JZL184, as a monoacylglycerol lipase inhibitor, down-regulates inflammation in a cannabinoid pathway dependent manner.

INTRODUCTION: Stroke is a prevalent disorder which is associated with several complications including inflammation. JZL-184 (JZL) inhibits arachidonic acid (AA) production and consequently results in two-arachidonoylglycerol (2-AG) accumulation. Both reduced production of AA metabolic products and increased 2-AG, the agonist of type 1 cannabinoid receptor (CB1), can result in reduced inflammation. In this study, we investigated the mechanisms of JZL in the improvement of stroke complications in mouse permanent cerebral ischemia (PPMCAO) model using AM251, the antagonist of CB1. MATERIAL AND METHODS: PMCAO mice were divided into six groups including intact, controls, vehicle, JZL, AM251 and JZL plus AM251 administrated groups. Brain infarction and edema, brain levels of matrix metalloperoteinase-9 (MMP9), interleukin (IL)-10 and tumor necrosis factor-α (TNF-α) and behavioral functions have been examined in all groups. RESULTS: The results showed that JZL lowered brain infarction, neurological disorders, TNF-α and MMP9 more effectively than JZL plus AM251. JZL and JZL plus AM251 reduced brain edema and increased brain IL-10. JZL, AM251 and JZL plus AM251 improve behavioral functions. DISCUSSION: JZL reduces brain infarction and brain pro-inflammatory molecules in CB1 pathway dependent manner. JZL also reduces brain edema and increased IL-10 in CB1 pathways or decreased AA metabolites. Further, AM251 improves behavioral functions via unknown mechanisms.

Study on cerebroprotective actions of Clerodendron glandulosumleaves extract against long term bilateral common carotid artery occlusion in rats.

Stroke is a major cause of death and disability worldwide. The resulting burden on the society continues to grow, with increase in the incidence of stroke. Oxidative stress has been involved in the pathogenesis of several neurological diseases including acute stroke.Focal and global cerebral ischemia represents diseases that are common in the human population.In recent years much attention is being paid towards the exploration of herbal preparation, antioxidant agents and combination therapies including COX-2 inhibitors in experimental model of stroke.Possible effect of a hydroalcoholic leaf extract of Clerodendron glandulosumColeb (C. glandulosum)on oxidant-antioxidant status in ischemia-hypoperfusion injury in the rat forebrain has been investigated.Healthy adult male Wistar albino rats were divided into five groups (n=8). Group I was served as Sham control (normal saline 1ml/kg, orally), group II was served hypoperfusion control (normal saline 1ml/kg, orally), group III, group IV were served as hydroalcoholic extract treated (200 and 400mg/kg, orally) and group V was treated with Quercetin (10mg/kg, orally) for 14days to assess preventive and curative effects of C. glandulosum. Flavonoid and phenolic compounds exhibit a broad spectrum of biological activity, including antioxidant. C. glandulosum extract (200 and 400mg/kg, p.o) was administered orally, once daily for a period of 2 weeks after the occlusion of BCCA. After 14th days rats were subjected to behavioral studies. After behavioral studies animals were sacrificed and brain was removed and homogenized. Estimation of Lipid peroxidation (LPO) Myeloperoxidase (MPO), estimation of protein levels and the activities of Superoxide dismutase (SOD), Catalase (CAT), were performed. Infarct size and histopathological changes were observed in treated groups.

Aag-initiated base excision repair promotes ischemia reperfusion injury in liver, brain, and kidney.

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag(-/-) mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag(-/-) mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag(-/-) liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.

Neuroprotective effect of calcitriol on ischemic/reperfusion injury through the NR3A/CREB pathways in the rat hippocampus.

Calcitriol has been demonstrated to provide neuroprotection against ischemia/reperfusion (I/R) injury. However, the exact mechanism of this protection remains unknown. In the present study, the neuroprotective effect of calcitriol was investigated in rats exposed to cerebral I/R injury induced by middle cerebral artery occlusion (MCAO). In addition, the involvement of NR3A, extracellular signal­regulated kinase 1/2 (ERK1/2), and phosphorylated cAMP/Ca2+­response element binding protein (p­CREB) in this protective action was determined in the hippocampal neurons. Western blot analysis was conducted to analyze the protein levels of NR3A, mitogen­activated protein kinase kinase (MEK) and p­CREB. The immunoreactivity of p­CREB and NR3A were measured by quantum dot­based immunofluorescence analysis. Results showed that MCAO rats exhibited large cortical infarct volumes. By contrast, intraperitoneal administration of calcitriol significantly reduced infarct volumes seven days following reperfusion, and these results were accompanied by elevated NR3A and p­CREB activity in the hippocampal neurons. The inhibition of MEK by the addition of PD98059 led to attenuation of the neuroprotective effects of calcitriol and a correlated decrease in CREB activity. The results also demonstrated that calcitriol protected the brain from I/R injury through the NR3A­MEK/ERK­CREB pathway.

Matrix metalloproteinase-2 deletions protect against hemorrhagic transformation after 1 h of cerebral ischemia and 23 h of reperfusion.

Although elevated matrix metalloproteinase (MMP)-2 levels were highly related to the degradation of tight junction (TJ) proteins and basal lamina and neuronal injury after ischemia, until very recently, little experimental evidence was available to test the role of the MMP-2 knockout (KO) in blood-brain-barrier (BBB) injury and the development of hemorrhage transformation (HT). Here, we assessed the role of the MMP-2 KO in BBB injury, HT and other brain injuries after 1h of ischemia and 23 h of reperfusion. Middle cerebral artery occlusion (MCAO) was performed in MMP-2 KO mice. Reperfusion was started 1h after the onset of MCAO. All mice were sacrificed 24h after the MCAO. MMP-2 deficiency reduced the decrease in protein levels of collagen IV and cellular membrane occludin (p<0.01 and 0.05 vs. wild-type (WT), respectively) and attenuated increase in cytosol occludin level in ischemic brain (p<0.01 vs. WT). The hemorrhage volume and brain infarction were significantly decreased in both the cortex and striatum in the MMP-2 KO mice (p<0.01 vs. WT). The MMP-2 KO also had reduced brain swelling in the cortex and improved neurological deficits (p<0.01 vs. WT). These studies provide direct evidence that targeting MMP-2 will effectively protect against collagen and occludin loss and HT after ischemia and reperfusion.

Neuroprotection after stroke by targeting NOX4 as a source of oxidative stress.

SIGNIFICANCE: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. RECENT ADVANCES: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. CRITICAL ISSUES: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. FUTURE DIRECTIONS: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy.

The association between small vessel infarcts and the activities of amyloid-ß peptide degrading proteases in apolipoprotein E4 allele carriers.

Small vessel (SV) and large vessel (LV) brain infarcts are distinct pathologies. Using a homebound elderly sample, the numbers of either infarct subtypes were similar between those apolipoprotein E4 allele (ApoE4) carriers (n = 80) and noncarriers (n = 243). We found that the higher the number of SV infarcts, but not LV infarcts, a participant had, the higher the activity of substrate V degradation in serum especially among ApoE4 carriers (ß = +0.154, SE = 0.031, P < .0001) after adjusting for the confounders. Since substrate V degradation could be mediated by insulin-degrading enzyme (IDE) or/and angiotensin-converting enzyme (ACE), but no relationship was found between SV infarcts and specific ACE activities, blood IDE may be a useful biomarker to distinguish the brain infarct subtypes. Insulin-degrading enzyme in blood may also imply an important biomarker and a pathological event in Alzheimer disease through SV infarcts in the presence of ApoE4.

PHLPP1 gene deletion protects the brain from ischemic injury.

A recently discovered protein phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase) has been shown to dephosphorylate Akt on its hydrophobic motif (Ser473) thereby decreasing Akt kinase activity. We generated PHLPP1 knockout (KO) mice and used them to explore the ability of enhanced in vivo Akt signaling to protect the brain against ischemic insult. Brains from KO mice subjected to middle cerebral artery occlusion (MCAO) for 2 hours showed significantly greater increases in Akt activity and less neurovascular damage after reperfusion than wild-type (WT) mice. Remarkably, infarct volume in the PHLPP1 KO was significantly reduced compared with WT (12.7±2.7% versus 22.9±3.1%) and this was prevented by Akt inhibition. Astrocytes from KO mice and neurons in which PHLPP1 was downregulated showed enhanced Akt activation and diminished cell death in response to oxygen-glucose deprivation. Thus, deletion of PHLPP1 can enhance Akt activation in neurons and astrocytes, and can significantly increase cell survival and diminish infarct size after MCAO. Inhibition of PHLPP could be a therapeutic approach to minimize damage after focal ischemia.

Dual action of NO synthases on blood flow and infarct volume consecutive to neonatal focal cerebral ischemia.

Research into neonatal ischemic brain damage is impeded by the lack of a complete understanding of the initial hemodynamic mechanisms resulting in a lesion, particularly that of NO-mediated vascular mechanisms. In a neonatal stroke rat model, we recently show that collateral recruitment contributes to infarct size variability. Non-specific and selective NO synthase (NOS) inhibition was evaluated on cerebral blood-flow changes and outcome in a P7 rat model of arterial occlusion (left middle cerebral artery electrocoagulation with 50 min occlusion of both common carotid arteries). Blood-flow changes were measured by using ultrasound imaging with sequential Doppler recordings in both internal carotid arteries and basilar trunk. Cortical perfusion was measured by using laser Doppler flowmetry. We showed that global NOS inhibition significantly reduced collateral support and cortical perfusion (collateral failure), and worsened the ischemic injury in both gender. Conversely, endothelial NOS inhibition increased blood-flows and aggravated volume lesion in males, whereas in females blood-flows did not change and infarct lesion was significantly reduced. These changes were associated with decreased phosphorylation of neuronal NOS at Ser(847) in males and increased phosphorylation in females at 24h, respectively. Neuronal NOS inhibition also increased blood-flows in males but not in females, and did not significantly change infarct volumes compared to their respective PBS-treated controls. In conclusion, both nNOS and eNOS appear to play a key role in modulating arterial blood flow during ischemia mainly in male pups with subsequent modifications in infarct lesion.

Occlusion of middle cerebral artery induces apoptosis of cerebellar cortex neural cells via caspase-3 in rats.

AIM: Occlusion of the middle ferebral artery in rats may cause secondary injury that is not associated with middle ferebral artery feeding zone. This entity has been investigated very rarely. MATERIAL AND METHODS: HE staining method observed the changes of cerebellar cortex after MCAO operation. Electron Microscopy and TUNEL methods observed the apoptosis of neural cells of cerebellar cortex after MCAO in rats. Immunohistochemical analyses method observed the caspase-3 in neural cells of cerebellar cortex. RESULTS: The results of HE staining indicated that no ischemia-necrosis changes of cerebellar cortex tissue were observed after MCAO operation by HE staining. Further experiments by Electron Microscopy and TUNEL assay revealed that the apoptosis of neural cells of cerebellar cortex were induced after MCAO in rats. Furthermore, immunohistochemical analyses showed that caspase-3 played an important role on MCAO-induced apoptosis of neural cells of cerebellar cortex. CONCLUSION: These data showed for the first time that the role of caspase-3 in the mechanism of secondary injury of separated infarction in cerebellar cortex after middle cerebral artery occlusion in rats and it might give a new treatment strategy for individuals with human ischemic stroke.

Brief anoxia preconditioning and HIF prolyl-hydroxylase inhibition enhances neuronal resistance in organotypic hippocampal slices on model of ischemic damage.

It is well known that a brief anoxia or hypoxia episodes can render brain resistant to a subsequent ischemia. Recent investigations indicate that mechanisms of such stimulated endogenous neuroprotection are related to the family of hypoxia-inducible factors (HIF), however there are still little data available on the role of HIF family members in hippocampus-a brain structure, highly sensitive to oxygen deficiency. We have used the model of cultured hippocampal slices and single-cell quantitative RT-PCR to study HIF-1α and HIF-3α mRNA expression following triple 5-min mild anoxia, 30-min oxygen-glucose deprivation and their combination. We also tested the effects of HIF prolyl-hydroxylase inhibition with 2,4-pyridinedicarboxylic acid diethyl ester pre-treatment followed by a 30-min oxygen-glucose deprivation. It was found that neuronal damage induced by oxygen-glucose deprivation was accompanied by a significant decrease in both HIF-1α and HIF-3α mRNA levels in CA1 but not CA3 neurons. Anoxia preconditioning did not affect cell viability and HIF mRNA levels but applied before oxygen-glucose deprivation prevented neuronal damage and suppression of HIF-1α and HIF-3α mRNA expression. It was also found that effects of the prolyl-hydroxylase inhibitor were similar to anoxia preconditioning. These results suggest that anoxia preconditioning increases anti-ischemic neuronal resistance which to a certain extent correlates with the changes of HIF-1α and HIF-3α expression.

PTEN deletion prevents ischemic brain injury by activating the mTOR signaling pathway.

It is increasingly clear that the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a negative regulator of neuronal cell survival. However, its molecular mechanisms remain poorly understood. Here we found that PTEN/mTOR is critical for controlling neuronal cell death after ischemic brain injury. Male rats were subjected to MCAO (middle cerebral artery occlusion) followed by pretreating with bpv (pic), a potent inhibitor for PTEN, or by intra-cerebroventricular infusion of PTEN siRNA. bpv (pic) significantly decreased infarct volume and reduced the number of TUNEL-positive cells. We further demonstrated that although bpv (pic) did not affect brain injury-induced mTOR protein expression, bpv (pic) prevented decrease in phosphorylation of mTOR, and the subsequent decrease in S6. Similarly, down-regulation of PTEN expression also reduced the number of TUNEL-positive cells, and increased phospho-mTOR. These data suggest that PTEN deletion prevents neuronal cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by increasing the injury-induced mTOR phosphorylation.

Pre-ischemic treadmill training induces tolerance to brain ischemia: involvement of glutamate and ERK1/2.

Physical exercise has been shown to be beneficial in stroke patients and animal stroke models. However, the exact mechanisms underlying this effect are not yet very clear. The present study investigated whether pre-ischemic treadmill training could induce brain ischemic tolerance (BIT) by inhibiting the excessive glutamate release and event-related kinase 1/2 (ERK1/2) activation observed in rats exposed to middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were divided into three groups (n = 12/group): sham surgery without prior exercise, MCAO without prior exercise and MCAO following three weeks of exercise. Pre-MCAO exercise significantly reduced brain infarct size (103.1 +/- 6.7 mm3) relative to MCAO without prior exercise (175.9 +/- 13.5 mm3). Similarly, pre-MCAO exercise significantly reduced neurological defects (1.83 +/- 0.75) relative to MCAO without exercise (3.00 +/- 0.63). As expected, MCAO increased levels of phospho-ERK1/2 (69 +/- 5%) relative to sham surgery (40 +/- 5%), and phospho-ERK1/2 levels were normalized in rats exposed to pre-ischemic treadmill training (52 +/- 6%) relative to MCAO without exercise (69% +/- 5%). Parallel effects were observed on striatal glutamate overflow. This study suggests that pre-ischemic treadmill training might induce neuroprotection by inhibiting the phospho-ERK1/2 over-activation and reducing excessive glutamate release.

NADPH oxidase Nox1 contributes to ischemic injury in experimental stroke in mice.

Reactive oxygen species (ROS) are mediators of brain injury in ischemia/reperfusion. An involvement of the NADPH oxidase Nox2 has been demonstrated. In contrast, only little is known about the contribution of the Nox1 homologue in this context. Thus, we studied the role of Nox1 in early cerebral reperfusion injury in the middle cerebral artery filament occlusion model using Nox1 knockout mice. Genetic deletion of a functional Nox1 lead to a 55% attenuation in lesion size at 24h after induction of 1h ischemia (p<0.05). This result was paralleled by a significant improvement of neurological outcome, preservation of blood-brain barrier integrity and reduced cerebral edema in Nox1(y/)(-) compared to WT mice. Interestingly, no difference in infarct size between WT and Nox1(y/)(-) was observed with an occlusion time of 2h and longer. Apoptosis rate as measured by TUNEL staining was similar between the groups. Moreover, infusion of the antioxidant TEMPOL as well as of the unspecific NO-synthase inhibitor l-NAME elicited similar changes with respect to ischemic tissue damage between WT and Nox1-deficient mice. In conclusion, Nox1 is involved in the pathophysiology of cerebral ischemia. Our data however indicate that ROS-mediated direct cellular injury is unlikely to explain the protective effect achieved by genetic deletion of the enzyme.

The cyclooxygenase site, but not the peroxidase site of cyclooxygenase-2 is required for neurotoxicity in hypoxic and ischemic injury.

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.

Neuroprotection by baicalein in ischemic brain injury involves PTEN/AKT pathway.

Recently more evidences support baicalein (Bai) is neuroprotective in models of ischemic stroke. This study was conducted to determine the molecular mechanisms involved in this effect. Either permanent or transient (2 h) middle cerebral artery occlusion (MCAO) was induced in rats in this study. Permanent MCAO led to larger infarct volumes in contrast to transient MCAO. Only in transient MCAO, Bai administration significantly reduced infarct size. Baicalein also markedly reduced apoptosis in the penumbra of transient MCAO rats. Additionally, oxygen and glucose deprivation (OGD) was used to mimic ischemic insult in primary cultured cortical neurons. A rapid increase in the intracellular reactive oxygen species level and nitrotyrosine formation induced by OGD was counteracted by Bai, which is parallel with attenuated cell injury. The reduction of phosphorylation Akt and glycogen synthase kinase-3beta (GSK3beta) induced by OGD was restored by Bai, which was associated with preserved levels of phosphorylation of PTEN, the phophatase that negatively regulates Akt. As a consequence, Bcl-2/Bcl-xL-associated death protein phosphorylation was increased and the protein level of Bcl-2 in motochondria was maintained, which subsequently antagonize cytochrome c released in cytosol. LY294002 blocked the increase in phospho-AKT evoked by Bai and abolished the associated protective effect. Together, these findings provide evidence that Bai protects neurons against ischemia injury and this neuroprotective effect involves PI3K/Akt and PTEN pathway.

Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, alpha(IIb)beta(3) integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate-binding protein or immunoreceptor tyrosine-based activation motif-coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1(-/-) mice displayed impaired alpha(IIb)beta(3) integrin activation in response to major agonists and defective glycoprotein Ib-dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

Serine racemase deletion protects against cerebral ischemia and excitotoxicity.

D-Serine, formed from L-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for D-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.

Identification and preliminary characterization of ubiquitin C terminal hydrolase 1 (UCHL1) as a biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage.

By using two different approaches, ubiquitin C-terminal hydrolase 1 (UCHL1) was identified as a potential cerebrospinal fluid (CSF) biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage (ASAH) and presumably other CNS damage and disease states. Appropriate antibodies and a sensitive ELISA were generated, and the release of UCHL1 into CSF was compared with that of pNF-H and S100beta in a cohort of 30 ASAH patients. Both UCHL1 and pNF-H showed persistent release into CSF in almost all patients in the second week postaneurysmal rupture (AR), and S100beta levels rapidly declined to baseline levels in 23 of 30 patients. Seven of thirty patients showed persistently elevated S100beta levels over the first 5 days post-AR and also had relatively higher levels of pNF-H and UCHL1 higher compared with the rest. These patients proved to have very poor outcomes, with 6 of 7 expiring. Patients who did reduce S100beta levels tended to have a better outcome if pNF-H and UCHL1 levels were also lower, and elevated UCHL1 levels in the second week post-AR were particularly predictive of poor outcome. Acute coordinated releases of large amounts of UCHL1, pNF-H, and S100beta in 16 of 30 patients were observed, suggesting sudden loss of brain tissues associated with secondary events. We conclude that measurement of the CSF levels of these proteins reveals details of ASAH progression and recovery and predicts patient outcome.