JM-20 Treatment After MCAO Reduced Astrocyte Reactivity and Neuronal Death on Peri-infarct Regions of the Rat Brain.

Stroke is frequently associated with severe neurological decline and mortality, and its incidence is expected to increase due to aging population. The only available pharmacological treatment for cerebral ischemia is thrombolysis, with narrow therapeutic windows. Efforts aimed to identify new therapeutics are crucial. In this study, we look into plausible molecular and cellular targets for JM-20, a new hybrid molecule, against ischemic stroke in vivo. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) following 23 h of reperfusion. Animals treated with 8 mg/kg JM-20 (p.o., 1 h after reperfusion) showed minimal neurological impairment and lower GABA and IL-1ß levels in CSF when compared to damaged rats that received vehicle. Immunocontent of pro-survival, phosphorylated Akt protein decreased in the cortex after 24 h as result of the ischemic insult, accompanied by decreased number of NeuN+ cells in the peri-infarct cortex, cornu ammonis 1 (CA1) and dentate gyrus (DG) areas. Widespread reactive astrogliosis in both cortex and hippocampus (CA1, CA3, and DG areas) was observed 24 h post-ischemia. JM-20 prevented the activated Akt reduction, neuronal death, and astrocytes reactivity throughout the brain. Overall, the results reinforce the pharmacological potential of JM-20 as neuroprotective agent and provide important evidences about its molecular and cellular targets in this model of cerebral ischemia.

Cytokines do play a role in pathogenesis of tuberculous meningitis: A prospective study from a tertiary care center in India.

BACKGROUND: Though animal studies have suggested a role for proinflammatory cytokines in pathogenesis their exact role in pathogenesis of human meningeal tuberculosis continues to be controversial with different studies yielding contradictory results. AIM AND OBJECTIVES: To study the levels of proinflammatory cytokines in serum and cerebrospinal fluid (CSF) of patients with tubercular meningitis (TBM) and to determine whether these correlate with disease severity. PATIENTS AND METHODS: Present study included 146 patients with TBM (90- Definite TBM; 56- Probable TBM), diagnosed according to criteria laid by Ahuja et al. which were modified to include CSF nucleic acid based tests. Serum (n=146) and CSF (n=140) levels of various proinflammatory cytokines (IL-1ß, IL-2, IL-6, TNF-α and IFNγ) were compared between TBM patients and healthy volunteers (n=99). These levels were correlated with various clinical, radiological and CSF parameters of TBM patients. RESULTS: Proinflammatory cytokines include cytokines which promote systemic inflammation. In current study, the serum and CSF levels of various cytokines (IL-2, IL-4, IL-6, IL-1ß, IFN-γ and TNF-α) were significantly elevated in TBM patients compared to controls. A significant correlation was found between a) Higher stage of TBM and various cytokines (except for serum IL-6 and CSF IFN-γ); b) High CSF TNF-α, IL-4 and IL-1ß with severity of hydrocephalus; c) High CSF IL1ß and IFN-γ with presence of exudates on MRI; d) Serum and CSF levels of all cytokines with poor outcome as determined by death or as defined by S and E ADL (Schwab and England activities of daily living) score or by GOS (Glasgow outcome scale) (except for interferon gamma); and e) Serum and CSF IL-4 and IL1ß with presence of infarcts on MRI brain. CONCLUSION: Proinflammatory cytokines play an important role in the pathogenesis of TBM and contribute significantly towards severity of disease.

Possible role of transforming growth factor ß in tuberculous meningitis.

BACKGROUND: Transforming growth factor ß (TGF-ß) is an anti-inflammatory cytokine and its role in hydrocephalus and stoke has been suggested. Tuberculous meningitis (TBM) is associated with exudates, stroke, hydrocephalus and tuberculoma, but the role of TGF-ß has not been evaluated in relation to these changes. AIM: To evaluate the cerebrospinal fluid (CSF) TGF-ß level in the patients with TBM, and correlate these with clinical findings, MRI changes, paradoxical response and outcome at 6months. METHODS: TBM patients diagnosed on the basis of clinical, CSF and MRI criteria were prospectively included. The clinical details including duration of illness, seizures, focal motor deficit, Glasgow Coma Scale (GCS) score and stage of TBM were noted. Presence of exudate, hydrocephalus, tuberculoma and infarction in MRI was also noted. MRI was repeated at 3months and presence of paradoxical response was noted. Cerebrospinal fluid TGF-ß was measured using ELISA on admission and repeated at 3months and these were compared with 20 controls. RESULTS: TGF-ß level was significantly higher in TBM compared to the controls (385.76±249.98Vs 177.85±29.03pg/ml, P<0.0001). TGF-ß correlated with motor deficit, infarction and tuberculoma on admission but did not correlate with CSF abnormalities, drug induced hepatitis, paradoxical response and outcome. TGF-ß level at 3months was significantly lower than the baseline but remained higher than the controls. CONCLUSION: CSF TGF-ß levels are elevated in TBM and correlate with infarction and tuberculoma.

CSF Volumetric Analysis for Quantification of Cerebral Edema After Hemispheric Infarction.

BACKGROUND: Malignant cerebral edema (CED) complicates at least 20 % of large hemispheric infarcts (LHI) and may result in neurological deterioration or death. Midline shift (MLS) is a standard but crude measure of edema severity. We propose that volumetric analysis of shifts in cerebrospinal fluid (CSF) over time provides a reliable means of quantifying the spectrum of edema severity after LHI. METHODS: We identified 38 patients from 2008 to 2014 with NIHSS ≥8, baseline CT <6 h after stroke onset, at least 1 follow-up (FU) CT, and no parenchymal hematoma. The volumes of CSF (sulci, ventricles, and cisterns) ipsilateral (IL) and contralateral (CL) to infarct on baseline and FU CTs were quantified by manually assisted outlining with MIPAV image analysis software, as was infarct volume and MLS on FU CTs. Percentage change in CSF volumes (∆CSF) from baseline to FU scans was correlated with MLS and compared in those with vs. without malignant edema (defined as hemicraniectomy, osmotic therapy, or death/neurological deterioration with MLS ≥5 mm). RESULTS: 11 of 38 subjects (29 %) developed malignant edema. Neither baseline NIHSS nor CSF volume differed between those with and without edema (median NIHSS 18 vs. 13, p = 0.12, CSF volume 102 vs. 124 ml, p = 0.16). Inter-rater reliability for CSF measurements was excellent (intraclass correlation coefficient 0.97). ∆CSF correlated strongly with MLS at peak edema (r = -0.75), even adjusting for infarct volume (p = 0.009). ∆CSF was also greater in those with malignant edema [-55 % (IQR -49 to -62) vs. -36 % (-27 to -45), p = 0.004]. ∆CSF was the greatest within IL sulci [-97 % (-86 to -99) vs. -71 % (-41 to -79), p = 0.002] but also significantly greater within CL sulci in those with malignant edema [-50 % (-29 to -65) vs. -25 % (0 to -31), p = 0.014]. More than half this CSF volume reduction occurred by the time of first FU CT around 24 h after stroke, while MLS rose later. CONCLUSIONS: Volumetric CSF analysis reliably quantifies CED and distinguishes those with malignant edema and MLS from those with a more benign course after LHI. ∆CSF may provide an earlier and more sensitive indicator of edema severity across a broader dynamic range than MLS.

Evidence that a panel of neurodegeneration biomarkers predicts vasospasm, infarction, and outcome in aneurysmal subarachnoid hemorrhage.

Biomarkers for neurodegeneration could be early prognostic measures of brain damage and dysfunction in aneurysmal subarachnoid hemorrhage (aSAH) with clinical and medical applications. Recently, we developed a new panel of neurodegeneration biomarkers, and report here on their relationships with pathophysiological complications and outcomes following severe aSAH. Fourteen patients provided serial cerebrospinal fluid samples for up to 10 days and were evaluated by ultrasonography, angiography, magnetic resonance imaging, and clinical examination. Functional outcomes were assessed at hospital discharge and 6-9 months thereafter. Eight biomarkers for acute brain damage were quantified: calpain-derived α-spectrin N- and C-terminal fragments (CCSntf and CCSctf), hypophosphorylated neurofilament H,14-3-3 ß and ζ, ubiquitin C-terminal hydrolase L1, neuron-specific enolase, and S100ß. All 8 biomarkers rose up to 100-fold in a subset of patients. Better than any single biomarker, a set of 6 correlated significantly with cerebral vasospasm, brain infarction, and poor outcome. Furthermore, CSF levels of 14-3-3ß, CCSntf, and NSE were early predictors of subsequent moderate-to-severe vasospasm. These data provide evidence that a panel of neurodegeneration biomarkers may predict lasting brain dysfunction and the pathophysiological processes that lead to it following aSAH. The panel may be valuable as surrogate endpoints for controlled clinical evaluation of treatment interventions and for guiding aSAH patient care.

Correlation of pre-morbid alcoholism and changes in the level of biogenic amine metabolites in cerebrospinal fluid of accute brain infarction patients.

BACKGROUND: The disorder of biogenic amine metabolism (serotonin - 5-HT and dopamine - DA) is expected in the brain (neuron) damage caused by acute ischemia. It is known that long-term abuse of ethyl-alcohol damages the quality of neurons diffusely in the brain. Cerebrospinal fluid (CSF) and its biochemical content, 5-HT and DA, are reliable indicators of the vitality of neurons. The main objective of this research was to demonstrate that the elevated content of metabolites 5-HT and DA in the CSF in patients with acute brain infarction, who were pre-morbid alcohol-dependent patients, is additionally emphasized by diffusive damage of neuron vitality caused by alcoholism. SUBJECTS AND METHODS: Study sample consists of two groups - 50 alcohol-dependent patients with acute brain infarction under the age of 65 (group A) and 50 patients with acute brain infarction who were not alcohol-dependent (group B). All subjects underwent the same procedure - CSF was taken during admission to the hospital and history was obtained through anamnesis, heteroanamnesis and clinical examinations. RESULTS: Metabolism of DA and metabolic turnover of DA (3, 4 dihydroxyphenylacetic acid + homovanilic acid; DOPAC + HVA) was elevated in the liquor of both patient groups. The statistically significant difference between the groups was found in metabolic turnover of 5-HT (p<0.05), and metabolic turnover of DA (p<0.001). CONCLUSIONS: The metabolic neuron disbalance, i.e. their pathophysiological-biochemical dysfunction as a result of acute brain infarction, is present in a higher degree in patients with pre-morbid long-term alcohol abuse.

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.

Blood-brain barrier permeability is increased in normal-appearing white matter in patients with lacunar stroke and leucoaraiosis.

BACKGROUND AND AIM: The pathogenesis of cerebral small-vessel disease (SVD) is incompletely understood. Endothelial dysfunction has been implicated and may result in increased blood-brain barrier (BBB) permeability with leakage of blood constituents into the vessel wall and white matter. We used contrast-enhanced MRI to determine whether there was any evidence for BBB permeability in the white matter of patients with SVD, and whether this was present not only in areas of leucoaraiosis (white-matter lesions) but also in normal-appearing white matter (NAWM). METHODS: Subjects underwent T1 volumetric MRI before and after bolus injection of contrast. Scanning was continued for 30 min postinjection to determine the contrast-enhancement time course. The mean signal intensity change was plotted against time to calculate the area under the curve values, a parameter related to BBB permeability. Automated brain segmentation and regions of interest analysis were performed to determine 'permeability' in different brain compartments. RESULTS: Compared with controls (n=15), the SVD patient group (n=24) had signal changes consistent with increased BBB permeability in NAWM (p=0.033). Multivariate regression analyses identified leucoaraiosis grade as an independent predictor of these permeability related signal changes in NAWM after adjustment for age, gender, weight, brain volume, area under the curve in the internal carotid arteries and cardiovascular risk factors. CONCLUSION: This study provides evidence for increased BBB permeability in SVD, and this is particularly seen in SVD with leucoaraiosis. Its presence in NAWM would be consistent with it playing a causal role in disease pathophysiology.

Remarkable increase in 3-nitrotyrosine in the cerebrospinal fluid in patients with lacunar stroke.

The goal of our study was whether free radicals contribute to the pathogenesis of the lacunar stroke to investigate the day after hospitalization, the concentrations of 3-nitrotyrosine and tyrosine in the cerebrospinal fluid (CSF) from living patients. The subjects included 20 living patients with lacunar stroke and 20 controls. The NIH stroke scale score was used to assess the severity of the stroke, including that the patients were mild cases. There was no expansion of the infarct lesion in the brain, as assessed by CT on the day following admission. The concentration of 3-nitrotyrosine was significantly higher in patients with lacunar stroke. In contrast, the concentration of tyrosine did not differ between the two groups. Furthermore, the 3-nitrotyrosine/tyrosine ratio was significantly higher in patients with lacunar stroke than in controls. Our results show that free radicals are produced in the CSF of lacunar stroke patients and that nitration of neuronal proteines is enhanced under this condition. These obsetvations suggest that lacunar stroke patients should be treated with edaravon, which is a free radical scavenger usually prescribed for cases of major strokes, as it will likely improve the prognosis of these patients.

Association of white matter lesions and lacunar infarcts with executive functioning: the SMART-MR study.

The authors investigated the association of white matter lesions and lacunar infarcts with cognitive performance and whether brain atrophy mediates these associations. Within the Second Manifestations of Arterial Disease-Magnetic Resonance study (2001-2005, the Netherlands), cross-sectional analyses of 522 patients were performed (mean age, 57 years (standard deviation, 10); 76% male). Brain segmentation was used to quantify volumes of brain tissue, cerebrospinal fluid, and white matter lesions. Infarcts were rated visually. Brain volume, ventricular volume, and gray matter volume were divided by intracranial volume to obtain indicators of brain atrophy. Neuropsychological tests assessing executive functioning and memory were performed, and scores were transformed into z scores. The authors used linear regression analyses, adjusted for age, sex, education, intelligence, and vascular risk factors, to investigate the association of white matter lesions and number of lacunar infarcts with cognitive performance. A 1-standard-deviation higher volume of white matter lesions (beta = -0.12, 95% confidence interval: -0.20, -0.04) and the presence of >or=2 lacunar infarcts (beta = -0.48, 95% confidence interval: -0.87, -0.09) were associated with worse executive functioning. These associations remained after adjusting for brain atrophy. Both were not associated with worse memory. Results suggest that subcortical ischemic vascular lesions are associated with decreased executive functioning, but not with memory functioning, independent of brain atrophy.

Interleukin-1beta is increased in the cerebrospinal fluid of patients with small infarcts.

BACKGROUND AND PURPOSE: Interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are involved in inflammatory responses during large vessel occlusion in animal models. The aim of this study was to investigate the intrathecal levels of cytokines in patients with acute small infarcts. METHODS: Forty patients with acute minor stroke and 32 non-stroke patients (including 29 age- and gender-matched subjects) who received operations with spinal anesthesia were studied prospectively and underwent measurements of cerebrospinal fluid (CSF) IL-1beta and IL-6 levels. RESULTS: After an age- and gender-matched analysis of 58 patients (29 pairs), the mean intrathecal levels of IL-1beta were 0.80 pg/ml in patients with small infarcts and 0.59 pg/ml in non-stroke patients (P < 0.0001). In addition, the mean CSF levels of IL-6 were 21.54 pg/ml and 7.52 pg/ml in the stroke and control groups, respectively (P = 0.38). These results were consistent with the data without matching. The CSF levels of IL-1beta in the 40 stroke patients were significantly higher than in the 32 non-stroke controls (P < 0.0001). CONCLUSIONS: The proinflammatory cytokine IL-1beta, but not IL-6, remained elevated in the CSF of patients in the acute stage of small infarcts.

Endothelin-1 in plasma, cisternal CSF and microdialysate following aneurysmal SAH.

OBJECTIVE: Endothelin-1 (ET-1) is postulated to play an important role in the development of cerebral vasospasm (CVS) following SAH. This study was conducted to investigate the time course of ET-release in three different sources: CSF, plasma and microdialysate. METHODS: In a prospective study ET-1-concentrations were measured in plasma, cisternal CSF and microdialysate in 20 patients with aneurysmal SAH for at least 8 days after hemorrhage. RESULTS: ET-1 concentration in microdialysate was almost four times higher compared to CSF and plasma. (p<0.001) Only in CSF ET-1-release showed a significant increase over time with highest values on day 5 post ictus (p = 0.03). This was parallel to the increase of transcranial Doppler velocities. ET-1 in plasma and microdialysate did not change over time. CONCLUSION: ET-1 may have a different biological function in different biological tissues. Only ET-1 in CSF seemed to be associated with CVS.

The increase of neuron-specific enolase in cerebrospinal fluid and plasma as a marker of neuronal damage in patients with acute brain infarction.

Our goal was to determine the neuron-specific enolase (NSE) concentration in cerebrospinal fluid (CSF) and plasma in patients with the acute brain infarction (BI) and analyze the correlation between the measured NSE concentration and infarct volume and the degree of neurological and functional deficit. The study included 55 patients aged 56-68 with BI in the acute phase. The control group consisted of 16 patients subjected to diagnostic radiculography. The results showed a significant increase of NSE concentration within the first seven days in patients compared to the controls (2.838 +/- 0.504 ng/ml CSF and 4.479 +/- 0.893 ng/ml plasma). A significant correlation was found between NSE concentration and infarction volume and the degree of neurological and functional deficit both in the CSF (r = 0.828, r = 0.735, r = 0.796; p < 0.001) and in plasma (r = 0.810, r = 0.681, r = 0.783; p < 0.001). The results suggest that an early determination of this marker in CSF and plasma in patients with BI could be a valuable diagnostic factor.

Tau protein in the cerebrospinal fluid is a marker of brain injury after aortic surgery.

BACKGROUND: Tau is a protein localized primarily in neurons, especially in the axonal compartment. Cerebrospinal fluid tau levels are elevated in acute stroke and head traumas. The purpose of this study is to elucidate the alterations of cerebrospinal fluid tau levels in patients with or without neurologic complication after aortic surgery. METHODS: Twenty-eight patients undergoing descending thoracic (n = 8) or thoracoabdominal (n = 20) aortic surgery were enrolled. Cerebrospinal fluid tau levels were measured before operation and at seven time points up to the 72nd postoperative hour, and were compared with cerebrospinal fluid S100B levels. RESULTS: Two patients developed brain infarction, including the one with paraplegia. In these patients, 20-fold to 100-fold tau elevation was observed, but S100B elevation was less evident in the patient without paraplegia. Three other patients developed spinal cord injury. Additional three patients suffered from temporary neurologic dysfunction of the brain. Tau levels in the latter three patients showed tenfold elevation and were higher than those in the three patients with spinal cord injury or those in the patients without neurologic complication up to 24 postoperative hours. The S100B levels were also higher in the three patients with temporary neurologic dysfunction of the brain than in the patients without neurologic complication at the conclusion of surgery. From 6 to 24 postoperative hours, they were higher in the three patients with spinal cord injury than in the patients without neurologic complication. CONCLUSIONS: These preliminary results suggest that cerebrospinal fluid tau levels reflect brain injury. Because tau levels may separate the patients with temporary neurologic dysfunction, they may serve as a useful marker of brain injury.

Neuroprotective effects of aspirin in patients with acute cerebral infarction.

Aspirin may reduce ischemic brain injury. The aim of this study was to explore the effect of aspirin on glutamate release after acute stroke. We studied 238 patients with a first episode of hemispheric ischemic stroke of less than 24 h duration. Early neurological deterioration was diagnosed when the Canadian Stroke Scale dropped 1 or more points between admission and 48 h. Glutamate was determined on cerebrospinal fluid (CSF) samples obtained at admission. Sixty-three patients were undergoing treatment with 75-500 mg/day of aspirin at the time of stroke onset. CSF glutamate concentrations were higher in the group of patients not taking aspirin (8.9+/-5.2 vs. 4.9+/-3.1 microM/l, P< 0.0001). Aspirin treatment at stroke onset had a 97% risk reduction of early neurological deterioration, and this effect remained unchanged after a further adjustment for glutamate concentrations. These findings suggest that low doses of aspirin may be useful in the management of patients with cerebral ischemia, not only for its antithrombotic properties, but also by direct neuroprotective effects.