Rolf Kalff

Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis

Contemporary brain tumor research focuses on two challenges: First, tumor typing and grading by analyzing excised tissue is of utmost importance for choosing a therapy. Second, for prognostication the tumor has to be removed as completely as possible. Nowadays, histopathology of excised tissue using haematoxylin-eosine staining is the gold standard for the definitive diagnosis of surgical pathology specimens. However, it is neither applicable in vivo, nor does it allow for precise tumor typing in those cases when only nonrepresentative specimens are procured. Infrared and Raman spectroscopy allow for very precise cancer analysis due to their molecular specificity, while nonlinear microscopy is a suitable tool for rapid imaging of large tissue sections. Here, unstained samples from the brain of a domestic pig have been investigated by a multimodal nonlinear imaging approach combining coherent anti-Stokes Raman scattering, second harmonic generation, and two photon excited fluorescence microscopy. Furthermore, a brain tumor specimen was additionally analyzed by linear Raman and Fourier transform infrared imaging for a detailed assessment of the tissue types that is required for classification and to validate the multimodal imaging approach. Hence label-free vibrational microspectroscopic imaging is a promising tool for fast and precise in vivo diagnostics of brain tumors.

Expression of voltage-gated potassium channels Kv1.3 and Kv1.5 in human gliomas

K(+) channels play an important role in glial cell proliferation and are functionally expressed in glial tumors. Because voltage-gated K(+) channel (Kv) subtypes Kv1.3 and Kv1.5 have been shown to contribute to growth-related properties of normal glia rather specifically, we investigated different human glioma samples for the expression of these channel subtypes using reverse transcriptase-PCR. Kv1.5 expression correlated with glioma entities and malignancy grades, i.e. expression was high in astrocytomas, moderate in oligodendrogliomas, and low in glioblastomas. No such correlation was evident for Kv1.3 expression. This study shows a clear differential expression of Kv1.5 in gliomas according to subtype and malignancy grade. This result corresponds to previous data on the expression of voltage-gated sodium channels in gliomas, which likewise showed a low or absent expression of channel subtypes in high-grade tumors.

Outcome after decompressive craniectomy in patients with severe ischemic stroke

SummaryDecompressive craniectomy after space occupying infarction of the middle cerebral artery (MCA) tends to decrease mortality and increase functional outcome. The aim of this retrospective study was to evaluate mortality rates and functional outcome in our centre and to identify predictors of prognosis. The charts of 30 consecutive patients (6 women, 24 men, mean age 59.3 ± 11.0 years) who underwent craniectomy after space occupying MCA-infarction from 1996 to 2002 were analyzed. Functional outcome was assessed by semistructured telephone interview as Barthel-Index, modifed Rankin scale and extended Barthel-Index. Five patients (mean age 67.2 ± 6.1 years) died within 5.2 ± 2.4 days (range 2–8 days) after the first symptoms due to herniation. Nine patients (mean age 63.1 ± 7.1 years) died 141.0 ± 92.5 days (range 40–343) after stroke onset due to internal complications. 16 patients survived (mean surviving time 2.1 ± 1.5 years, mean age 54.1 ± 11.4 years). Mortality was related to age and the number of risk factors/comorbidity, and functional outcome was dependent on the number of risk factors/comorbidity. Our small observational, retrospective study suggests that hemicraniectomy in patients with space occupying MCA- infarction decreases mortality rate and increases functional outcome. Further randomized trials may prove useful to better define the indications, timing and prognosis for this procedure.

Expression of ether à go-go potassium channels in human gliomas

Ether à go-go (EAG) K(+) channels have been shown to be involved in tumor generation and malignant growth. Gliomas have not been investigated thus far. Using RT-PCR we investigated healthy human brain and human gliomas of different subtypes and malignancy grades for the expression of human EAG1 and eag-related gene (ERG) 1 channels. mRNA of both channels was detected in all tissues. Expression was strong in normal brain, moderate in high-grade and high in low-grade gliomas. Our findings suggest a differential expression of hEAG1 and hERG1 in gliomas depending on the malignancy grade and nature of the tumor cells. However, the hypothesis that EAG channels are related to the oncogenic process itself is only partly supported by this study.

Readmission to a surgical intensive care unit: incidence, outcome and risk factors

IntroductionWe investigated the incidence of, outcome from and possible risk factors for readmission to the surgical intensive care unit (ICU) at Friedrich Schiller University Hospital, Jena, Germany.MethodsWe conducted an analysis of prospectively collected data from all patients admitted to the postoperative ICU between September 2004 and July 2006.ResultsOf 3169 patients admitted to the ICU during the study period, 2852 were discharged to the hospital floor and these patients made up the study group (1828 male (64.1%), mean patient age 62 years). The readmission rate was 13.4% (n = 381): 314 (82.4%) were readmitted once, 39 (10.2%) were readmitted twice and 28 (7.3%) were readmitted more than twice. The first readmission to the ICU occurred within a median of seven days (range 5 to 14 days). Patients who were readmitted to the ICU had a higher simplified acute physiology II score (37 +/- 16 versus 33 +/- 16; p < 0.001) and sequential organ failure score (6 +/- 3 versus 5 +/- 3; p = 0.001) on initial admission to the ICU than those who were not readmitted. In-hospital mortality was significantly higher in patients readmitted to the ICU (17.1% versus 2.9%; p < 0.001) than in other patients. In a multivariate analysis, age (odds ratio (OR) = 1.13 per 10 years; 95% confidence interval (CI) = 1.03 to 1.24; p = 0.04), maximum sequential organ failure score (OR = 1.04 per point; 95% CI = 1.01 to 1.08; p = 0.04) and C-reactive protein levels on the day of discharge to the hospital floor (OR = 1.02; 95% CI = 1.01 to 1.04; p = 0.035) were independently associated with a higher risk of readmission to the ICU.ConclusionsIn this group of surgical ICU patients, readmission to the ICU was associated with a more than five-fold increase in hospital mortality. Older age, higher maximum sequential organ failure score and higher C-reactive protein levels on the day of discharge to the hospital floor were independently associated with a higher risk of readmission to the ICU.

Neuronavigation in surgery of intracranial and spinal tumors

Purpose: To demonstrate the new possibilities and advantages of neuronavigation in the surgery of intracranial and spinal tumors, based on patient populations treated in our hospital. Materials and methods: An infrared navigation system with integrated microscope guidance was used for frameless intracranial neuronavigation. The biopsies of intracranial tumors were carried out using a frame-based stereotactic technique. Intracranial navigation was, in part, combined with the use of an intraoperative CT scanner and a three-dimensional ultrasound system for data acquisition, correction of brain shifts, and intraoperative quality control. The navigation was also supported by presurgical brain mapping with magnetic source imaging. Navigation in spinal surgery was exclusively performed using an infrared navigation system in combination with an intraoperative CT scanner. Results: The stereotactic tumor biopsies (n=57) were carried out with an accuracy of 91.4% as compared with the histological diagnosis. The work flow of stereotactic procedures could be increased by using the intraoperative CT scanner. Fifty-seven patients with intracranial tumors were treated with the aid of neuronavigation between July 1997 and December 1999. These patients showed an improvement from 80% to 86% on the Karnofsky index 8 weeks postoperatively. The majority of intracranial cases were primary brain tumors (n=30) and metastases (n=13) in functionally important areas of the brain. In four patients, a significant brain shift was observed during neuronavigation, and could be corrected by an image update using either the intraoperative CT scanner (n=2) or the three-dimensional ultrasound system (n=2). The presurgical brain mapping with magnetoencephalography was shown to be reliable in the sensory cortex (n=25). Eleven patients with a thoracic or lumbar tumor were treated by open surgery or stabilization, using a combination of spinal neuronavigation and the intraoperative CT scanner. Two patients with spinal tumors underwent navigated biopsies. Neither of them showed a reduction in the clinical stage, but the Karnofsky index improved from 63% up to 72% 8 weeks postoperatively. Conclusion: Neuronavigation allows very precise intracranial and spinal surgery. The problem of brain shift during the navigation procedures has been solved by intraoperative image acquisition. The use of neuronavigation was shown to improve the postoperative quality of life of patients suffering from brain and spinal tumors.

Pyogenic infections of the central nervous system secondary to dental affections—a report of six cases

Pyogenic infections of the central nervous system of dental origin are quite uncommon in industrialized countries. We report six cases with intracerebral (n=4) and intraspinal (n=2) infections treated in our hospital. The microbial pathogen was successfully isolated in all patients. Fusobacterium nucleatum as well as Streptococcus species were found in three cases. Bacillus species were identified in two patients. Actinomyces was the etiologic agent in one case. All patients suffered from dental pathologies, so that after clinical and radiological exclusion of other sources an oral focus was presumed. Therapeutic management consisted of an operative procedure in order to obtain decompression, as well as evacuation of the pus on the one hand, followed by targeted antibiotics on the other. Clinical improvement was achieved in all patients, with one patient lost to follow-up. On magnetic resonance tomography, the inflammatory changes also disappeared in all cases. We recommend that oral infection with recurrent bacteraemia should always be considered in the pathogenesis of the so-called “cryptic” intracerebral and intraspinal infections.

Tumor margin identification and prediction of the primary tumor from brain metastases using FTIR imaging and support vector machines.

Infrared spectroscopy enables the identification of tissue types based on their inherent vibrational fingerprint without staining in a nondestructive way. Here, Fourier transform infrared microscopic images were collected from 22 brain metastasis tissue sections of bladder carcinoma, lung carcinoma, mamma carcinoma, colon carcinoma, prostate carcinoma and renal cell carcinoma. The scope of this study was to distinguish the infrared spectra of carcinoma from normal tissue and necrosis and to use the infrared spectra of carcinoma to determine the primary tumor of brain metastasis. Data processing follows procedures that have previously been developed for the analysis of Raman images of these samples and includes the unmixing algorithm N-FINDR, segmentation by k-means clustering, and classification by support vector machines (SVMs). Upon comparison with the subsequent hematoxylin and eosin stained tissue sections of training specimens, correct classification rates of the first level SVM were 98.8% for brain tissue, 98.4% for necrosis and 94.4% for carcinoma. The primary tumors were correctly predicted with an overall rate of 98.7% for FTIR images of the training dataset by a second level SVM. Finally, the two level discrimination models were applied to four independent specimens for validation. Although the classification rates are slightly reduced compared to the training specimens, the majority of the infrared spectra of the independent specimens were assigned to the correct primary tumor. The results demonstrate the capability of FTIR imaging to complement histopathological tools for brain tissue diagnosis.

Interpreting CARS images of tissue within the C–H-stretching region

Single band coherent anti-Stokes Raman scattering (CARS) microscopy is one of the fastest implementation of nonlinear vibrational imaging allowing for video-rate image acquisition of tissue. This is due to the large Raman signal in the C-H-stretching region. However, the chemical specificity of such images is conventionally assumed to be low. Nonetheless, CARS imaging within the C-H-stretching region enables detection of single cells and nuclei, which allows for histopathologic grading of tissue. Relevant information such as nucleus to cytoplasm ratio, cell density, nucleus size and shape is extracted from CARS images by innovative image processing procedures. In this contribution CARS image contrast within the C-H-stretching region is interpreted by direct comparison with Raman imaging and correlated to the tissue composition justifying the use of CARS imaging in this wavenumber region for biomedical applications.

Fatty acid synthase as a novel target for meningioma therapy

High levels of fatty acid synthase (FAS) expression have been reported in hormone receptor-positive tumors, including prostate, breast, and ovarian cancers, and its inhibition reduces tumor growth in vitro and in vivo. Similar to other hormone receptor-positive tumor types, meningiomas are progesterone receptor- and estrogen receptor-immunoreactive brain tumors. To define the role of FAS in human meningioma growth control, we first analyzed the FAS expression using a tissue microarray containing 38 meningiomas and showed increased FAS expression in 70% of atypical WHO grade II and anaplastic WHO grade III meningiomas compared with 10% of benign WHO grade I tumors. We next confirmed this finding by real-time PCR and Western blotting. Second, we demonstrated that treatment with the FAS inhibitor, cerulenin (Cer), significantly decreased meningioma cell survival in vitro. Third, we showed that Cer treatment reduced FAS expression by modulating Akt phosphorylation (activation). Fourth, we demonstrated that Cer treatment of mice bearing meningioma xenografts resulted in significantly reduced tumor volumes associated with increased meningioma cell death. Collectively, our data suggest that the increased FAS expression in human meningiomas represents a novel therapeutic target for the treatment of unresectable or malignant meningioma.