Role of cathepsin K in the development of chronic subdural hematoma.

Despite extensive investigations, the process of development of chronic subdural hematoma (CSDH) is not known. The present study aims to investigate CSDH by measuring biomarkers in it, gas analysis, and immunohistochemical examination. A total of 42 patients with symptomatic CSDH who underwent burr-hole drainage were enrolled. Intraoperatively, hematoma fluid and peripheral venous blood (PVCSDH) were simultaneously collected. As controls, peripheral venous blood (PVControl) and intracranial cerebrospinal fluid (CSF) were collected from other subjects during other surgeries. CatK, lipocalin-type prostaglandin D synthase (PGDS), and cystatin C (CysC) present in these specimens were measured using enzyme-linked immunosorbent assay. Data obtained were statistically analyzed after age correction. In 15 patients, gas analysis was performed for CSDH and PVCSDH. Furthermore, immunohistochemical examination for the outer membrane was performed for four patients. CatK, PGDS, and CysC levels were markedly elevated in the CSF and CSDH. CatK levels in PVCSDH were significantly higher than in PVControl (P<0.0001). In contrast, CysC levels in PVCSDH were significantly lower than in PVControl (P=0.004). The gas analysis revealed that the internal environment of CSDH is characterized by marked hypoxia, hypoglycemia, and lactic acidosis. Furthermore, the outer membrane consistently showed a diffuse staining for CatK. Based on these, CatK was thought to play a role in the development of CSDH, with the levels in peripheral venous blood elevated in patients with CSDH.

Chronic Subdural Hematoma Preceded by High-Impact Trauma: Does the Intensity of Trauma Influence the Pathogenesis of Traumatic Chronic Subdural Hematoma?

AIM: The purpose of this study was to investigate whether the intensity of trauma influences the pathogenesis of traumatic chronic subdural hematoma (CSDH). MATERIAL AND METHODS: Thirty-one patients treated surgically for traumatic CSDH were divided into high-impact and lowimpact groups according to the intensity of trauma. They were respectively evaluated with respect to clinical and radiological findings at presentation, and the subdural concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and beta-trace protein (ΒTP) [a highly specific protein in the cerebrospinal fluid (CSF)] related to the pathogenesis of CSDH. If ΒTP (subdural fluid/serum) was > 2, an admixture of CSF to the subdural fluid was indicated. RESULTS: The ΒTP (subdural fluid/serum) was > 2 in all patients with a traumatic CSDH. The mean concentration of subdural ΒTP in the high-impact group was higher than in the low-impact group (6.1 mg/L versus 3.9 mg/L), and the difference was statistically significant (p=0.02). In addition, mean concentrations of IL-6, IL-8 and VEGF were higher in the high-impact group, as compared to the low-impact group, though the differences did not reach statistical significance. CONCLUSION: Trauma may be related to CSF leakage into the subdural space in CSDH, and the intensity of trauma may influence the amount of CSF leakage. Although there is no direct correlation between the amount of CSF leakage and other subdural molecules, the intensity of trauma may be associated with larger concentrations of molecules in traumatic CSDH.

Cerebrospinal fluid biomarkers for prognosis of long-term cognitive treatment outcomes in patients with idiopathic normal pressure hydrocephalus.

The prognosis of cognitive improvement after cerebrospinal fluid (CSF) shunting in idiopathic normal pressure hydrocephalus (iNPH) remains uncertain, with no reports on CSF biomarkers related to long-term cognitive prognosis. We performed a preliminary study of CSF biomarker protein levels for cognitive outcome prognostication of two-year outcomes after shunt treated iNPH in 36 patients (13 women) with a median age of 75years (IQR 69-78). CSF biomarkers included soluble amyloid precursor proteins (sAPP, sAPPα, sAPPß), amyloid ß (Aß)1-38, Aß1-42 and phosphorylated tau (p-tau), lipocalin-type prostaglandin D synthase (L-PGDS)/ß-trace, and cystatin C. The results clearly showed that p-tau levels (sensitivity of 71.4%, specificity of 77.8%, cut-off value of 22.0pg/mL), Aß1-38/Aß1-42 ratio (77.8%, 81%, 3.58), and the Aß1-42/p-tau ratio (76%, 72.7%, 14.6) in preoperative CSF have the potential to determine postoperative prognosis. Improved cognition may be associated with the improvement in CSF circulation after LPS, which likely induces cystatin C and L-PGDS and switches synthesis from Aß1-42 to Aß1-38.

Lipocalin-type prostaglandin D synthase scavenges biliverdin in the cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the second major protein in human cerebrospinal fluid (CSF) and belongs to the lipocalin superfamily composed of various secretory lipophilic ligand transporter proteins. However, the endogenous ligand of L-PGDS has not yet been elucidated. In this study, we purified L-PGDS from the CSF of aneurysmal subarachnoid hemorrhage (SAH) patients. Lipocalin-type PG D synthase showed absorbance spectra with major peaks at 280 and 392 nm and a minor peak at around 660 nm. The absorbance at 392 nm of L-PGDS increased from 1 to 9 days and almost disappeared at 2 months after SAH, whereas the L-PGDS activity decreased from 1 to 7 days and recovered to normal at 2 months after SAH. These results indicate that some chromophore had accumulated in the CSF after SAH and bound to L-PGDS, thus inactivating it. Matrix assisted laser desorption ionization time-of-flight mass spectrometry of L-PGDS after digestion of it with endoproteinase Lys-C revealed that L-PGDS had covalently bound biliverdin, a by-product of heme breakdown. These results suggest that L-PGDS acted as a scavenger of biliverdin, which is a molecule not found in normal CSF. This is the first report of identification of a pathophysiologically important endogenous ligand for this lipocalin superfamily protein in humans.

Lipocalin 2 in cerebrospinal fluid as a marker of acute bacterial meningitis.

BACKGROUND: Early differential diagnosis between acute bacterial and viral meningitis is problematic. We aimed to investigate whether the detection of lipocalin 2, a protein of the acute innate immunity response, may be used as a marker for acute bacterial meningitis. METHODS: Transgenic mice expressing the human transferrin were infected by intraperitoneal route and were imaged. Cerebrospinal fluid (CSF) was sampled up to 48hours post- infection to measure lipocalin 2. We also tested a collection of 90 and 44 human CSF with confirmed acute bacterial or acute viral meningitis respectively. RESULTS: Lipocalin 2 was detected after 5 h in CSF during experimental infection in mice. Lipocalin 2 levels were significantly higher (p < 0.0001) in patients with confirmed acute bacterial meningitis (mean 125 pg/mL, range 106-145 pg/mL) than in patients with acute viral meningitis (mean 2 pg/mL, range 0-6 pg/mL) with a sensitivity of 81%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 71% in diagnosing acute bacterial meningitis. CONCLUSIONS: Increased levels of lipocalin 2 in cerebrospinal fluid may discriminate between acute bacterial and viral meningitis in patients with clinical syndrome of meningitis.

The role of neutrophil gelatinase-associated lipocalin (NGAL) in cerebrospinal fluids for screening of acute bacterial meningitis.

BACKGROUND: Acute bacterial meningitis is a rare but extremely severe disease. The aim of this study was to investigate whether neutrophil gelatinase-associated lipocalin (NGAL) is present and measurable in cerebrospinal fluid (CSF) and if its assessment may be useful for identifying patients with bacterial meningitis. METHODS: Eligible specimens were all consecutive CSFs of patients with suspect acute bacterial meningitis that were referred from the Unit of Infectious Diseases for routine chemical and morphological analysis over a three months period. CSF measurements consisted in NGAL, glucose, and total protein concentrations, along with cell count and differential. RESULTS: Eighty eight CSFs were received throughout the study period, 58 (66%) with CSF findings compatible with bacterial meningitis. The values of white blood cells (WBC), polymorphonuclear (PMN) and mononuclear (MONO) leukocytes, red blood cells (RBC), total proteins, and NGAL were significantly increased in positive CSFs, whereas that of glucose did not significantly differ. A significant correlation was found between CSF concentration of NGAL and CSF values of PMN, WBC, RBC and total proteins, but not with that of glucose and MONO. The concentration of NGAL in CSF showed an area under the curve (AUC) of 0.94 for identifying positive CSFs, with specificity and sensitivity of 1.00 and 0.741 at a diagnostic threshold of 13 ng/mL. CONCLUSIONS: NGAL is present in CSF of patients with bacterial meningitis and its measurement may be helpful for identifying positive CSFs.

Lipocalin 2: novel component of proinflammatory signaling in Alzheimer's disease.

Alzheimer's disease (AD) is associated with an altered immune response, resulting in chronic increased inflammatory cytokine production with a prominent role of TNF-α. TNF-α signals are mediated by two receptors: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Signaling through TNFR2 is associated with neuroprotection, whereas signaling through TNFR1 is generally proinflammatory and proapoptotic. Here, we have identified a TNF-α-induced proinflammatory agent, lipocalin 2 (Lcn2) via gene array in murine primary cortical neurons. Further investigation showed that Lcn2 protein production and secretion were activated solely upon TNFR1 stimulation when primary murine neurons, astrocytes, and microglia were treated with TNFR1 and TNFR2 agonistic antibodies. Lcn2 was found to be significantly decreased in CSF of human patients with mild cognitive impairment and AD and increased in brain regions associated with AD pathology in human postmortem brain tissue. Mechanistic studies in cultures of primary cortical neurons showed that Lcn2 sensitizes nerve cells to ß-amyloid toxicity. Moreover, Lcn2 silences a TNFR2-mediated protective neuronal signaling cascade in neurons, pivotal for TNF-α-mediated neuroprotection. The present study introduces Lcn2 as a molecular actor in neuroinflammation in early clinical stages of AD.

Beta-trace protein in ascites and pleural effusions: limits of CSF leakage detection.

Rhino- and/or otoliquorrhea can be diagnosed by detecting beta-trace protein (ß-TP) in nasal or ear secretions, as ß-TP is found in high concentrations in cerebrospinal fluid (CSF) but not in serum. CSF fistulae following trauma or surgery can also occur at other anatomical sites, resulting in CSF leakage into the thoracic and abdominal cavities. By analogy, determination of ß-TP has also been used to diagnose CSF admixture in pleural effusions and ascites. However, no systematic study has yet evaluated the concentrations of ß-TP in such fluids in the absence of CSF. To determine the validity of ß-TP determination as a marker for the presence of CSF, we investigated ß-TP concentrations in pleural effusions and ascites without CSF admixture. Patients from whom samples of ascites or pleural effusion and a paired plasma sample were available were investigated. One hundred sixty-four patients were prospectively recruited. ß-TP concentrations were determined by nephelometry. Mass spectrometric proteome analysis confirmed the presence of ß-TP in the samples. Median ß-TP concentrations detected in ascites and pleural effusions (range, 0.014-26.5 mg/L, median 2.29 mg/L) exceeded the corresponding plasma concentrations 2.6-fold. According to cutoffs published to diagnose rhino- and otoliquorrhea, between 6.1% and 95.7% of the specimens would have been erroneously rated CSF-positive. Protein analysis confirmed the presence of ß-TP in pleural effusion and ascites. Ascites and pleural effusion contain high concentrations of ß-TP that exceed the levels in corresponding plasma. Therefore, ß-TP is not a specific marker for the presence of CSF in these fluids.

Proteomic profiling of cerebrospinal fluid identifies prostaglandin D2 synthase as a putative biomarker for pediatric medulloblastoma: A pediatric brain tumor consortium study.

The aims of this study were to demonstrate the feasibility of centrally collecting and processing high-quality cerebrospinal fluid (CSF) samples for proteomic studies within a multi-center consortium and to identify putative biomarkers for medulloblastoma in CSF. We used 2-DE to investigate the CSF proteome from 33 children with medulloblastoma and compared it against the CSF proteome from 25 age-matched controls. Protein spots were subsequently identified by a combination of in-gel tryptic digestion and MALDI-TOF TOF MS analysis. On average, 160 protein spots were detected by 2-DE and 76 protein spots corresponding to 25 unique proteins were identified using MALDI-TOF. Levels of prostaglandin D2 synthase (PGD2S) were found to be six-fold decreased in the tumor samples versus control samples (p<0.00001). These data were further validated using ELISA. Close examination of PGD2S spots revealed the presence of complex sialylated carbohydrates at residues Asn(78) and Asn(87) . Total PGD2S levels are reduced six-fold in the CSF of children with medulloblastoma most likely representing a host response to the presence of the tumor. In addition, our results demonstrate the feasibility of performing proteomic studies on CSF samples collected from patients at multiple institutions within the consortium setting.

Neurochemical measures co-vary with personality traits: forensic psychiatric findings replicated in a general population sample.

Neurobiological markers in cerebrospinal fluid (CSF) and in serum, previously found to co-vary with destructive personality traits in violent offenders, were explored in a general population sample of 21 patients undergoing knee surgery. Results on the Karolinska Scales of Personality (KSP) and the Temperament and Character Inventory (TCI) were compared with CSF/serum albumin ratios and serum concentrations of beta-trace protein (betaTP) (as markers for blood-brain barrier (BBB) permeability), to CSF/serum albumin ratios between the dopamine and serotonin metabolites homovanillic acid (HVA)/5-hydroxyindoleacetic acid (HIAA) and to CSF and serum ratios between activated thyroid hormone (T3) and its precursor T4. Serum betaTP concentrations correlated with CSF/serum albumin ratios (P=0.018), but not with preoperative serum creatinine concentrations. Serum betaTP correlated significantly with Monotony Avoidance and Impulsiveness; CSF HVA/5-HIAA ratios with Irritability and low Cooperativeness. The betaTP is a potential serum marker for the integrity of the BBB that does not necessitate lumbar puncture. Thyroid hormones did not correlate with personality traits. As reported in forensic psychiatric patients, aggressive, unempathic personality traits were thus associated with increased dopaminergic activity in relation to the serotonergic activity and impulsivity to increased BBB permeability also in a general population group.

L-PGDS (betatrace protein) inhibits astrocyte proliferation and mitochondrial ATP production in vitro.

L-PGDS is the most abundant protein present in the cerebrospinal fluid (CSF). Although CSF was believed to be homogenous in content, a previous study has showed that a marked concentration gradient of L-PGDS exists between the spinal CSF and the CSF in the subarachnoid space of patients with optic nerve disease (papilledema and normal-tension glaucoma). Astrocytes play a critical role in maintaining the integrity of axon function in the central nervous system and specifically in the optic nerve, and we therefore investigated the biochemical effects of L-PGDS on the proliferation of astrocytes and on the production of adenosine triphosphate (ATP) by astrocyte mitochondria. We found an inhibitory effect of L-PGDS on both proliferation of astrocytes and production of astrocyte ATP. The concentrations that inhibited astrocyte proliferation and ATP production were in the range measured in patients with idiopathic intracranial hypertension and in patients with normal-tension glaucoma. As the CSF is in contact with axons and mitochondria of the optic nerve (Bristow et al. Archives of Ophthalmology, 120, 791-796, 2002), we postulate that a change in the concentration of CSF protein such as L-PGDS could exercise a harmful effect on these structures.

Reduced levels of amyloid-beta-binding proteins in cerebrospinal fluid from Alzheimer's disease patients.

Amyloid-beta(Abeta) aggregation is a major hallmark of Alzheimer's disease (AD). Previous studies have suggested that only unbound Abeta can take part in the aggregation process. Therefore, endogenous Abeta-binding proteins may have an important role in preventing AD. Here, we analyzed cerebrospinal fluid (CSF) samples from 35 subjects with AD, 18 subjects with frontotemporal dementia (FTD) and 29 non-demented controls to test if reduced Abeta-binding capacity in CSF is a specific feature of AD. A panel of known Abeta-binding CSF proteins, including beta-trace/prostaglandin D2 synthase (beta-trace), transthyretin (TTR), cystatin C (CysC) and alpha(1)-antitrypsin (AAT), were quantified and related to diagnosis and CSF levels of Abeta(1-38), Abeta(1-40) and Abeta(1-42). AD patients displayed a mild reduction in the CSF levels of beta-trace (p=0.020), CysC (p=0.017), AAT (p=0.019) and TTR (p=0.012) compared with controls. While the reductions in AAT and TTR were AD-specific, the levels of beta-trace and CysC were also reduced in FTD. As expected, CSF Abeta(1-42) was reduced in AD compared with controls (p=0.00005) and with FTD patients (p=0.015). Positive correlations between Abeta(1-42) and beta-trace, CysC and TTR, respectively, were seen only in the AD group, suggesting that deficient Abeta-binding capacity in CSF may contribute to the amyloidogenic process in AD.

Urinary biomarkers predict brain tumor presence and response to therapy.

PURPOSE: A major difficulty in treating brain tumors is the lack of effective methods of identifying novel or recurrent disease. In this study, we have evaluated the efficacy of urinary matrix metalloproteinases (MMP) as diagnostic biomarkers for brain tumors. EXPERIMENTAL DESIGN: Urine, cerebrospinal fluid, and tissue specimens were collected from patients with brain tumors. Zymography, ELISA, and immunohistochemistry were used to characterize the presence of MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL), and vascular endothelial growth factor (VEGF). Results were compared between age- and sex-matched controls and subjected to univariate and multivariate statistical analyses. RESULTS: Evaluation of a specific panel of urinary biomarkers by ELISA showed significant elevations of MMP-2, MMP-9, MMP-9/NGAL, and VEGF (all P < 0.001) in samples from brain tumor patients compared with controls. Multiplexing MMP-2 and VEGF provided superior accuracy compared with any other combination or individual biomarker. Receiver-operating characteristics curves for MMP-2 and VEGF showed excellent discrimination. Immunohistochemistry identified these same proteins in the source tumor tissue. A subset of patients with longitudinal follow-up revealed subsequent clearing of biomarkers after tumor resection. CONCLUSION: We report, for the first time, the identification of a panel of urinary biomarkers that predicts the presence of brain tumors. These biomarkers correlate with presence of disease, decrease with treatment, and can be tracked from source tissue to urine. These data support the hypothesis that urinary MMPs and associated proteins are useful predictors of the presence of brain tumors and may provide a basis for a novel, noninvasive method to identify new brain tumors and monitor known tumors after treatment.

Cerebrospinal fluid leakage into the subdural space: possible influence on the pathogenesis and recurrence frequency of chronic subdural hematoma and subdural hygroma.

OBJECT: The purpose of this study was to clarify whether cerebrospinal fluid (CSF) leakage into the subdural space is involved in the genesis of chronic subdural hematoma (CSDH) and subdural hygroma (SH) and to clarify whether this leakage of CSF into the subdural space influences the postoperative recurrence rate of CSDH and SH. METHODS: In this prospective observational study, 75 cases involving patients treated surgically for CSDH (67 patients) or SH (8 patients) were evaluated with respect to clinical and radiological findings at presentation, the content of beta -trace protein (beta TP) in the subdural fluid (betaTPSF) and serum (betaTPSER), and the CSDH/SH recurrence rate. The betaTPSF was considered to indicate an admixture of CSF to the subdural fluid if betaTPSF/betaTPSER>2. RESULTS: The median beta TPSF level for the whole patient group was 4.29 mg/L (range 0.33-51 mg/L). Cerebrospinal fluid leakage, as indicated by betaTPSF/betaTPSER>2, was found to be present in 93% of the patients with CSDH and in 100% of the patients with SH (p=0.724). In patients who later had to undergo repeated surgery for recurrence of CSDH/SH, the betaTPSF concentrations (median 6.69 mg/L, range 0.59-51 mg/L) were significantly higher (p=0.04) than in patients not requiring reoperation (median 4.12 mg/L, range 0.33-26.8 mg/L). CONCLUSIONS: As indicated by the presence of betaTP in the subdural fluid, CSF leakage into the subdural space is present in the vast majority of patients with CSDH and SH. This leakage could be involved in the pathogenesis of CSDH and SH. Patients who experience recurrences of CSDH and SH have significantly higher concentrations of betaTPSF at initial presentation than patients not requiring reoperation for recurrence. These findings are presented in the literature for the first time and have to be confirmed and expanded upon by further studies.

CSF proteomic analysis reveals persistent iron deficiency-induced alterations in non-human primate infants.

Iron deficiency (ID) anemia during infancy results in long-term neurological consequences, yet the mediating mechanisms remain unclear. Infant monkeys often become naturally anemic during the first 6 months of life, presenting an opportunity to determine the effect of developmental iron deficiency. After weaning, animals were chosen randomly for supplementation with oral iron or, fed a standard commercial chow diet. The control group was never iron deficient. ID anemia was corrected by 12 months in both groups, as indicated by hematological parameters. CSF was collected for proteomic analysis at 12 months of age to assess the impact of developmental ID on the brain. The CSF proteome for both formerly iron deficient groups was similar and revealed 12 proteins with expression levels altered at least twofold. These proteins were identified by matrix assisted laser desorption ionization time-of-flight spectrometry and included prostaglandin D synthase, olfactory receptors and glial fibrillary acidic protein. Thus the proteomic analysis reveals a persistent effect of ID and provides insights into reports of disturbed sleep, hypomyelination and other behavioral alterations associated with ID. Furthermore, alterations in the CSF proteome despite normal hematologic parameters indicate that there is a hierarchical system that prioritizes repletion of red cell mass at the expense of the brain.