Ortrud Uckermann

Sedative and anticonvulsant drugs suppress postnatal neurogenesis

Sedative and anticonvulsant drugs, which inhibit N‐methyl‐D‐aspartate receptor–mediated excitation or enhance GABA‐mediated action, may cause apoptotic neurodegeneration in the developing mammalian brain. Here we explored whether such agents influence early postnatal neurogenesis.

Expression of glutamate receptor subunits in human cancers

Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

Vibrational Spectroscopic Imaging and Multiphoton Microscopy of Spinal Cord Injury

Spinal cord injury triggers a series of complex biochemical alterations of nervous tissue. Up to now, such cellular events could not be studied without conventional tissue staining. The development of optical, label-free imaging techniques could provide powerful monitoring tools with the potential to be applied in vivo. In this work, we assess the ability of vibrational spectroscopy to generate contrast at molecular level between normal and altered regions in a rat model of spinal cord injury. Using tissue sections, we demonstrate that Fourier transform infrared (FT-IR) spectroscopy and spontaneous Raman spectroscopy are able to identify the lesion, the surrounding scar, and unharmed normal tissue, delivering insight into the biochemical events induced by the injury and allowing mapping of tissue degeneration. The FT-IR and Raman spectroscopic imaging provides the basis for fast multimodal nonlinear optical microscopy (coherent anti-Stokes Raman scattering, endogenous two-photon fluorescence, and second harmonic generation). The latter proves to be a fast tool for imaging of the lesion on unstained tissue samples, based on the alteration in lipid content, extracellular matrix composition, and microglia/macrophages distribution pattern. The results establish these technologies in the field of regeneration in central nervous system, with the long-term goal to extend them to intravital use, where fast and nonharmful imaging is required.

Label-Free Delineation of Brain Tumors by Coherent Anti-Stokes Raman Scattering Microscopy in an Orthotopic Mouse Model and Human Glioblastoma

Background Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors. Methods Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumors localization, cell proliferation and vascularization. Results The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma. Conclusions CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.

Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

Effects of tissue fixation on coherent anti-Stokes Raman scattering images of brain

Abstract. Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging multiphoton technique for the label-free histopathology of the central nervous system, by imaging the lipid content within the tissue. In order to apply the technique on standard histology sections, it is important to know the effects of tissue fixation on the CARS image. Here, we report the effects of two common fixation methods, namely with formalin and methanol–acetone, on mouse brain and human glioblastoma tissue. The variations induced by fixation on the CARS contrast and intensity were compared and interpreted using Raman microspectroscopy. The results show that, whenever unfixed cryosections cannot be used, fixation with formalin constitutes an alternative which does not deteriorate substantially the contrast generated by the different brain structures in the CARS image. Fixation with methanol–acetone strongly modifies the tissue lipid content and is therefore incompatible with the CARS imaging.

Infrared spectroscopic studies of cells and tissues: triple helix proteins as a potential biomarker for tumors.

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders.

Matrix metalloproteinase 9 regulates cell death following pilocarpine-induced seizures in the developing brain.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.

Label-free identification of the glioma stem-like cell fraction using Fourier-transform infrared spectroscopy

Abstract Purpose: Vibrational spectroscopy enables the label-free characterization of cells and tissue by probing the biochemical composition. Here, we evaluated these techniques to identify glioblastoma stem cells. Materials and methods: The biochemical fingerprints of glioblastoma cells were established in human cell lines with high and low content of CD133 (cluster of differentiation 133)-positive cells using attenuated total reflection Fourier-transform infrared (ATR FT-IR) on vital cells and FT-IR mapping, which delivers spatially resolved spectroscopic datasets. After data preprocessing, unsupervised cluster analysis was applied. CD133 was addressed with flow cytometry and immunohistochemistry and used as a stemness marker. Results: In all preparations, the algorithm was able to correctly classify the spectra, differentiating CD133-rich and -poor populations. The main spectral differences were found in the region of 1000 cm− 1 to 1150 cm− 1 that can be assigned to vibrations of chemical bonds of DNA, RNA, carbohydrates and phospholipids. Interestingly, this spectral region is a key feature to discern glioblastoma from normal brain parenchyma, as FT-IR spectroscopic mapping of experimental brain tumors demonstrated. Conclusions: We were able to show biochemical differences between glioblastoma cell populations with high and low content of cancer stem cells that are presumably related to changes in the RNA/DNA content.

Biochemical Monitoring of Spinal Cord Injury by FT-IR Spectroscopy—Effects of Therapeutic Alginate Implant in Rat Models

Spinal cord injury (SCI) induces complex biochemical changes, which result in inhibition of nervous tissue regeneration abilities. In this study, Fourier-transform infrared (FT-IR) spectroscopy was applied to assess the outcomes of implants made of a novel type of non-functionalized soft calcium alginate hydrogel in a rat model of spinal cord hemisection (n = 28). Using FT-IR spectroscopic imaging, we evaluated the stability of the implants and the effects on morphology and biochemistry of the injured tissue one and six months after injury. A semi-quantitative evaluation of the distribution of lipids and collagen showed that alginate significantly reduced injury-induced demyelination of the contralateral white matter and fibrotic scarring in the chronic state after SCI. The spectral information enabled to detect and localize the alginate hydrogel at the lesion site and proved its long-term persistence in vivo. These findings demonstrate a positive impact of alginate hydrogel on recovery after SCI and prove FT-IR spectroscopic imaging as alternative method to evaluate and optimize future SCI repair strategies.