Eva-Maria Stoerr

Function of carbonic anhydrase IX in glioblastoma multiforme.

Carbonic anhydrase (CA) IX is over-expressed in glioblastoma; however, its functions in this context are unknown. Metabolically, glioblastomas are highly glycolytic, leading to a significant lactic acid load. Paradoxically, the intracellular pH is alkaline. We hypothesized that CAIX contributes to the extrusion of hydrogen ions into the extracellular space, thereby moderating intra- and extracellular pH and creating an environment conductive to enhanced invasion. We investigated the role of CAIX as a prognostic marker in patients with glioblastoma and its biological function in vitro. CAIX expression was analyzed in 59 patients with glioblastoma by immunohistochemistry. The expression levels were correlated to overall survival. In vitro, U251 and Ln 18 glioblastoma cells were incubated under hypoxia to induce CAIX expression, and RNA interference (RNAi) was used to examine the function of CAIX on cell attachment, invasion, intracellular energy transfer, and susceptibility to adjuvant treatment. High CAIX expression was identified as an independent factor for poor survival in patients with glioblastoma. In vitro, cell attachment and invasion were strongly reduced after knockdown of CAIX. Finally, the effects of radiation and chemotherapy were strongly augmented after CAIX interference and were accompanied by a higher rate of apoptotic cell death. CAIX is an independent prognostic factor for poor outcome in patients with glioblastoma. Cell attachment, invasion, and survival during adjuvant treatment are significantly influenced by high CAIX expression. These results indicate that inhibition of CAIX is a potential metabolic target for the treatment of patients with glioblastoma.

Neuropeptide Y - an early biomarker for cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

Abstract Objectives: In the human brain, the potent vasoconstrictive neuropeptide Y (NPY) is abundantly expressed. Neuropeptide Y, which is stored in perivascular nerve fibers of the cerebral arteries, regulates the cerebral vascular diameter as well as cerebral blood flow. However, the role of NPY in the pathogenesis of cerebral vasospasm (CV) related to subarachnoid hemorrhage (SAH) is unclear. We prospectively analyzed and compared the release of endogenous NPY in the cerebrospinal fluid (CSF) of 66 patients with SAH to NPY release in a control group. Additionally, we correlated the levels of NPY with CV and consecutive ischemic stroke. Methods: Sixty-six consecutive patients (40 women, 26 men; mean age 53·1 years) with aneurysmal SAH were included. In the SAH group, CSF was drawn daily from day 1 to day 10 after the onset of SAH. The CSF of 29 patients undergoing spinal anesthesia for orthopedic surgery served as control samples. The NPY levels were determined in duplicate CSF samples by means of a competitive enzyme immunoassay (EIA). The levels of NPY in CSF were correlated with the development of CV over the 10-day period after the onset of SAH and to the occurrence of consecutive ischemic stroke. To evaluate CSF NPY levels as a predictive biomarker for vasospasm, we calculated the sensitivity and specificity as well as the positive and negative predictive values. Results: The NPY levels were significantly higher in the SAH group than in the control group (p < 0·001). The treatment modality (clip versus coil) did not influence the level of NPY in CSF (p > 0·05). Patients with CV showed significantly higher NPY levels than patients without CV during the entire observation period. The NPY levels of the non-CV group dissipated over time, whereas the CV group showed continuously increasing values. The NPY levels from day 4 to 10 were significantly higher in patients with CV-related stroke than in non-stroke patients. Using 0·3 ng/ml as a cut-off value, NPY levels on day 3 predicted the occurrence of CV with a sensitivity and specificity of 82% and 72%, respectively. High NPY levels, starting on day 4, significantly correlated with poor Glasgow Outcome Score grading at the follow-up (p < 0·05). Discussion: Our data indicate that NPY is involved in the pathogenesis of SAH-related CV and ischemia. Neuropeptide Y represents an early and reliable biomarker for the prediction of CV and consecutive stroke due to aneurysmal SAH.

The possible role of neuropeptide Y after spontaneous subarachnoid hemorrhage.

ObjectiveNeuropeptide Y (NPY), a highly potent vasoconstrictive neuropeptide, is widely expressed in the human brain, regulating vessel diameter and cerebral blood flow. Earlier studies focusing on the possible role of NPY in the context of aneurismal subarachnoid hemorrhage (SAH) and vasospasm have produced conflicting results. However, despite extensive research efforts, the pathophysiological mechanisms underlying the SAH-related vasospasm and delayed cerebral ischemia (DCI) have not been clarified. We, therefore, attempted to investigate the role of NPY in SAH-induced vasospasm in a larger, well documented patient population utilizing modern analytical tools. We focused on the release of the potent vasoconstrictor NPY in cerebrospinal fluid (CSF) and blood, and its correlation to vasospasm and stroke in the early clinical stage.MethodsThirty-seven patients with SAH and a control group consisting of 29 patients were included. Eighteen patients developed stroke, 21 patients met the Doppler sonographical criteria for vasospasm. Twenty-nine patients had aneurysms of the anterior circulation and four patients of the posterior circulation. All patients had ventricular drainage inserted and an arterial catheter. Blood and CSF were drawn daily for NPY analysis during a 10-day interval.ResultsThe levels of NPY in CSF and plasma were significantly higher after SAH than in the control group (p = 0.001). The vasospasm group showed NPY levels in CSF which continuously ranged above the NPY levels of the non-vasospasm group (p = 0.001). Patients with stroke caused by vasospasm had significantly higher levels of NPY (p = 0.001).DiscussionNPY is released excessively into blood and CSF following SAH. Patients with cerebral infarction caused by vasospasm had significantly higher levels of NPY. Our results indicate a certain role for NPY in the pathophysiology of vasospasm due to SAH and justify further studies in this area of research.

Calcitonin-gene related peptide and cerebral vasospasm

The pathophysiology of arterial vasospasm following subarachnoid hemorrhage (SAH) is poorly understood and the contribution of endogenous neuropeptides has not been sufficiently elucidated. Recently, we detected an excessive release of vasoconstrictive neuropeptide Y (NPY) in SAH patients and identified a significant correlation of NPY cerebrospinal fluid (CSF) levels with vasospasm-related ischemia. Here, we present the results of an experimental study on the possible role of the potent endogenous vasodilator calcitonin-gene related peptide (CGRP) in the acute stage of SAH. Twelve consecutive patients with SAH were included. Seven patients had severe arterial vasospasm, confirmed by transcranial doppler-sonography (TCD). Prospectively, CSF was collected from day 1 to day 10 after onset of the SAH. The levels of CGRP were determined in a competitive enzyme immunoassay and were correlated with the clinical course and hemodynamic changes. A cohort of 29 patients without CNS disease served as a control. CGRP was significantly higher in SAH patients compared with the control group (p<0.05). From day 1 to day 4, the CGRP levels in patients without vasospasm were significantly higher than the levels of CGRP in patients with vasospasm (p<0.05). These patients did not develop cerebral ischemia. The significantly increased levels of the CGRP during the first days after onset of the SAH in the non-vasospasm group indicate a potential protective role of CGRP. CGRP may alleviate arterial vasoconstriction and thus protect the brain from vasospasm and subsequent ischemia.

Expression of carbonic anhydrase IX in craniopharyngiomas

OBJECT In craniopharyngiomas, cystic growth causes pressure on vital structures of the adjacent brain, leading to significant morbidity. However, the molecular pathogenesis of this cyst formation remains unknown. Carbonic anhydrase IX (CA IX) is a tumor-associated, hypoxia-inducible enzyme, which can cause fluid production and development of cysts. The authors investigated CA IX expression in craniopharyngiomas and its correlation with the extent of cyst formation. In addition, the major pathways of CA IX regulation, hypoxia and p53 mutation, were analyzed. METHODS Expression of CA IX was analyzed in 20 craniopharyngioma patients by means of in situ hybridization and immunohistochemistry. Preoperative imaging was used to quantify cyst volume. To analyze putative hypoxic induction of CA IX, immunohistochemical staining for HIF-1α and VEGF was performed. Since p53 negatively regulates CA IX expression, we also analyzed the tumors for p53 mutation by direct sequencing. RESULTS Significant CA IX was found in 85% of the 20 cases. The extent of CA IX expression was significantly correlated with cyst volume. HIF-1α expression was largely absent in all tissue samples, whereas moderate VEGF expression was present in a subset of cases but without correlation to cyst volume. No p53 mutation was found in any of the analyzed tumors. CONCLUSIONS Carbonic anhydrase IX, which is virtually absent in normal brain, is significantly upregulated in craniopharyngiomas and shows a significant association with cyst size. The mechanisms of regulation remain unknown, since neither hypoxia nor p53 appears to play a role. These results indicate that inhibition of CA IX may be a potential target for the adjuvant treatment in patients with cystic craniopharyngiomas.

Simultaneous imaging of cortical partial oxygen pressure and anatomic structures using a transparent optical sensor foil.

Background/Purpose Reliable information of cerebral oxygenation is—besides the monitoring of the intracranial pressure—of eminent interest when treating patients with brain injuries. In this study, we introduce a new, fast, and sensitive method capable of determining the cortical partial oxygen pressure on the surface of the cortex using a special sensor foil. Methods The introduced method exploits the O2-dependent phosphorescence of a thin sensor foil, which is excited by a short light-emitting diode flash. The optical signal is registered by a charge-coupled device camera and analyzed with PC-based software. The adequacy of this method was tested in 10 animals. The sensor device was placed directly over the cortex after craniotomy and removal of the dura. Arterial oxygen pressure was systematically varied by modifying the ventilation gas mixture. A total of 225 measurements were performed within 4 regions of interest. Results Obtained results were sufficient in each case. The pO2 over the cortex correlated well with arterial pO2. Measurements over arteries showed a correlation coefficient of 0.72 (P<0.001), over veins 0.58 (P<0.001), over cortical parenchyma 0.46 (P<0.001), and in a larger region of interest containing vessels and cortical tissue 0.59 (P<0.001). The frequency of the measurements was 7 Hz with a single measurement covering an area of 30×30 μm. Conclusions For the first time, nearly online pO2 maps of a brain cortex can be generated, allowing simultaneously also separate measurements over distinct anatomic structures yielding a good spatial resolution.

Visualisation of cortical pO(2) during an epidural mass lesion in rodents.

Monitoring p(bt)O(2) is a valuable supplemental -procedure for neurocritically ill patients. Here, we utilise an opto-chemical method for measuring cortical pO(2) during a reversibly introduced epidural mass lesion in a rat model. The sensor was placed in a cortical window of 17 ventilated Wistar rats. Inflating the balloon device over the contralateral hemisphere increased ICP. Physiological parameters and cortical pO(2) were recorded. The ICP increased from 6.2 ± 4.6 to 44.6 ± 12.6 mmHg (p < 0.001). Cortical pO(2) over arterioles changed from 28.9 ± 2.1 to 19.0 ± 2.1 mmHg (p < 0.001), over venules from 14.8 ± 1.2 to 9.9 ± 1.5 mmHg (p = 0.002) and over parenchyma from 4.1 ± 0.7 to 2.4 ± 0.8 mmHg respectively (p < 0.001), while basic physiological parameters remained constant (p > 0.05). During baseline, arterial pO(2) correlated significantly with cortex arteriole and venole pO(2), but not with cortex parenchyma pO(2). While ICP was raised, cortical pO(2) did not correlate with arterial pO(2). In this model, a moderate epidural mass lesion causes a significant decrease in cortical pO(2). Cortex parenchyma pO(2) appeared to be independent from arterial pO(2). The correlation of cortex vessel pO(2) with arterial pO(2) disappeared during the epidural mass lesion. These findings show the capability of the device to elucidate the behaviour of functionally different cortex areas at pathophysiological conditions.

A new semi-invasive method for two dimensional pO2 measurements of cortical structures

BACKGROUND Measuring brain oxygenation in patients with TBI or SAH is of major interest. We present a new semi-invasive method for two dimensional measurements of cortical pO2. METHODS For this feasibility study, a porphyrin containing sensor foil was placed directly on the cortex of intubated and variably ventilated Wistar rats. The sensor was excited with a light pulse and pictures of the foils pO2 dependant emissions were captured with a CCD camera. After online data processing, two-dimensional maps of cortex oxygenation were displayed and analyzed using ROIs (here: arteriole, vein, parenchyma) with a display rate of 7 Hz. The size of one single measurement pixel was 0.03 x 0.03 mm2. FINDINGS The mean pO2 over cortex arterioles was 20.3 +/- 0.69, over veins 17.1 +/- 0.5 and over parenchyma 9.1 +/- 0.6 (mmHg +/- SD). The arterial pO2 showed a good correlation to the pO2 in the ROIs (r = 0.46-0.72, p < 0.0001, n = 198). Comparing groups with different paO2 and paCO2 we found significantly different pO2 values in the ROIs of the cortex. CONCLUSIONS This prototype is capable of obtaining cortical pO2 maps with excellent temporal and spatial resolution and provides simultaneous imaging of the cortex structures.

Excessive release of endogenous neuropeptide Y into cerebrospinal fluid after treatment of spontaneous subarachnoid haemorrhage and its possible impact on self-reported neuropsychological performance – results of a prospective clinical pilot study on good-grade patients

ABSTRACT Objectives: Neuropsychological dysfunction after treatment of spontaneous subarachnoid haemorrhage (sSAH) is common but underreported. The vasoconstrictor neuropeptide Y (NPY) is excessively released after sSAH and in psychiatric disorders. We prospectively analysed the treatment-specific differences in the secretion of endogenous cerebrospinal fluid (CSF) NPY during the acute stage after sSAH and its impact on cognitive processing. Methods: A total of 26 consecutive patients (f:m = 13:8; mean age 50.6 years) with good-grade sSAH were enrolled (drop out n = 5): n = 9 underwent endovascular aneurysm occlusion, n = 6 microsurgery, and n = 6 patients with perimesencephalic SAH received standardized intensive medical care. Ventricular CSF was drawn daily from day 1–10. CSF NPY levels were determined with competitive enzyme immunoassay. All patients underwent neuropsychological self-report assessment [36-Item Short Form Health Survey (SF-36) and ICD-10-Symptom-Rating questionnaire (ISR)] after the onset of sSAH (day 11–35; t1) and at the 6-month follow-up (t2). Results: At t1, increased mean levels of NPY in CSF significantly correlated with impaired performance in most ISR scores (ISR total p = .018, depression p = .035, anxiety p = .008, nutrition disorder p = .047, supplementary items p = .038) and in several psychological SF-36 items (vitality p = .019, general mental health p = .001, mental component summary p = .025). Discussion: To the best of our knowledge, this study is the first to correlate the levels of endogenous NPY in supratentorial CSF with cognitive outcome in good-grade sSAH patients. Excessive NPY release into CSF may have a short-term influence on the pathogenesis of neuropsychological deficits. The impact of cerebrovascular manipulation on NPY release has to be further elucidated. Abbreviations: ANOVA: analysis of variance; aSAH: aneurysmal subarachnoid haemorrhage; AUC: area under the curve; CBF: cerebral blood flow; CSF: cerebrospinal fluid; CT (scan): computed tomography (scan); CV: cerebral vasospasm; DIND: delayed ischemic neurological deficit; DSA: digital subtraction angiography; EIA: enzyme immunoassay; EV: endovascular aneurysm occlusion; EVD: external ventricular drainage; FU: 6-month follow-up; GCS: Glasgow Coma Scale; Ghp: general health perceptions; GOS: Glasgow Outcome Scale; h: hour/s; HH: Hunt and Hess; ICU: intensive care unit; ISR: ICD-10-Symptom-Rating questionnaire; MCS: mental component summary; Mhi: general mental health; min: minute/s; min-max: minimum – maximum; ml: millilitre; mRS: modified Ranking Scale; MS: microsurgical clipping, microsurgical aneurysm occlusion; ng: nanograms; no. [n]: number; NPY: Neuropeptide Y; p: p value; Pain: bodily pain; PCS: physical component summary; Pfi: physical functioning; pSAH: perimesencephalic subarachnoid haemorrhage; PTSD: posttraumatic stress disorder; QoL: quality of life; Rawhtran: health transition item; Rolem: role limitations because of emotional problems; Rolph: role limitations due to physical health problems; SAH: subarachnoid haemorrhage; SD: standard deviation; SF-36: 36-Item Short Form Health Survey; Social: social functioning; sSAH: spontaneous subarachnoid haemorrhage; TCD: trans-cranial Doppler ultrasound; (test) t1: test in the sub-acute phase after the onset of bleeding (between day 11 and 35 after subarachnoid haemorrhage); (test) t2: test in the short-term (chronic phase) after treatment at 6-month follow-up; test t1 - t2: intergroup development from t1 to t2; Vital: vitality; vs: versus.