Knockdown of the AIM2 molecule attenuates ischemia-reperfusion-induced spinal neuronal pyroptosis by inhibiting AIM2 inflammasome activation and subsequent release of cleaved caspase-1 and IL-1ß.

Ischemia-reperfusion (IR) injury induces activation of several inflammasomes that widely affect neuroinflammation and, subsequently, neuronal viability. The absent in melanoma 2 (AIM2) inflammasome is highly expressed in neurons after traumatic injury. This study was performed to investigate whether the AIM2 molecule acts as an initiator to trigger AIM2 inflammasome activation and regulate neuronal pyroptosis in a mouse IR model. The early motor dysfunction that occurred within the first 8 h post-IR injury was closely associated with a massive increase in dsDNA in serum and cerebrospinal fluid (CSF) at the same observed timepoints. However, the subsequent persistent dysfunction was consistent with the continuously increasing protein levels of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), cleaved caspase-1 and IL-1ß with time. Upregulated AIM2 immunoreactivity was primarily visualized in neurons. The si-AIM2 treatment in vivo preserved part of motor function, accompanied by decreased protein levels of AIM2, ASC, cleaved caspase-1 and IL-1ß. In vitro, the direct interactions between the AIM2 molecule and caspase-1 were demonstrated by immunofluorescence staining and coimmunoprecipitation. In this context, both si-AIM2 and Ac-YVAD-CMK treatments effectively maintained neuronal viability, as demonstrated by the decreased percentage of cells with pyroptosis and release of lactate dehydrogenase (LDH), accompanied by weak immunoreactivity and a decreased number of AIM2-caspase-1 positive neurons. By contrast, poly(dA-dT) treatment exacerbated neuronal pyroptosis by reversing the above-mentioned effects. However, no significant differences were observed after si-Con treatment. These results suggest AIM2 molecule played an important role in initiating AIM2 inflammasome activation through IR-induced release of ectopic dsDNA.

Combined proteomic and metabolomic analyses of cerebrospinal fluid from mice with ischemic stroke reveals the effects of a Buyang Huanwu decoction in neurodegenerative disease.

Ischemic stroke is one of the most common causes of death worldwide and is a major cause of acquired disability in adults. However, there is still a need for an effective drug for its treatment. Buyang Huanwu decoction (BHD), a traditional Chinese medicine (TCM) prescription, has long been used clinically to aid neurological recovery after stroke. To establish potential clinical indicators of BHD efficacy in stroke treatment and prognosis, we conducted a combined proteomic and metabolomic analysis of cerebrospinal fluid (CSF) samples in a mouse stroke model. CSF samples were obtained from male mice with acute ischemic stroke induced by middle cerebral ischemic/reperfusion (CI/R) injury, some of which were then treated with BHD. Label-free quantitative proteomics was conducted using nano-LC-MS/MS on an LTQ Orbitrap mass and metabolomic analysis was performed using nanoprobe NMR and UHPLC-QTOF-MS. The results showed that several proteins and metabolites were present at significantly different concentrations in the CSF samples from mice with CI/R alone and those treated with BHD. These belonged to pathways related to energy demand, inflammatory signaling, cytoskeletal regulation, Wnt signaling, and neuroprotection against neurodegenerative diseases. In conclusion, our in silico data suggest that BHD treatment is not only protective but can also ameliorate defects in pathways affected by neurological disorders. These data shed light on the mechanism whereby BHD may be effective in the treatment and prevention of stroke-related neurodegenerative disease.

1H NMR metabolic signature of cerebrospinal fluid following repetitive lower-limb remote ischemia preconditioning.

Background OBJECTIVE: The cerebral ischemia/reperfusion greatly influences brain metabolism. Remote ischemia preconditioning (RIPC) is reported to confer neuroprotective effects against cerebral ischemia in animal models and human. This study aims to investigate the metabolomic profiles of cerebrospinal fluid (CSF) in patients treated with repetitive lower limb RIPC and provides an insight into possible mechanism underlying RIPC-induced neuroprotection. METHOD: Fifty healthy patients undergoing minor surgery under spinal anesthesia were randomly allocated to 2 groups: control group (Group C, n = 25) and RIPC treatment group (Group T,n = 25). Repetitive limb RIPC were performed 3 sessions, consisting of three 5-min cycles per session from the day before surgery to the morning on the surgery day. The CSF samples were collected from 48 patients before intrathecal injection of local anesthetic. A proton nuclear magnetic resonance (1H NMR)-based metabonomics approach was used to obtain the CSF metabolic profiles of the samples (n = 24 each). The acquired data were processed with MestReNova and followed by statistical analysis with SIMCA-P. RESULTS: The model obtained with the orthogonal partial least-squares discriminant analysis (OPLS-DA) identified difference of metabolite profiles between two groups. The validation of the discriminant analysis showed that the accuracy of the OPLS-DA model was 81.3%. Sixteen metabolites including glucose, amino-acids and organic acids et al. were identified as the most influential CSF biomarkers for the discrimination between two groups, which are involved in pathways of energy metabolism and amino-acids metabolism. CONCLUSION: 1H NMR spectra combined with pattern recognition analysis offers a new and promising platform to investigate metabolic signatures in patients treated with RIPC. Our results suggest repetitive RIPC mainly changes energy metabolism and amino-acid metabolism in brain, which provides a potential mechanistic understanding of RIPC-induced tolerance to cerebral ischemia.

Effect of Buyang Huanwu decoction on amino acid content in cerebrospinal fluid of rats during ischemic/reperfusion injury.

The inhibitory effect of Buyang Huanwu decoction (BYHWD) on ischemic injury has been proven, but it is not clear how amino acid levels in cerebrospinal fluid (CSF) are associated with BYHWD treatment, nor the mechanism by which BYHWD protects the brain from ischemia/reperfusion injury. We investigated the effect of BYHWD on the amino acid content of CSF in rats during ischemia-reperfusion injury. Ischemia was imposed by right middle cerebral artery occlusion (MCAO). CSF was continuously collected from the striatum via brain microdialysis before and after ischemia/reperfusion. We used on-line derivatization combined with high-performance liquid chromatography with fluorescence detection (HPLC-FD) to determine levels of glutamate (Glu), aspartate (Asp), glycine (Gly), taurine (Tau), and γ-aminobutyric acid (GABA) in CSF. The MCAO model displayed an infarct lesion in the ipsilateral hemisphere and nerve injuries, as the left upper limb was unable to extend and turn leftward. Significant increases in excitatory and inhibitory amino acids were observed in the CSF of the ischemic rats relative to the sham-operated group (P<0.01). Treatment with BYHWD reduced the areas of cerebral infarction and improved the neurological behavior scores of rats after MCAO. BYHWD treatment was also associated with a significant decrease in excitatory amino acids and increase in inhibitory amino acids in the CSF. Only the higher dose of BYHWD (20mg/kg) affected all these levels significantly. Attenuated excitatory toxicity and reduced areas of cerebral infarction associated with BYHWD treatment might be due to a protective mechanism induced by BYHWD against ischemia/reperfusion injury.

(1)H NMR-based metabolomics exploring biomarkers in rat cerebrospinal fluid after cerebral ischemia/reperfusion.

In our study, metabolomics was used to investigate biochemical changes in the early stages of rats focal cerebral ischemia/reperfusion (I/R) injury. Cerebrospinal fluid (CSF) samples at 0, 0.5, 1, 3, and 6 h of reperfusion (n = 10), based on (1)H NMR spectroscopy and multivariate data analyses, were tested to analyze the changing of metabolites during the early disease process. Partial least squares-discriminant analysis scores plots of the (1)H NMR data revealed clear differences among the experiment groups. Combining the results of the loading plot and t-test, we found that twenty-seven metabolites were changed significantly (p < 0.05) in the CSF samples among the different groups. Among that, the potential biomarkers in CSF of ischemic rats were: acetic acid, 3-hydroxyisovaleric acid, 3-hydroxybutyric acid, choline, l-alanine, creatine, creatinine, glycine, pyruvic acid, glycerol, glutamic acid, d-fructose, l-lactic acid and acetone. These findings help us understand the biochemical metabolite changes in CSF of I/R rats in early stages. What's more, metabolomics may, therefore, have the potential to be developed into a clinically useful diagnostic tool of ischemic brain injury.

Protective effect of intrathecal ketorolac in spinal cord ischemia in rats: a microdialysis study.

BACKGROUND: The prevention of ischemic paraplegia after thoracoabdominal aortic surgery is challenging for both anesthesiologists and surgeons. In a previous study, we showed that intrathecal ketorolac pre-treatment protects rats against ischemic spinal cord injury. In the present study, using a microdialysis method, we investigated whether this neuroprotective effect was related to changes in the spinal cord release of nitric oxide (NO) or the excitatory amino acids (EAAs) aspartate and glutamate. METHODS: Rats were randomized to receive either intrathecal saline or ketorolac 60 microg (10 rats per group), 1 h before spinal cord ischemic injury induced by balloon inflation of a 2F Fogarty catheter in the thoracic aorta with maintenance of the proximal arterial blood pressure at 40 mmHg for 11 min, followed by reperfusion. Another 10 animals were used as the sham-operated control group. Ischemic injury was assessed by hind limb motor function. Cerebrospinal fluid dialysates were collected at baseline (before ischemia) and at 1, 2, 3, 4, 6, 12 and 24 h after the start of reperfusion, and were analyzed for EAAs using high-performance liquid chromatography and for NO metabolites using an NO analyzer. RESULTS: The results showed that intrathecal ketorolac attenuated spinal cord ischemic injury. Dialysate concentrations of NO and EAAs were increased after spinal cord ischemia, and this effect was inhibited by intrathecal administration of ketorolac. CONCLUSIONS: The results of this study suggest that the neuroprotective effect of intrathecal ketorolac in spinal cord ischemia in rats may be caused by a decrease in the spinal cord release of NO and EAAs.

Accumulation of calpain and caspase-3 proteolytic fragments of brain-derived alphaII-spectrin in cerebral spinal fluid after middle cerebral artery occlusion in rats.

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Monitoring of intrathecal oxygen tension during experimental aortic occlusion predicts ultrastructural changes in the spinal cord.

OBJECTIVE: To study the correlation between intrathecal PO2 and ultrastructural changes in the spinal cord during thoracic aortic occlusion in pigs. MATERIAL AND METHODS: In 18 pigs, online intrathecal oxygenation was monitored by a multiparameter Paratrend catheter (Biomedical Sensors, High Wycombe, United Kingdom) during 60 minutes' clamping of the proximal and distal descending thoracic aorta. The animals were randomly divided into 2 groups (A and B) depending on the level of distal aortic clamping. Distal aortic perfusion was restored through an aorto-iliac shunt, which also maintained low thoracic segmental perfusion of the spinal cord in group B. Perfusion-fixation technique was used before harvesting the spinal cord specimens, which later were evaluated with light and electron microscopy by an independent observer. Intrathecal parameters were interpreted as normal if PO2 was more than 0.8 kPa and PCO2 was less than 12 kPa, as intermediate ischemia if PO2 was 0.8 or less or PCO (2) was more than 12 kPa, and as absolute ischemia if PO2 was 0.8 or less and PCO2 was more than 12 kPa. RESULTS: Among 6 animals with ultrastructural changes of absolute spinal cord ischemia-reperfusion injury, 5 also had absolute ischemia according to variables derived by the Paratrend catheter. The 2 methods were in agreement in 3 of 5 animals with intermediate ischemia-reperfusion changes and in 5 of 6 animals with normal findings. The accuracy of cerebrospinal fluid PO2 and PCO2 to predict electron microscopy-verified intermediate or absolute ischemia-reperfusion injury was 94%. CONCLUSIONS: Monitoring of intrathecal PO2 after clamping of the descending aorta correlated with ultrastructural changes in the spinal cord in this pig model.

Neuroprotective effects of phencyclidine on acute cerebral ischemia and reperfusion injury of rabbits.

Acute cerebral ischemia and reperfusion injury of rabbits was produced by permanently occluding the vertebral arteries and temporarily clamping the common carotid arteries for 30 min. Phencyclidine [1-(phenylcyclohexyl)piperidine, PCP] 40-80 micrograms.kg-1 icv 30 min before ischemia significantly attenuated the decrease of the total power of electroencephalogram (EEG) within 30 min of ischemia and improved the recovery of brain electric activity following reperfusion. PCP 20-80 micrograms.kg-1 dose-dependently suppressed the creatine kinase (CK) release during cerebral ischemia and reperfusion, and PCP 40-80 micrograms.kg-1 reduced brain ischemic damage. These improvements indicated that PCP has protective effects on acute cerebral ischemia and reperfusion injury.