From Research to Diagnostic Application of Raman Spectroscopy in Neurosciences: Past and Perspectives

In recent years, Raman spectroscopy has been more and more frequently applied to address research questions in neuroscience. As a non-destructive technique based on inelastic scattering of photons, it can be used for a wide spectrum of applications including neurooncological tumor diagnostics or analysis of misfolded protein aggregates involved in neurodegenerative diseases. Progress in the technical development of this method allows for an increasingly detailed analysis of biological samples and may therefore open new fields of applications. The goal of our review is to provide an introduction into Raman scattering, its practical usage and also commonly associated pitfalls. Furthermore, intraoperative assessment of tumor recurrence using Raman based histology images as well as the search for non-invasive ways of diagnosis in neurodegenerative diseases are discussed. Some of the applications mentioned here may serve as a basis and possibly set the course for a future use of the technique in clinical practice. Covering a broad range of content, this overview can serve not only as a quick and accessible reference tool but also provide more in-depth information on a specific subtopic of interest

Differentiation of primary CNS lymphoma and glioblastoma using Raman spectroscopy and machine learning algorithms

Objective and Methods: Timely discrimination between primary CNS lymphoma (PCNSL) and glioblastoma is crucial for diagnostics and therapy, but most importantly also determines the intraoperative surgical course. Advanced radiological methods allow this to a certain extent but ultimately, biopsy is still necessary for final diagnosis. As an upcoming method that enables tissue analysis by tracking changes in the vibrational state of molecules via inelastic scattered photons, we used Raman Spectroscopy (RS) as a label free method to examine specimens of both tumor entities intraoperatively, as well as postoperatively in formalin fixed paraffin embedded (FFPE) samples. Results: We applied and compared statistical performance of linear and nonlinear machine learning algorithms (Logistic Regression, Random Forest and XGBoost), and found that Random Forest classification distinguished the two tumor entities with a balanced accuracy of 82,4% in intraoperative tissue condition and with 94% using measurements of distinct tumor areas on FFPE tissue. Taking a deeper insight into the spectral properties of the tumor entities, we describe different tumor-specific Raman shifts of interest for classification. Conclusions: Due to our findings, we propose RS as an additional tool for fast and non-destructive, perioperative tumor tissue discrimination, which may augment treatment options at an early stage. RS may further serve as a useful additional tool for neuropathological diagnostics with little requirements for tissue integrity

TGF-Beta Modulates the Integrity of the Blood Brain Barrier In Vitro, and Is Associated with Metabolic Alterations in Pericytes

The blood–brain barrier (BBB) is a selectively permeable boundary that separates the circulating blood from the extracellular fluid of the brain and is an essential component for brain homeostasis. In glioblastoma (GBM), the BBB of peritumoral vessels is often disrupted. Pericytes, being important to maintaining BBB integrity, can be functionally modified by GBM cells which induce proliferation and cell motility via the TGF-β-mediated induction of central epithelial to mesenchymal transition (EMT) factors. We demonstrate that pericytes strengthen the integrity of the BBB in primary endothelial cell/pericyte co-cultures as an in vitro BBB model, using TEER measurement of the barrier integrity. In contrast, this effect was abrogated by TGF-β or conditioned medium from TGF-β secreting GBM cells, leading to the disruption of a so far intact and tight BBB. TGF-β notably changed the metabolic behavior of pericytes, by shutting down the TCA cycle, driving energy generation from oxidative phosphorylation towards glycolysis, and by modulating pathways that are necessary for the biosynthesis of molecules used for proliferation and cell division. Combined metabolomic and transcriptomic analyses further underscored that the observed functional and metabolic changes of TGF-β-treated pericytes are closely connected with their role as important supporting cells during angiogenic processes

Neurodegeneration and neuroinflammation are linked, but independent of a-synuclein inclusions, in a seeding/spreading mouse model of Parkinson's disease

A key pathological process in Parkinson's disease (PD) is the transneuronal spreading of α-synuclein. Alpha-synuclein (α-syn) is a presynaptic protein that, in PD, forms pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of α-syn particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used a model of spreading of α-syn induced by striatal injection of α-syn preformed fibrils into the mouse striatum to address this question. We performed quantitative analysis for α-syn inclusions, neurodegeneration, and microgliosis in different brain regions, and generated gene expression profiles of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without α-syn inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. Using longitudinal gene expression profiling, we observed early gene expression changes, linked to neuroinflammation, that preceded neurodegeneration, indicating an active role of microglia in this process. Altered gene pathways overlapped with those typical of PD. Our observations indicate that α-syn inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that microglia, activated by diffusible, oligomeric α-syn, may play a key role in this process. Our findings uncover new features of α-syn induced pathologies, in particular microgliosis, and point to the necessity for a broader view of the process of α-syn spreading

Enhanced plasticity of mature granule cells reduces survival of newborn neurons in the adult mouse hippocampus

Dentate granule cells are born throughout life in the mammalian hippocampus. The integration of newborn neurons into the dentate circuit is activity-dependent and structural data characterizing synapse formation suggested that the survival of adult-born granule cells is regulated by competition for synaptic partners. Here we tested this hypothesis by using a mouse model with genetically enhanced plasticity of mature granule cells through temporally controlled expression of a nuclear inhibitor of protein phosphatase 1 (NIPP1*). Using thymidine analogues and retrovirus-mediated cell labeling, we show that synaptic integration and subsequent survival of newborn neurons is decreased in NIPP1*- expressing mice, suggesting that newborn neurons compete with pre-existing granule cells for stable integration. The data presented here provides experimental evidence for a long-standing hypothesis and suggest cellular competition as a key mechanism regulating the integration and survival of newborn granule cells in the adult mammalian hippocampus

Influence of low-intensity pulsed ultrasound on bone repair upon irradiation: a histomorphometric study in rabbits.

The present study evaluated the influence of LIPUS on regeneration processes of bone defects below the critical size in irradiated and non-irradiated rabbit tibia. The study was based on a total of six white New-Zealand adult female rabbits. Apart from surgery to create bone defects on all tibiae, the following four treatments were randomly added on: (1) C group: only the surgical procedure was applied with no additional treatment, serving as the control, (2) R group, the irradiated side received 15 Gy in single dose, (3) US group, treated with LIPUS, and (4) R+US group, irradiated with 15 Gy and treated with LIPUS (n=6 defects per group). The surgery control samples showed 83.10% ± 17.79% of bone repair after 9 weeks, while the irradiated bone had significantly (p 0.05) improve the response compared to the non-treated irradiated specimens. In the irradiated bones, ultrasound treatment produced only 3.89% less new bone compared to the untreated control group; this repair is insignificantly lower than the natural bone healing in the untreated control group. LIPUS treatment on non-irradiated bone, however, showed bone formations beyond the size defect (115.91% ± 33.69%), highly significantly different when compared to the control group or any irradiated group. It is noteworthy that the application of ultrasound to healthy bone produced highly significantly enhanced bone formations, with 36.70% more regenerated bone when comparing the same application on irradiated bone (79.21% ± 21.07%). LIPUS vibration stimuli may be considered as a promising complementary treatment approach in non-irradiated bone regeneration procedures to shorten the treatment and to enhance the bone healing. In irradiated bones, the effect of ultrasound application is less apparent and further studies are needed to refine the dynamics of the present results.Peer reviewe

Bioactive glass particles and deproteinized bovine bone mineral as scaffold in bone tissue regeneration:effects of Minocycline-HCL.

peer reviewedAim/Hypothesis: Bone tissue regeneration remains an important challenge in orthopaedic and maxillofacial surgery. The present study in rat was performed to determine if Minocycline could influence the behavior of Deproteinized Bovine Bone Mineral (DBBM) and bioactive glass particles when used as scaffold for Guided Bone Regeneration (GBR). Minocycline is a wide spectrum antibiotic with demonstrated effects on bone formation, bone resorption and connective tissue breakdown. We chose to investigate this molecule in relation with bioactive glasses and DBBM, both important classes of bone grafting materials. Material and methods: Two completely occlusive titanium bone augmentation caps were placed on each side of the sagittal cranial suture of the rat calvaria. One was filled with bioactive glass particles (BioGran-Biomet 3i, Plam Beach, FL, USA) and the second with DBBM particles (Bio-Oss®–Geistlich, Wolhusen, Switzerland). Bone filler particles were mixed with blood, collected from the animal’s tail, and a drop of balanced salt solution in order to acquire a moldable consistency of the particles and a perfectly adapted fill of the regeneration chambers. In Minocycline-Cl group (n=10) bone grafts were additionally placed into a minocycline solution at 0.1 mg/ml prior placement. The study protocol included animal accommodation with water ad libitum, regular rat pellet with artificial light with a night and day cycle. All surgeries were conducted under strict aseptic conditions. For each observation time (4, 8 and 16 weeks) two calvaria were embedded in methyl methacrylate resin and cut in thin serial sections (100-200 µm), grounded and polished to a thickness of 20-40 µm. A Giemsa (Gross & Strunz, 1977), Paragon and a combination of Stevenel’s blue with Van Gieson staining was used for light microscopy. The remaining calvaria were fixed, decalcified in an EDTA solution at 0.2 M (pH 7.4) for 30 days and dehydrated in ascending ethanol series for 96 h. The paraffin embedded blocks were sectioned (5 µm) and either hematoxylin eosin (H&E) or Toluidine blue stained for the histologic analysis. Results: This study has confirmed osteoconductive and osteoinductive response of BioGran. The addition of Minocycline has hardly influenced the result positively. In contact with blood, BioGran underwent a surface modification in form of a calcium phosphate precipitate, quite similar in composition and structure to hydroxyapatite. Cracks in the outer shell were to be recognized and the silica core tended to disappear leaving a calcium phosphate pouch for future bone growth. Likewise, first signs of osteoinduction in the form of ectopic mineralization foci could be observed after just 4 weeks at some distance from BioGran particles. These ossification islets were lined by a layer of osteoblasts for centrifugal expansion. The osteoconductivity and predictability of Bio-Oss®, are well documented. Some studies however found that Bio-Oss® inhibited new bone formation or interfered with new bone generation especially when used as an onlay graft under totally occlusive capsules in a GTR model. Our observations were similar: the major part of the biomaterial particles was embedded in connective tissue. Most of the new bone regenerated were lamellar bone formations in close contact and bonded to the DBBM particles. Islands of spontaneous ossification suggesting some osteoinductive activities could only be observed in Bio-Oss® sections treated with Minocycline. A possible correlation of these centres with the basal skull bone, outside of our observation field, cannot be excluded. Conclusions and clinical implications: The osteoconductive and osteoinductive properties of bioactive glass particles could be confirmed within the limitations of the present study. Minocycline-HCl may be considered as a promising complementary treatment approach and may add some osteoinductive properties to DBBM. Further investigations into the volume of the newly generated bone are needed to refine the present results

Minocycline hydrochloride as a potential adjuvant to improve osteoconductive and osteoinductive properties of bone substitutes in an extra-skeletal bone augmentation model: Preliminary observations in rats

Aim/Hypothesis: Bone tissue regeneration remains an important challenge in orthopedic and maxillofacial surgery. The present in vivo study was performed to determine if minocycline-hydrochloride (minocycline-HCl) could influence the behavior of Deproteinized Bovine Bone Mineral (DBBM) and bioactive glass particles when used as filler material for extra-skeletal bone generation in a Guided Bone Augmentation (GBA) model. Material and methods: An occlusive titanium cap was placed on each side of the sagittal cranial suture of the rat calvaria. One was filled with bioactive glass particles, the second with DBBM particles, both previously mixed with blood (control groups). In minocycline-HCl loaded groups (experimental groups), bone grafts were additionally placed into a minocycline solution. For each observation time (4, 8 and 16 weeks) samples were harvested and processed for histology. Half of the samples were embedded in methylmethacrylate for undecalcified histology whereas the other half was fixed, decalcified and embedded in paraffin for classical histologic analysis. Results: This study highlighted osteoconductive and osteoinductive responses associated to bioactive glass particles. However, the addition of minocycline-HCl had no measurable influence on the result. In the control groups, the major part of the space originally created by the caps was occupied by biomaterial particles surrounded by connective tissue. Only slight new bone formations could be seen in the bottom region close to the native skull bone. In sections of minocycline-HCl loaded DBBM, areas of spontaneous ossification could be observed both, after 8 weeks and 16 weeks. Conclusions and clinical implications: Our observations suggest osteoconductive and osteoinductive properties of bioactive glass particles within the limitations of the present study. Minocycline-HCl may be considered as a promising complementary treatment approach and may add some osteoinductive properties to DBBM. Further investigations as to the volume of newly generated bone are needed to refine the present results

Effects of Low Intensity Pulsed Ultrasound Stimulation on the Temporal Dynamics of Irradiated Bone Tissue Healing: A Histomorphometric Study in Rabbits.

peer reviewedThe present study evaluated the influence of Low-Intensity Pulsed Ultrasound (LIPUS) on the regeneration processes of non-critical-size bone defects in irradiated and non-irradiated rabbit tibias. Bone defects were surgically created on both tibiae of six rabbits. The control group had no additional treatment. In one intervention group, one tibia was irradiated with 15 Gy in a single dose. A second group was treated with LIPUS, and a third with a combination of both treatments. The control samples showed 83.10% ± 17.79% of bone repair after 9 weeks, while the irradiated bone had regenerated significantly less during the same period (66.42% ± 29.36%). The LIPUS treatment on irradiated bones performed a 79.21% ± 21.07% bone fill and could not significantly improve the response compared to the non-treated irradiated specimens. However, LIPUS treatment on non-irradiated bone showed bone formations beyond the size defect (115.91% ± 33.69%), which was a highly significant increase when compared to the control group or any irradiated group. The application of ultrasound to healthy bone produced highly significant and enhanced bone formations with 36.70% more regenerated bone when compared to the same application on irradiated bone. LIPUS vibration stimuli may be considered as a promising complementary treatment approach in non-irradiated bone regeneration procedures to shorten the treatment and enhance bone healing. In irradiated bones, the effect of ultrasound application is less clear, and further studies are needed to refine the dynamics of the present results

PATH-29. POTENTIAL OF RAMAN SPECTROSCOPY IN ONCOLOGICAL NEUROSURGERY

Raman spectroscopy (RS) has gained increasing interest for the analysis of biological tissues within the recent years. It is a label-free, non-destructive method providing insights in biochemical properties of tumor cells. It is possible to compare RS signals with histological properties of identical tissue parts. Therefore, RS bears promising potentials in neurosurgical neurooncology. On one hand, it could potentially be used for both intraoperative tumor diagnostics and resection control. On the other hand, it could provide important knowledge on tumor biochemistry and used for a subclassification of tumors with a potential impact on personalized therapy approaches. Within our group, we analyzed over 3000 measurement points in different brain tumors ex vivo with a robotized RS system and correlated the spectral curves with histopathological results. We separated and subclassified the data by AI-based methods. Additionally, we compared the latter results with those of a handheld probe, which is potentially navigatable for in vivo, intraoperative applications. We could demonstrate, that it is possible to separate distinct tumor groups only based on RS signals, especially by using computer-based signal analysis. Furthermore, we could demonstrate the differences of the spectra of deep-frozen and formalin-fixed tissues versus non-fixed tissues. Based on our results, we will highlight the potentials of RS for intraoperative neurosurgical application in resection control for brain tumors, as well as we will focus on the potentials for brain tumor diagnostics based purely on this method or by using it as an adjunct. Those methods bear additional potentials in the field of personalized chemotherapy approaches