Identification of distinctive features in human intracranial tumors by label-free nonlinear multimodal microscopy.

Nonlinear multimodal microscopy offers a series of label-free techniques with potential for intraoperative identification of tumor borders in situ using novel endoscopic devices. Here, we combined coherent anti-Stokes Raman scattering, two-photon excited fluorescence (TPEF) and second harmonic generation imaging to analyze biopsies of different human brain tumors, with the aim to understand whether the morphological information carried by single field of view images, similar to what delivered by present endoscopic systems, is sufficient for tumor recognition. We imaged 40 human biopsies of high and low grade glioma, meningioma, as well as brain metastases of melanoma, breast, lung and renal carcinoma, in comparison with normal brain parenchyma. Furthermore, five biopsies of schwannoma were analyzed and compared with nonpathological nerve tissue. Besides the high cellularity, the typical features of tumor, which were identified and quantified, are intracellular and extracellular lipid droplets, aberrant vessels, extracellular matrix collagen and diffuse TPEF. Each tumor type displayed a particular morphochemistry characterized by specific patterns of the above-mentioned features. Nonlinear multimodal microscopy performed on fresh unprocessed biopsies confirmed that the technique has the ability to visualize tumor structures and discern normal from neoplastic tissue likewise in conditions close to in situ.

An integrated genomic analysis of anaplastic meningioma identifies prognostic molecular signatures.

Anaplastic meningioma is a rare and aggressive brain tumor characterised by intractable recurrences and dismal outcomes. Here, we present an integrated analysis of the whole genome, transcriptome and methylation profiles of primary and recurrent anaplastic meningioma. A key finding was the delineation of distinct molecular subgroups that were associated with diametrically opposed survival outcomes. Relative to lower grade meningiomas, anaplastic tumors harbored frequent driver mutations in SWI/SNF complex genes, which were confined to the poor prognosis subgroup. Aggressive disease was further characterised by transcriptional evidence of increased PRC2 activity, stemness and epithelial-to-mesenchymal transition. Our analyses discern biologically distinct variants of anaplastic meningioma with prognostic and therapeutic significance.

Label-free multiphoton microscopy reveals relevant tissue changes induced by alginate hydrogel implantation in rat spinal cord injury.

The development of therapies promoting recovery after spinal cord injury is a challenge. Alginate hydrogels offer the possibility to develop biocompatible implants with mechanical properties tailored to the nervous tissue, which could provide a permissive environment for tissue repair. Here, the effects of non-functionalized soft calcium alginate hydrogel were investigated in a rat model of thoracic spinal cord hemisection and compared to lesioned untreated controls. Open field locomotion tests were employed to evaluate functional recovery. Tissue analysis was performed with label-free multiphoton microscopy using a multimodal approach that combines coherent anti-Stokes Raman scattering to visualize axonal structures, two-photon fluorescence to visualize inflammation, second harmonic generation to visualize collagenous scarring. Treated animals recovered hindlimb function significantly better than controls. Multiphoton microscopy revealed that the implant influenced the injury-induced tissue response, leading to decreased inflammation, reduced scarring with different morphology and increased presence of axons. Demyelination of contralateral white matter near the lesion was prevented. Reduced chronic inflammation and increased amount of axons in the lesion correlated with improved hindlimb functions, being thus relevant for locomotion recovery. In conclusion, non-functionalized hydrogel improved functional outcome after spinal cord injury in rats. Furthermore, label-free multiphoton microscopy qualified as suitable technique for regeneration studies.

IDH1 mutation in human glioma induces chemical alterations that are amenable to optical Raman spectroscopy.

INTRODUCTION: Mutations in the isocytrate dehydrogenase 1 (IDH1) gene are early genetic events in glioma pathogenesis and cause profound metabolic changes. Because this genotype is found in virtually every tumor cell, therapies targeting mutant IDH1 protein are being developed. The intraoperative administration of those therapies would require fast technologies for the determination of IDH1 genotype. As of today, there is no such diagnostic test available. Recently, infrared spectroscopy was shown to bridge this gap. Here, we tested Raman spectroscopy for analysis of IDH1 genotype in glioma, which constitutes an alternative contact-free technique with the potential of being applicable in situ. METHODS: Human glioma samples (n = 36) were obtained during surgery and cryosections were prepared. IDH1 mutations were assessed using DNA sequencing and 100 Raman spectra were obtained for each sample. RESULTS: Analysis of Raman spectra revealed increased intensities in spectral bands related to DNA in IDH1 mutant glioma while bands assigned to molecular vibrations of lipids were significantly decreased. Moreover, intensities of Raman bands assigned to proteins differed in IDH1 mutant and IDH1 wild-type glioma, suggesting alterations in the protein profile. The selection of five bands (498, 826, 1003, 1174 and 1337 cm-1) allowed the classification of Raman spectra according to IDH1 genotype with a correct rate of 89%. CONCLUSION: Raman spectroscopy constitutes a simple, rapid and safe procedure for determination of the IDH1 mutation that shows great promise for clinically relevant in situ diagnostics.

Non-functionalized soft alginate hydrogel promotes locomotor recovery after spinal cord injury in a rat hemimyelonectomy model.

BACKGROUND: Spinal cord injury (SCI) and the consecutive devastating neurological sequelae have an enormous individual and economic impact. Implantation of functionalized hydrogels is a promising approach, because they can serve as a matrix for the regenerating tissue, carry and release bioactive molecules and various cell types. We already demonstrated that non-functionalized soft alginate hydrogel supported axonal outgrowth and protected neurons against oxidative stress in vitro. Here, we investigated the effects of such soft alginate hydrogels on locomotor recovery in small and large spinal cord lesions. METHOD: Hemimyelonectomy of 2 mm or 4 mm length was performed in rats and soft alginate hydrogel was implanted. Functional recovery of the hindlimbs was assessed in the open field [Batto Beattie Bresnahan (BBB) score] and using swimming test [Louisville Swim score (LSS)] for 140 days post injury (DPI). Reference histology was performed. RESULTS: Rats that received an alginate implant into 2 mm spinal cord lesions demonstrated significantly improved locomotor recovery compared to controls detectable already at 10 DPI. At 140 DPI, they reached higher LSS and BBB scores in swimming and open field tests, respectively. However, this beneficial effect of alginate was lacking in animals with larger (4 mm) lesions. Histological examination suggested that fibrous scarring in the spinal cord was reduced after alginate implantation in comparison to controls. CONCLUSIONS: Implantation of soft alginate hydrogel in small spinal cord lesions improved functional recovery. Possible underlying mechanisms include the mechanical stabilization of the wound, reduction of secondary damage and inhibition of fibrous scarring.

Assessing the efficacy of coherent anti-Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples.

Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two-photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5-aminolevulinic acid-induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition. Experimental glioblastoma expressing green fluorescent protein.

Gene-activated fat grafts for the repair of spinal cord injury: a pilot study.

BACKGROUND: Spinal cord injury (SCI) is a complex disease requiring a concerted multi-target approach. The most appropriate combination of therapeutic gene, cellular vehicle, and space filling scaffold still has to be determined. We present an approach that employs syngeneic adipose tissue serving as a three-dimensional biological implant, source of progenitor cells, and delivery system for therapeutic genes. In this pilot experiment, we evaluated the feasibility and short-term effects using gene-activated autologous fat grafts after SCI. METHODS: An experimental SCI model was established in syngeneic Fischer 344 rats by a T9-T10 hemimyelonectomy. Fat tissue was harvested from two donor rats. Animals were divided into four groups and treated with either (i) fat grafts activated by an adenoviral vector carrying the human NT-3 cDNA, (ii) or BDNF, (iii) or with untreated fat grafts or (iv) remained untreated. Animals were euthanized either 7 or 21 days after surgery, and spinal cord tissue was investigated by histological and immunohistochemical methods. RESULTS: NT-3 and BDNF were produced by gene-activated fat grafts for at least 21 days in vitro and in vivo. Fat tissue grafts remained stable at the site of implantation at 7 days and at 21 days. Neither BDNF-activated nor NT-3-activated fat graft had a detectable limiting effect on the neuronal degeneration. BDNF recruited microglia to perilesional site and attenuated their inflammatory response. CONCLUSIONS: Gene-activated syngeneic fat tissue serves as a three-dimensional biological material delivering therapeutic molecules to the site of SCI over an extended period of time. The BDNF-fat graft attenuated the inflammatory response. Whether these findings translate into functional recovery will require extended observation times.

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.

Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord.

Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

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 tumor's 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.

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.