissected for fiber type evaluation. The electromyographic incidence of an accessory phrenic nerve was 76%. At day 1 after phrenicotomy, PeEn (normalized values) was significantly diminished in the sternal (median: 0.69; interquartile range: 0.66-0.75) and mid-costal area (0.68; 0.66-0.72) compared to the non-denervated side (0.84; 0.78-0.90) at threshold p<0.05. In the crural area, innervated by the accessory phrenic nerve, PeEn remained unchanged (0.79; 0.72-0.86). During reinnervation over 81 days, PeEn normalized in the mid-costal area (0.84; 0.77-0.86), whereas it remained reduced in the sternal area (0.77; 0.70-0.81). Fiber type grouping, a histological sign for reinnervation, was found in the mid-costal area in 20% after 27 days and in 80% after 81 days. Collateral reinnervation can restore diaphragm activity after phrenicotomy. Electromyographic PeEn represents a new, distinctive assessment characterizing intramuscular function following denervation and reinnervation.

A cardiopulmonary bypass with deep hypothermic circulatory arrest rat model for the investigation of the systemic inflammation response and induced organ damage.

BACKGROUND: Cardiopulmonary bypass (CPB) is a commonly used technique in cardiac surgery. CPB is however associated with a strong induction of systemic inflammatory response syndrome (SIRS) which in conjunction with ischemia and reperfusion may lead to multiple organ failure. The aim of the study was to establish and characterize a CPB rat model incorporating deep hypothermic circulatory arrest with a specific focus on the extent of the inflammatory reactions and organ damage as a groundwork for novel therapeutics against SIRS and I/R induced organ injury. MATERIALS AND METHODS: Male Wistar rats (n = 6) were cannulated for CPB, connected to a heart-lung-machine (HLM) and cooled to a temperature of 16°C before they underwent 45 minutes of deep hypothermic circulatory arrest with global ischaemia. Arrest was followed by rewarming and 60 minutes of reperfusion. Haemodynamic and vital parameters were recorded throughout the CPB procedure. Only animals displaying sinus rhythm throughout reperfusion were utilized for analysis. Rats were euthanized and tissue samples were harvested. Blood gas analysis was performed and blood samples were taken. Induction of organ damage was examined by analysis of protein levels and phosphorylation status of kinases and stress proteins. Results were compared to animals (n = 6) which did not undergo CPB. RESULTS: CPB induced leucocytosis and an increase of interleukin-6 and TNF-α plasma values indicating an inflammatory response. Markers of tissue damage and dysfunction, such as troponin T, creatinine and AST were elevated. Phosphorylation of STAT3 was induced in all examined organs. Activation of MAPK and induction of heat shock proteins occurred in an organ-specific manner with most pronounced effects in heart, lungs and kidneys. CONCLUSIONS: The presented CPB rat model shows the induction of SIRS and activation of specific signalling cascades. SIRS seems not to be provoked during DHCA and is elicited mainly during reperfusion. This model might be suitable to test the efficacy of therapeutics applied in major heart surgery with and without DHCA.

Sevoflurane anesthesia improves cognitive performance in mice, but does not influence in vitro long-term potentation in hippocampus CA1 stratum radiatum.

BACKGROUND: Whether the occurrence of postoperative cognitive dysfunction is a result of the effects of surgery or anesthesia is under debate. In this study, we investigated the impact of sevoflurane anesthesia on cognitive performance and cellular mechanisms involved in learning and memory. METHODS: Male C57Bl6/J mice (4-5 months) were exposed to one minimum alveolar concentration sevoflurane for two hours. After 24 h, cognitive performance of mice was assessed using the modified hole board test. Additionally, we evaluated hippocampal long-term potentiation and expression levels of different receptor subunits by recording excitatory postsynaptic field potentials and using the western blot technique, respectively. Non-anesthetized mice served as controls. RESULTS: In anesthetized mice, neither cognitive performance nor long-term potentiation was impaired 24 h after anesthesia. Interestingly, sevoflurane anesthesia induced even an improvement of cognitive performance and an elevation of the expression levels of N-methyl-D-aspartate (NMDA) receptor type 1 and 2B subunits in the hippocampus. CONCLUSIONS: Since NMDA receptor type 1 and 2B subunits play a crucial role in processes related to learning and memory, we hypothesize that sevoflurane-induced changes in NMDA receptor subunit composition might cause hippocampus-dependent cognitive improvement. The data of the present study are in favor of a minor role of anesthesia in mediating postoperative cognitive dysfunction.

Functional outcome in female rats after 45 minutes of deep hypothermic circulatory arrest: gender matters.

BACKGROUND: After adjusting for cofactors, female gender remains an independent risk factor. The aim of our study was to investigate the role of gender on functional outcome 14 days after 45 minutes of deep hypothermic circulatory arrest (DHCA) in rats. METHODS: After institutional animal care committee Institutional Review Board approval, 40 male and 40 female rats were randomly divided into two groups (40 DHCA, 40 controls). The rats were neutered or sham-neutered and 40 of them were subjected to DHCA with 40 controls remaining naïve. Postoperative functional performance was assessed with modified hole-board test. Brain morphology was assessed using hematoxylin and eosin (HE) staining and cerebral inflammation/apoptosis determined immunohistochemically. Data were analyzed using general linear models (post hoc analysis of variance [ANOVA] and Bonferroni t tests) and Kruskal-Wallis (post hoc Mann-Whitney U test) (p < 0.05). RESULTS: Cognition and behavior after 45 minutes of DHCA were comparable to that of four naïve rats in all four DHCA groups. Motor function and morphologic outcome were better in females. Nuclear factor kappa B (NFκB) was upregulated in the female normal group. Activated caspase-3 was higher in females whereas poly(ADP-ribose) polymerase was in males. Of note is the secondary finding of a high drop-out rate during the early postoperative phase in the female groups (16 out of 37). CONCLUSIONS: Postoperative motor skills were better in females, with less neuronal damage, more neuronal NFκB, and activated caspase-3. However, the chance finding of a high mortality rate in females warrants investigation with mortality as the principal aim, focusing on heart, liver, lung, kidney, and intestine with regard to its rate.

Evaluation of neurobehavioral deficits following different severities of cerebral ischemia in rats: a comparison between the modified hole board test and the Morris water maze test.

OBJECTIVE: While histological injury following cerebral ischemia has been extensively characterized in rodents, evidence on the effects on executive functioning is still missing. This study was designed to evaluate neuropsychological outcome following different severities of cerebral ischemia in rats with the modified hole board test or the Morris water maze. SETTING: With institutional review board approval, anesthetized rats were exposed to bilateral carotid artery occlusion (BCAO) for escalating time intervals (0-12.5 min). Postoperatively cognitive performance was assessed using either the modified hole board test (mHB) or the Morris water maze (MWM). On postoperative day 14 rats were euthanized and intact neurons in five cerebral regions were counted. RESULTS: Rats of the 0 and 5 min groups showed normal functional outcome with mild histological damage after 5 min of BCAO. Following 7.5 min of BCAO the mHB test showed cognitive deficits reflecting histological damage of the hippocampus while the MWM revealed normal functional outcome. Rats of the 10 and 12.5 min groups showed cognitive deficits in both tests, motor dysfunction and behavioral alterations in the mHB test and profound histological damage. CONCLUSIONS: The results indicate that the mHB is not inferior to the MWM for the evaluation of cognitive impairment following incomplete forebrain ischemia in rats. As the mHB additionally investigates a variety of behavioral dimensions and motor parameters in the same test environment, it is advantageous for the evaluation of interacting and potentially confounding behavioral changes following cerebral ischemia in rats.

A mobile phone based alarm system for supervising vital parameters in free moving rats.

BACKGROUND: Study protocols involving experimental animals often require the monitoring of different parameters not only in anesthetized, but also in free moving animals. Most animal research involves small rodents, in which continuously monitoring parameters such as temperature and heart rate is very stressful for the awake animals or simply not possible. Aim of the underlying study was to monitor heart rate, temperature and activity and to assess inflammation in the heart, lungs, liver and kidney in the early postoperative phase after experimental cardiopulmonary bypass involving 45 min of deep hypothermic circulatory arrest in rats. Besides continuous monitoring of heart rate, temperature and behavioural activity, the main focus was on avoiding uncontrolled death of an animal in the early postoperative phase in order to harvest relevant organs before autolysis would render them unsuitable for the assessment of inflammation. FINDINGS: We therefore set up a telemetry-based system (Data Science International, DSI™) that continuously monitored the rat's temperature, heart rate and activity in their cages. The data collection using telemetry was combined with an analysis software (Microsoft excel™), a webmail application (GMX) and a text message-service. Whenever an animal's heart rate dropped below the pre-defined threshold of 150 beats per minute (bpm), a notification in the form of a text message was automatically sent to the experimenter's mobile phone. With a positive predictive value of 93.1% and a negative predictive value of 90.5%, the designed surveillance and alarm system proved a reliable and inexpensive tool to avoid uncontrolled death in order to minimize suffering and harvest relevant organs before autolysis would set in. CONCLUSIONS: This combination of a telemetry-based system and software tools provided us with a reliable notification system of imminent death. The system's high positive predictive value helped to avoid uncontrolled death and facilitated timely organ harvesting. Additionally we were able to markedly reduce the drop out rate of experimental animals, and therefore the total number of animals used in our study. This system can be easily adapted to different study designs and prove a helpful tool to relieve stress and more importantly help to reduce animal numbers.

Xenon administration immediately after but not before or during cardiopulmonary bypass with cerebral air embolism impairs cerebral outcome in rats.

CONTEXT: The neuroprotective properties of xenon might improve cerebral outcome after cardiac surgery using cardiopulmonary bypass. However, in the presence of cerebral air emboli, xenon impaired cognitive and histological outcome in a rat cardiopulmonary bypass model, a result which is due to the property of xenon to expand air bubbles. OBJECTIVE: The current study was designed to assess whether cerebral outcome in the setting of cardiopulmonary bypass with cerebral air embolism could be altered by administration of xenon restricted to periods when the occurrence of cerebral air embolism is unlikely. DESIGN: With institutional review board approval, 40 rats were allocated randomly to one of four groups (n = 10) which determined the period of xenon inhalation: 'before', 'during' or 'after' cardiopulmonary bypass or 'none'. SETTING: Rats were subjected to 90  min of normothermic cardiopulmonary bypass combined with 10 small cerebral air emboli. Xenon was administered according to group assignment: the 'none' group received no xenon; in the other groups, the lungs were ventilated with 56% xenon before, during or after cardiopulmonary bypass and cerebral air embolism. MAIN OUTCOME MEASURES: Motor and cognitive outcomes were tested using the modified hole-board test. Cerebral infarction volumes were determined on postoperative day 21. RESULTS: Animals that received xenon after cardiopulmonary bypass and cerebral air embolism had impaired motor function scores [after: median 6.6 (range 0.25-8), before: 0.5 (0-3), during: 1.5 (0.25-2.75), none: 1 (0-1.75)] and cognitive performance [after: 9 (6.5-9), before: 0 (0-5.5), during: 1 (0-5.5), none: 1 (0-4)] compared with all other groups (P < 0.05). Administration of xenon after cardiopulmonary bypass and cerebral air embolism also led to larger cerebral infarction volumes [after: 74 µl (54-157), before: 45 µl (20-82), during: 33 µl (23-54), none: 22 µl (17-78)] compared with the groups that received xenon during cardiopulmonary bypass and cerebral air embolism or no xenon at all (P < 0.05). CONCLUSION: Xenon administered immediately after cardiopulmonary bypass and cerebral air embolism impaired motor, cognitive and histological outcome in rats. At no time did inhalation of xenon lead to any beneficial effects on cerebral outcome when compared with inhalation of nitrogen.

Perioperative moxifloxacin treatment in rats subjected to deep hypothermic circulatory arrest: reduction in cerebral inflammation but without improvement in cognitive performance.

OBJECTIVE: Moxifloxacin reduces infectious complications after cerebral damage, such as ischemia and stroke. This study investigated whether moxifloxacin treatment influences cerebral inflammation and improves cognitive outcome after cardiopulmonary bypass with deep hypothermic circulatory arrest in rats. METHODS: Rats were randomly assigned to deep hypothermic circulatory arrest (n = 40), sham operation (n = 40), and untreated control (n = 20) groups. Deep hypothermic circulatory arrest and sham groups were equally subdivided into moxifloxacin and placebo subgroups, receiving 6 × 100 mg/kg moxifloxacin or saline solution every 2 hours intraperitoneally. Hippocampal tumor necrosis factor α, nuclear factor κB, cyclooxygenase 2, and macrophages were assessed immunohistochemically. Histologic outcome was determined with hematoxylin and eosin. Neurologic outcome was assessed preoperatively and postoperatively. Cognitive performance was tested with the modified hole board test for 14 postoperative days. RESULTS: On postoperative day 14, deep hypothermic circulatory arrest moxifloxacin group was lower than deep hypothermic circulatory arrest placebo group in hippocampal neurons positive for tumor necrosis factor α (1.33, 0.73-2.37, vs 4.10, 2.42-18.67), nuclear factor κB (3.03, 1.33-5.20, vs 9.32, 2.53-24.14), and cyclooxygenase 2 (3.16, 0.68-6.04, vs 8.07, 3.27-19.91) and also had fewer macrophages than all other groups (72, 60-90, vs deep hypothermic circulatory arrest placebo 128, 76-203, sham moxifloxacin 89, 48-96, and sham placebo 81, 47-87). On postoperative day 14, both deep hypothermic circulatory arrest groups showed impaired motor, cognitive, and histologic outcomes relative to sham-operated groups, with no difference between deep hypothermic circulatory arrest subgroups. CONCLUSIONS: Moxifloxacin transiently reduces cerebral inflammatory reaction, but without impact on neurologic function, histologic outcome, or long-term cognitive performance.

Fast rewarming after deep hypothermic circulatory arrest in rats impairs histologic outcome and increases NFκB expression in the brain.

OBJECTIVE: Deep hypothermia is used as a neuroprotectant during cardiac surgery utilizing deep hypothermic circulatory arrest (DHCA), although the ideal rewarming strategy is not known yet. Some of the neuroprotective properties of hypothermia seem to be mediated by Nuclear Factor Kappa B (NFκB) as an important transcription factor. The current study was designed to investigate the effect of the rewarming rate on histologic outcome and cerebral NFκB expression one day following DHCA in rats. METHODS: With IRB approval, 20 rats were cannulated for cardiopulmonary bypass (CPB), cooled to a rectal temperature of 15-18°C, subjected to 45min of DHCA and randomly assigned to either a slow (40 min) or a fast (20 min) rewarming protocol. At 24 hours post DHCA, the number of eosinophilic neurons was analyzed with hematoxylin and eosin (HE) staining, and NFκB expression immunohistochemically. The two experimental groups were compared with untreated control rats. RESULTS: HE staining showed more eosinophilic neurons in the motor cortex following fast rewarming (60 [15-388]) compared to slow rewarming (15 [10-21]) (p<0.05). Neuronal expression of NFκB was increased in the fast rewarming group in both brain areas, the motor cortex (fast: 258 [135-393]; slow: 165 [80-212]; control: 73 [44-111]) as well as the hippocampus (fast: 243 [209-314]; slow: 202 [187-239]; control: 86 [68-108]) (p<0.05). Hyperthermic episodes were strictly avoided. CONCLUSIONS: Fast rewarming with strict avoidance of hyperthermia after DHCA in rats was accompanied by pronounced histologic damage and accentuated cerebral NFκB expression.

Avoiding stroke during cardiac surgery.

The life saving benefits of cardiac surgery are frequently accompanied by negative side effects such as stroke, that occurs with an incidence of 2%-13% dependent to type of surgery. The etiology is most likely multifactorial with embolic events considered as main contributor. Although stroke presents a common complication, no guidelines for any routine use of pharmacological substances or non-pharmacological strategies exist to date. Non-pharmacological strategies include monitoring of brain oxygenation and perfusion with devices such as near infrared spectroscopy and Transcranial Doppler help. Epiaortic and transesophageal echocardiography visualize aorta pathology, enabling the surgeon to sidestep atheromatous segments. Additionally can the use of specially designed aortic cannulae and filters help to reduce embolization. Brain perfusion can be improved by using antero- or retrograde cerebral perfusion during deep hypothermic circulatory arrest, by tightly monitoring mean arterial blood pressure and hemodilution. Controlling perioperative temperature and glucose levels may additionally help to ameliorate secondary damage. Many pharmacological compounds have been shown to be neuroprotective in preclinical models, but clinical studies failed to confirm these results so far. Remacemide, an NMDA-receptor-antagonist showed a significant drug-based neuroprotection during cardiac surgery. Other substances currently assessed in clinical trials whose results are still pending are acadesine, an adenosine-regulating substance, the free radical scavenger edaravone and the local anesthetic lidocaine. Stroke remains as significant complication after cardiac surgery. Non-pharmacological strategies allow perioperative caregivers to detect injurious events and to ameliorate stroke and its sequelae. Considering the multi-factorial etiology though, stroke prevention will likely have to be addressed with an individualistic combination of different strategies and substances.

The impact of cardiopulmonary bypass on systemic interleukin-6 release, cerebral nuclear factor-kappa B expression, and neurocognitive outcome in rats.

BACKGROUND: Neurocognitive deficits after cardiac surgery with cardiopulmonary bypass (CPB) continue to affect patients' quality of life, and an inflammatory reaction may be one of the contributors. We designed this experiment to study perioperative systemic interleukin-6 (IL-6) concentrations, cerebral expression of nuclear factor-kappa B (NF-kappaB), and neurocognitive outcome after CPB in young rats. The impact of oxygenator size on these outcomes was also assessed. METHODS: Rats were randomly assigned to 1 of 4 groups: control (n = 7, nonanesthetized), sham-operated rats (n = 10, anesthetized, cannulated, and not connected to CPB), and 2 CPB groups, anesthetized, cannulated, and subjected to 90 min of CPB, using either a small-volume rat oxygenator (CPB/rat oxygenator, n = 10) or a neonate oxygenator (CPB/neonate oxygenator, n = 10). Systemic IL-6 was determined before, at the end of, and 2 h after CPB or at equivalent times. Hippocampal NF-kappaB expression was assessed on postoperative day 21 using immunohistochemistry. Neurocognitive performance was assessed with the modified hole-board test at baseline and for 21 postoperative days. RESULTS: Both CPB groups had increased systemic IL-6 levels compared with sham, with the neonate oxygenator causing a substantially larger increase at 2 h after CPB compared with the rat oxygenator group (CPB/rat oxygenator: 220 pg/mL [16-415]; CPB/neonate oxygenator: 1400 pg/mL [592-5812]) (P < 0.05). Hippocampal NF-kappaB was increased in experimental groups compared with controls (10 +/- 4). CPB resulted in more NF-kappaB-positive neurons (271 +/- 57 CPB/neonate oxygenator and 269 +/- 72 CPB/rat oxygenator) compared with sham operation (173 +/- 24). Neurocognitive and behavioral performances were unaltered and comparable among all groups. CONCLUSIONS: Pronounced systemic inflammatory responses to experimental CPB associated with increased hippocampal expression of NF-kappaB were not accompanied by neurocognitive impairment. This suggests that other factors beyond CPB and inflammatory responses might contribute to adverse neurocognitive outcomes after cardiac surgery.

Cerebral tumor necrosis factor alpha expression and long-term neurocognitive performance after cardiopulmonary bypass in rats.

OBJECTIVE: Cerebral inflammatory reaction is discussed as a contributor to adverse cerebral outcome after cardiac surgery with cardiopulmonary bypass. This study was designed to determine the effect of cardiopulmonary bypass on both cerebral expression of tumor necrosis factor alpha and neurocognitive outcome in rats. METHODS: With institutional review board approval, 50 rats were randomly assigned to one of 3 groups: rats of the cardiopulmonary bypass group were subjected to 75 minutes of normothermic cardiopulmonary bypass. Sham-operated animals underwent identical preparation but were not connected to cardiopulmonary bypass, whereas rats of the control group were neither anesthetized nor cannulated. Ten rats per group survived 4 hours after cardiopulmonary bypass or the sham operation for immediate postoperative determination of tumor necrosis factor alpha-expressing cells (immunohistochemistry) and cerebral tumor necrosis factor alpha mRNA levels (polymerase chain reaction). The remaining animals survived 10 days for neurocognitive assessment by using the modified hole-board test and for analysis of cerebral tumor necrosis factor alpha activation in the late postoperative period. RESULTS: Expression of tumor necrosis factor alpha mRNA was increased 4 hours after cardiopulmonary bypass and the sham operation, with higher expression in the cardiopulmonary bypass group (chi(2) [2] = 25.08, P < .001). Both experimental groups demonstrated larger numbers of tumor necrosis factor alpha-positive cells in the early and late postoperative periods (F [1] = 13.08, P < or = .001) and an impaired neurocognitive performance on the first postoperative days compared with that seen in the control group (F [2, 24] = 4.26, P = .02). CONCLUSIONS: Cerebral tumor necrosis factor alpha activation in both experimental groups during the early postoperative period was accompanied by transient neurocognitive impairment. Therefore cardiopulmonary bypass alone demonstrated no effect on cerebral inflammation and neurocognitive outcome.

Long-term assessment of NFkappaB expression in the brain and neurologic outcome following deep hypothermic circulatory arrest in rats.

OBJECTIVES: Inflammatory response is discussed as a contributor to neurologic deficits following cardiac surgery using deep hypothermic circulatory arrest (DHCA). Nuclear Factor Kappa B (NFkappaB) presents a central transcription factor whose expression pattern and subsequent role very much depend on the type and manner of cerebral injury. This study was designed to assess the time course of cerebral NFkappaB expression in relation to neurologic performance over 28 days following 45min of DHCA in rats. METHODS: With Institutional Review Board approval, 30 rats were subjected to cardiopulmonary bypass (CPB) with 45min of DHCA (rectal temperature 15-18 degrees Celsius) and randomly assigned to 1, 3, 7, 14 and 28 days of postoperative survival. Untreated animals served as control (n=6). Cerebral NFkappaB expression was analyzed immunohistochemically, cyclooxygenase-2 (COX-2) and inhibitor of kappa B-alpha (IkappaBalpha) using Western Blot and the number of eosinophilic neurons with hematoxylin and eosin (HE) staining. Neurologic outcome was assessed pre- and postoperatively. RESULTS: Neuronal expression of NFkappaB in the hippocampus peaked at day one, remaining elevated in the motor cortex until day 28. Rats showed neurologic deficits on postoperative day one. Cerebral COX-2 was increased during the first postoperative week and IkappaBalpha peaked on day 14. Histologic damage in the motor cortex and hippocampus persisted until day 28. No systemic inflammation was detectable postoperatively. CONCLUSIONS: Postoperative day one presents with the highest NFkappaB-expression in the ischemia-sensitive hippocampus, accompanied by neurologic dysfunction and histologic damage following 45min of DHCA in rats.

Cerebral air emboli differentially alter outcome after cardiopulmonary bypass in rats compared with normal circulation.

BACKGROUND: Cerebral air emboli (CAE) are thought to contribute to adverse cerebral outcomes following cardiac surgery with cardiopulmonary bypass (CPB). This study was designed to investigate the effect of escalating volumes of CAE on survival and neurologic and histologic outcomes. In addition, the effect of xenon administration during CAE on these outcomes was determined. METHODS: With institutional review board approval, four groups were studied (n = 15). In two CPB-CAE groups, rats were subjected to 90 min CPB with 10 repetitively administered CAE. Rats in two sham-CAE groups were also exposed to CAE but not to CPB. Rats were randomly assigned to sequential dose cohorts receiving CAE ranging from 0.2 to 10 microl in a dose-escalating fashion. Groups were further subdivided into xenon (56%) and nitrogen groups. Rats with severe neurologic damage were killed; others were neurologically tested until postoperative day 7, when infarct volumes were determined. Survival and neurologic and histologic outcomes were tested with logistic regression analyses (P < 0.05). RESULTS: This study demonstrates a dose-dependent relation between CAE volumes and survival, neurologic outcome, and histologic outcome. For all outcomes, CPB adversely affected the dose-effect curves compared with sham-CAE groups (P < 0.05). Xenon demonstrated no impact on either outcome. CONCLUSIONS: This study describes the successful incorporation of CAE in a rodent CPB model and allows identifying suitable CAE volumes for subsequent studies. CAE exhibit a differential effect on outcome in rats undergoing CPB versus those not exposed to CPB. Perioperative administration of xenon remained without any effect on outcome.

The long-term effect of sevoflurane on neuronal cell damage and expression of apoptotic factors after cerebral ischemia and reperfusion in rats.

We investigated the long-term effects of sevoflurane on histopathologic injury and key proteins of apoptosis in a rat hemispheric ischemia/reperfusion model. Sixty-four male Sprague-Dawley rats were randomly assigned to Group 1 (fentanyl and N2O/O2; control) and Group 2 (2.0 vol% sevoflurane and O2/air). Ischemia (45 min) was produced by unilateral common carotid artery occlusion plus hemorrhagic hypotension (mean arterial blood pressure 40 mm Hg). Animals were killed after 1, 3, 7, and 28 days. In hematoxylin and eosin-stained brain sections eosinophilic hippocampal neurons were counted. Activated caspase-3 and the apoptosis-regulating proteins Bax, Bcl-2, Mdm-2, and p53 were analyzed by immunostaining. No eosinophilic neurons were detected in sevoflurane-anesthetized rats over time, whereas 9%-38% of the hippocampal neurons were eosinophilic (days 1-28) in control animals. On days 1 and 3, the concentration of Bax was 140%-200% larger in fentanyl/N2O-anesthetized animals compared with sevoflurane. Bcl-2 was 100% less in control animals during the first 3 days. Activated caspase-3 was detected in neurons of both groups (0.75%-2.2%). These data support a sustained neuroprotective potency of sevoflurane related to reduced eosinophilic injury after cerebral ischemia/reperfusion.

Influence of propofol on neuronal damage and apoptotic factors after incomplete cerebral ischemia and reperfusion in rats: a long-term observation.

BACKGROUND: Propofol reduces neuronal damage from cerebral ischemia when investigated for less than 8 postischemic days. This study investigates the long-term effects of propofol on neuronal damage and apoptosis-related proteins after cerebral ischemia and reperfusion. METHODS: Male Sprague-Dawley rats were randomly assigned as follows: group 1 (n = 32, control): fentanyl and nitrous oxide-oxygen; group 2 (n = 32, propofol): propofol and oxygen-air. Ischemia (45 min) was induced by carotid artery occlusion and hemorrhagic hypotension. Pericranial temperature and arterial blood gases were maintained constant. After 1, 3, 7, and 28 postischemic days, brains were removed, frozen, and sliced. Hippocampal eosinophilic cells were counted. The amount of apoptosis-related proteins Bax, p53, Bcl-2, and Mdm-2 and neurons positive for activated caspase-3 were analyzed. RESULTS: In propofol-anesthetized rats, no eosinophilic neurons were detected, whereas in control animals, 16-54% of hippocampal neurons were eosinophilic (days 1-28). In control animals, the concentration of Bax was 70-200% higher after cerebral ischemia compared with that in animals receiving propofol over time. Bcl-2 was 50% lower in control animals compared with propofol-anesthetized rats during the first 3 days. In both groups, a maximal 3% of the hippocampal neurons were positive for activated caspase-3. CONCLUSIONS: These data show sustained neuroprotection with propofol. This relates to reduced eosinophilic and apoptotic injury. Activated caspase-3-dependent apoptotic pathways were not affected by propofol. This suggests the presence of activated caspase-3-independent apoptotic pathways.