Elucidation of GPR55-Associated Signaling behind THC and LPI Reducing Effects on Ki67-Immunoreactive Nuclei in Patient-Derived Glioblastoma Cells.

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB1 and CB2 receptors, THC modulates cellular responses among others via GPR55. Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67+ nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gßγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids.

A toolbox to analyze collective cell migration, proliferation and cellular organization simultaneously.

BACKGROUND: Analyses of collective cell migration and orientation phenomena are needed to assess the behavior of multicellular clusters. While some tools to the authors' knowledge none is capable to analyze collective migration, cellular orientation and proliferation in phase contrast images simultaneously. METHODS: We provide a tool based to analyze phase contrast images of dense cell layers. PIV is used to calculatevelocity fields, while the structure tensor provides cellular orientation. An artificial neural network is used to identify cell division events, allowing to correlate migratory and organizational phenomena with cell density. CONCLUSION: The presented tool allows the simultaneous analysis of collective cell behavior from phase contrast images in terms of migration, (self-)organization and proliferation.

MACC1-induced migration in tumors: Current state and perspective.

Malignant tumors are still a global, heavy health burden. Many tumor types cannot be treated curatively, underlining the need for new treatment targets. In recent years, metastasis associated in colon cancer 1 (MACC1) was identified as a promising biomarker and drug target, as it is promoting tumor migration, initiation, proliferation, and others in a multitude of solid cancers. Here, we will summarize the current knowledge about MACC1-induced tumor cell migration with a special focus on the cytoskeletal and adhesive systems. In addition, a brief overview of several in vitro models used for the analysis of cell migration is given. In this context, we will point to issues with the currently most prevalent models used to study MACC1-dependent migration. Lastly, open questions about MACC1-dependent effects on tumor cell migration will be addressed.

MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics.

Metastasis-associated in colon cancer 1 (MACC1) is a marker for metastasis, tumor cell migration, and increased proliferation in colorectal cancer (CRC). Tumors with high MACC1 expression show a worse prognosis and higher invasion into neighboring structures. Yet, many facets of the pro-migratory effects are not fully understood. Atomic force microscopy and single cell live imaging were used to quantify biomechanical and migratory properties in low- and high-MACC1-expressing CRC cells. Furthermore, collective migration and expansion of small, cohesive cell colonies were analyzed using live cell imaging and particle image velocimetry. Lastly, the impact of proliferation on collective migration was determined by inhibition of proliferation using mitomycin. MACC1 did not affect elasticity, cortex tension, and single cell migration of CRC cells but promoted collective migration and colony expansion in vitro. Measurements of the local velocities in the dense cell layers revealed proliferation events as regions of high local speeds. Inhibition of proliferation via mitomycin abrogated the MACC1-associated effects on the collective migration speeds. A simple simulation revealed that the expansion of cell clusters without proliferation appeared to be determined mostly by single cell properties. MACC1 overexpression does not influence single cell biomechanics and migration but only collective migration in a proliferation-dependent manner. Thus, targeting proliferation in high-MACC1-expressing tumors may offer additional effects on cell migration.

Microglia-Dependent and Independent Brain Cytoprotective Effects of Mycophenolate Mofetil During Neuronal Damage.

Acute lesions of the central nervous system often lead to permanent limiting deficits. In addition to the initial primary damage, accompanying neuroinflammation is responsible for progression of damage. Mycophenolate mofetil (MMF) as a selective inhibitor of inosine 5-monophosphate dehydrogenase (IMPDH) was shown to modulate the inflammatory response and promote neuronal survival when applied in specific time windows after neuronal injury. The application of brain cytoprotective therapeutics early after neuronal damage is a fundamental requirement for a successful immunomodulation approach. This study was designed to evaluate whether MMF can still mediate brain cytoprotection when applied in predefined short time intervals following CNS injury. Furthermore, the role of microglia and changes in IMPDH2 protein expression were assessed. Organotypic hippocampal slice cultures (OHSC) were used as an in vitro model and excitotoxically lesioned with N-methyl-aspartate (NMDA). Clodronate (Clo) was used to deplete microglia and analyze MMF mediated microglia independent effects. The temporal expression of IMPDH2 was studied in primary glial cell cultures treated with lipopolysaccharide (LPS). In excitotoxically lesioned OHSC a significant brain cytoprotective effect was observed between 8 and 36 h but not within 8 and 24 h after the NMDA damage. MMF mediated effects were mainly microglia dependent at 24, 36, 48 h after injury. However, further targets like astrocytes seem to be involved in protective effects 72 h post-injury. IMPDH2 expression was detected in primary microglia and astrocyte cell cultures. Our data indicate that MMF treatment in OHSC should still be started no later than 8-12 h after injury and should continue at least until 36 h post-injury. Microglia seem to be an essential mediator of the observed brain cytoprotective effects. However, a microglia-independent effect was also found, indicating involvement of astrocytes.

Nimodipine Exerts Time-Dependent Neuroprotective Effect after Excitotoxical Damage in Organotypic Slice Cultures.

During injuries in the central nervous system, intrinsic protective processes become activated. However, cellular reactions, especially those of glia cells, are frequently unsatisfactory, and further exogenous protective mechanisms are necessary. Nimodipine, a lipophilic L-type calcium channel blocking agent is clinically used in the treatment of aneurysmal subarachnoid haemorrhage with neuroprotective effects in different models. Direct effects of nimodipine on neurons amongst others were observed in the hippocampus as well as its influence on both microglia and astrocytes. Earlier studies proposed that nimodipine protective actions occur not only via calcium channel-mediated vasodilatation but also via further time-dependent mechanisms. In this study, the effect of nimodipine application was investigated in different time frames on neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures. Nimodipine, but not nifedipine if pre-incubated for 4 h or co-applied with NMDA, was protective, indicating time dependency. Since blood vessels play no significant role in our model, intrinsic brain cell-dependent mechanisms seems to strongly be involved. We also examined the effect of nimodipine and nifedipine on microglia survival. Nimodipine seem to be a promising agent to reduce secondary damage and reduce excitotoxic damage.

A computed tomography based survey study investigating the agreement of the therapeutic strategy for fragility fractures of the pelvis.

Treatment recommendations for fragility fractures of the pelvis (FFP) have been provided along with the good reliable FFP classification but they are not proven in large studies and recent reports challenge these recommendations. Thus, we aimed to determine the usefulness of the FFP classification determining the treatment strategy and favored procedures in six level 1 trauma centers. Sixty cases of FFP were evaluated by six experienced pelvic surgeons, six inexperienced surgeons in training, and one surgeon trained by the originator of the FFP classification during three repeating sessions using computed tomography scans with multiplanar reconstruction. The intra-rater reliability and inter-rater reliability for therapeutic decisions (non-operative treatment vs. operative treatment) were moderate, with Fleiss kappa coefficients of 0.54 (95% confidence interval [CI] 0.44-0.62) and 0.42 (95% CI 0.34-0.49). We found a therapeutic disagreement predominantly for FFP II related to a preferred operative therapy for FFP II. Operative treated cases were generally treated with an anterior-posterior fixation. Despite the consensus on an anterior-posterior fixation, the chosen procedures are highly variable and most plausible based on the surgeon's preference.

Interaction of Glia Cells with Glioblastoma and Melanoma Cells under the Influence of Phytocannabinoids.

Brain tumor heterogeneity and progression are subject to complex interactions between tumor cells and their microenvironment. Glioblastoma and brain metastasis can contain 30-40% of tumor-associated macrophages, microglia, and astrocytes, affecting migration, proliferation, and apoptosis. Here, we analyzed interactions between glial cells and LN229 glioblastoma or A375 melanoma cells in the context of motility and cell-cell interactions in a 3D model. Furthermore, the effects of phytocannabinoids, cannabidiol (CBD), tetrahydrocannabidiol (THC), or their co-application were analyzed. Co-culture of tumor cells with glial cells had little effect on 3D spheroid formation, while treatment with cannabinoids led to significantly larger spheroids. The addition of astrocytes blocked cannabinoid-induced effects. None of the interventions affected cell death. Furthermore, glial cell-conditioned media led to a significant slowdown in collective, but not single-cell migration speed. Taken together, glial cells in glioblastoma and brain metastasis micromilieu impact the tumor spheroid formation, cell spreading, and motility. Since the size of spheroid remained unaffected in glial cell tumor co-cultures, phytocannabinoids increased the size of spheroids without any effects on migration. This aspect might be of relevance since phytocannabinoids are frequently used in tumor therapy for side effects.

Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension.

Collective behavior of cells emerges from coordination of cell-cell-interactions and is important to wound healing, embryonic and tumor development. Depending on cell density and cell-cell interactions, a transition from a migratory, fluid-like unjammed state to a more static and solid-like jammed state or vice versa can occur. Here, we analyze collective migration dynamics of astrocytes and glioblastoma cells using live cell imaging. Furthermore, atomic force microscopy, traction force microscopy and spheroid generation assays were used to study cell adhesion, traction and mechanics. Perturbations of traction and adhesion were induced via ROCK or myosin II inhibition. Whereas astrocytes resided within a non-migratory, jammed state, glioblastoma were migratory and unjammed. Furthermore, we demonstrated that a switch from an unjammed to a jammed state was induced upon alteration of the equilibrium between cell-cell-adhesion and tension from adhesion to tension dominated, via inhibition of ROCK or myosin II. Such behavior has implications for understanding the infiltration of the brain by glioblastoma cells and may help to identify new strategies to develop anti-migratory drugs and strategies for glioblastoma-treatment.

Variants of Oxidized Regenerated Cellulose and Their Distinct Effects on Neuronal Tissue.

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp®, Equicel® and Equitamp®, have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B4 staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel® led to a neutral pH, Tabotamp® (pH 2.8) and Equitamp® (pH 4.8) caused a significant reduction of pH (p < 0.001). Equicel® and Tabotamp® increased cytotoxicity significantly in several cell lines (p < 0.01). On OHSC, Tabotamp® and Equicel® led to a stronger and deeper damage to the neuronal tissue than Equitamp® or gauze (p < 0.01). Equicel® increased strongly the number of microglia cells after 24 h (p < 0.001). Microglia cells were not detectable after Tabotamp® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel®, Tabotamp® or Equitamp® for specific applications in distinct clinical settings depending on their localization or tissue properties.

Assessment of Neuronal Damage in Brain Slice Cultures Using Machine Learning Based on Spatial Features.

Neuronal damage presents a major health issue necessitating extensive research to identify mechanisms of neuronal cell death and potential therapeutic targets. Commonly used models are slice cultures out of different brain regions extracted from mice or rats, excitotoxically, ischemic, or traumatically lesioned and subsequently treated with potential neuroprotective agents. Thereby cell death is regularly assessed by measuring the propidium iodide (PI) uptake or counting of PI-positive nuclei. The applied methods have a limited applicability, either in terms of objectivity and time consumption or regarding its applicability. Consequently, new tools for analysis are needed. Here, we present a framework to mimic manual counting using machine learning algorithms as tools for semantic segmentation of PI-positive dead cells in hippocampal slice cultures. Therefore, we trained a support vector machine (SVM) to classify images into either "high" or "low" neuronal damage and used naïve Bayes, discriminant analysis, random forest, and a multilayer perceptron (MLP) as classifiers for segmentation of dead cells. In our final models, pixel-wise accuracies of up to 0.97 were achieved using the MLP classifier. Furthermore, a SVM-based post-processing step was introduced to differentiate between false-positive and false-negative detections using morphological features. As only very few false-positive objects and thus training data remained when using the final model, this approach only mildly improved the results. A final object splitting step using Hough transformations was used to account for overlap, leading to a recall of up to 97.6% of the manually assigned PI-positive dead cells. Taken together, we present an analysis tool that can help to objectively and reproducibly analyze neuronal damage in brain-derived slice cultures, taking advantage of the morphology of pycnotic cells for segmentation, object splitting, and identification of false positives.

THC Reduces Ki67-Immunoreactive Cells Derived from Human Primary Glioblastoma in a GPR55-Dependent Manner.

Glioblastoma (GBM) is the most frequent malignant tumor of the central nervous system in humans with a median survival time of less than 15 months. ∆9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the best-characterized components of Cannabis sativa plants with modulating effects on cannabinoid receptors 1 and 2 (CB1 and CB2) and on orphan receptors such as GPR18 or GPR55. Previous studies have demonstrated anti-tumorigenic effects of THC and CBD in several tumor entities including GBM, mostly mediated via CB1 or CB2. In this study, we investigated the non-CB1/CB2 effects of THC on the cell cycle of GBM cells isolated from human tumor samples. Cell cycle entry was measured after 24 h upon exposure by immunocytochemical analysis of Ki67 as proliferation marker. The Ki67-reducing effect of THC was abolished in the presence of CBD, whereas CBD alone did not cause any changes. To identify the responsible receptor for THC effects, we first characterized the cells regarding their expression of different cannabinoid receptors: CB1, CB2, GPR18, and GPR55. Secondly, the receptors were pharmacologically blocked by application of their selective antagonists AM281, AM630, O-1918, and CID16020046 (CID), respectively. All examined cells expressed the receptors, but only in presence of the GPR55 antagonist CID was the THC effect diminished. Stimulation with the GPR55 agonist lysophosphatidylinositol (LPI) revealed similar effects as obtained for THC. The LPI effects were also inhibited by CBD and CID, confirming a participation of GPR55 and suggesting its involvement in modifying the cell cycle of patient-derived GBM cells.

Measuring Mechanical and Adhesive Properties of Single Cells Using an Atomic Force Microscope.

Atomic force microscopy allows the determination of both mechanical and adhesive properties of living single cells and generation of high-resolution surface images. Here, we describe a method to determine the Young's modulus of a cell and adhesion between a coated cantilever and a cell, as well as an overview of the analysis of the data obtained. Additionally, we point out typical and important pitfalls during the measurement and analysis.

Preparation and Culture of Organotypic Hippocampal Slices for the Analysis of Brain Metastasis and Primary Brain Tumor Growth.

Brain metastasis is a major challenge for therapy and defines the end stage of tumor progression with a very limited patients' prognosis. Experimental setups that faithfully mimic these processes are necessary to understand the mechanism of brain metastasis and to develop new improved therapeutic strategies. Here, we describe an in vitro model, which closely resembles the in vivo situation. Organotypic hippocampal brain slice cultures (OHSCs) prepared from 3- to 8-day-old mice are well suited for neuro-oncology research including brain metastasis. The original morphology is preserved in OHSCs even after culture periods of several days to weeks. Tumor cells or cells of metastatic origin can be seeded onto OHSCs to evaluate micro-tumor formation, tumor cell invasion, or treatment response. We describe preparation and culture of OHSCs including the seeding of tumor cells. Finally, we show examples of how to treat the OHSCs for life-dead or immunohistochemical staining.

MACC1 driven alterations in cellular biomechanics facilitate cell motility in glioblastoma.

BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) is an established marker for metastasis and tumor cell migration in a multitude of tumor entities, including glioblastoma (GBM). Nevertheless, the mechanism underlying the increased migratory capacity in GBM is not comprehensively explored. METHODS: We performed live cell and atomic force microscopy measurements to assess cell migration and mechanical properties of MACC1 overexpressing GBM cells. We quantified MACC1 dependent dynamics of 3D aggregate formation. For mechanistic studies we measured the expression of key adhesion molecules using qRT-PCR, and MACC1 dependent changes in short term adhesion to fibronectin and laminin. We then determined changes in sub-cellular distribution of integrins and actin in dependence of MACC1, but also in microtubule and intermediate filament organization. RESULTS: MACC1 increased the migratory speed and elastic modulus of GBM cells, but decreased cell-cell adhesion and inhibited the formation of 3D aggregates. These effects were not associated with altered mRNA expression of several key adhesion molecules or altered short-term affinity to laminin and fibronectin. MACC1 did neither change the organization of the microtubule nor intermediate filament cytoskeleton, but resulted in increased amounts of protrusive actin on laminin. CONCLUSION: MACC1 overexpression increases elastic modulus and migration and reduces adhesion of GBM cells thereby impeding 3D aggregate formation. The underlying molecular mechanism is independent on the organization of microtubules, intermediate filaments and several key adhesion molecules, but depends on adhesion to laminin. Thus, targeting re-organization of the cytoskeleton and cell motility via MACC1 may offer a treatment option to impede GBM spreading. Video Abstract.

Evaluation of machine learning models for automatic detection of DNA double strand breaks after irradiation using a γH2AX foci assay.

Ionizing radiation induces amongst other the most critical type of DNA damage: double-strand breaks (DSBs). Efficient repair of such damage is crucial for cell survival and genomic stability. The analysis of DSB associated foci assays is often performed manually or with automatic systems. Manual evaluation is time consuming and subjective, while most automatic approaches are prone to changes in experimental conditions or to image artefacts. Here, we examined multiple machine learning models, namely a multi-layer perceptron classifier (MLP), linear support vector machine classifier (SVM), complement naive bayes classifier (cNB) and random forest classifier (RF), to correctly classify γH2AX foci in manually labeled images containing multiple types of artefacts. All models yielded reasonable agreements to the manual rating on the training images (Matthews correlation coefficient >0.4). Afterwards, the best performing models were applied on images obtained under different experimental conditions. Thereby, the MLP model produced the best results with an F1 Score >0.9. As a consequence, we have demonstrated that the used approach is sufficient to mimic manual counting and is robust against image artefacts and changes in experimental conditions.

Abnormal Cannabidiol Affects Production of Pro-Inflammatory Mediators and Astrocyte Wound Closure in Primary Astrocytic-Microglial Cocultures.

Abnormal cannabidiol (abn-CBD) exerts neuroprotective effects in vivo and in vitro. In the present study, we investigated the impact of abn-CBD on the glial production of proinflammatory mediators and scar formation within in vitro models. Primary astrocytic-microglial cocultures and astrocytic cultures from neonatal C57BL/6 mice and CB2 receptor knockout mice were stimulated with lipopolysaccharide (LPS), and the concentrations of tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and nitrite were determined. Furthermore, we performed a live cell microscopy-based scratch-wound assay. After LPS stimulation, TNFα, IL-6 and nitrite production was more strongly increased in cocultures than in isolated astrocytes. Abn-CBD treatment attenuated the LPS-induced production of TNFα and nitrite in cocultures, while IL-6 production remained unaltered. In isolated astrocytes, only LPS-induced TNFα production was reduced by abn-CBD. Similar effects were observed after abn-CBD application in cocultures of CB2 knockout mice. Interestingly, LPS-induced TNFα and nitrite levels were far lower in CB2 knockout cultures compared to wildtypes, while IL-6 levels did not differ. In the scratch-wound assay, treatment with abn-CBD decelerated wound closure when microglial cells were present. Our data shows a differential role of abn-CBD for modulation of glial inflammation and astrocytic scar formation. These findings provide new explanations for mechanisms behind the neuroprotective potential of abn-CBD.

Opposite Effects of Neuroprotective Cannabinoids, Palmitoylethanolamide, and 2-Arachidonoylglycerol on Function and Morphology of Microglia.

Various studies performed in cultured cells and in in vivo models of neuronal damage showed that cannabinoids exert a neuroprotective effect. The increase in cannabinoids and cannabinoid like substances after stroke has been postulated to limit the content of neuronal injury. As well-accepted, inflammation, and neuronal damage are coupled processes and microglial cells as the main intrinsic immunological effector within the brain play a central role in their regulation. Treatment with the endocannabinoid, 2-arachidonoylglycerol (2-AG) or the endocannabinoid-like substance, palmitoylethanolamide (PEA) affected microglial cells and led to a decrease in the number of damaged neurons after excitotoxical lesion in organotypic hippocampal slice cultures (OHSC). 2-AG activated abnormal cannabidiol (abn-CBD) receptor, PEA was shown to mediate neuroprotection via peroxisome proliferator-activated receptor (PPAR)α. Despite the known neuroprotective and anti-inflammatory properties, the potential synergistic effect, namely possible entourage effect after treatment with the combination of these two protective cannabinoids has not been examined yet. After excitotoxical lesion OHSC were treated with PEA, 2-AG or a combination of both and the number of damaged neurons was evaluated. To investigate the role of microglial cells in PEA and 2-AG mediated protection, primary microglial cell cultures were treated with lipopolysaccharide (LPS) and 2-AG, PEA or a combination of those. Thereafter, we measured NO production, ramification index, proliferation and PPARα distribution in microglial cells. While PEA or 2-AG alone were neuroprotective, their co-application vanished the protective effect. This behavior was independent of microglial cells. Furthermore, PEA and 2-AG had contrary effects on ramification index and on NO production. No significant changes were observed in the proliferation rate of microglial cells after treatment. The expression of PPARα was not changed upon stimulation with PEA or 2-AG, but the distribution was significantly altered. 2-AG and PEA mediated neuroprotection was abolished when co-applied. Both cannabinoids exert contrary effects on morphology and function of microglial cells. Co-application of both cannabinoids with different targets did not lead to a positive additive effect as expected, presumably due to the contrary polarization of microglial cells.

Radiosensitization and a Less Aggressive Phenotype of Human Malignant Glioma Cells Expressing Isocitrate Dehydrogenase 1 (IDH1) Mutant Protein: Dissecting the Mechanisms.

The presence of an isocitrate dehydrogenase 1 (IDH1) mutation is associated with a less aggressive phenotype, increased sensitivity to radiation, and increased overall survival in patients with diffuse glioma. Based on in vitro experimentations in malignant glioma cell lines, the consequences on cellular processes of IDH1R132H expression were analyzed. The results revealed that IDH1R132H expression enhanced the radiation induced accumulation of residual γH2AX foci and decreased the amount of glutathione (GSH) independent of the oxygen status. In addition, expression of the mutant IDH1 caused a significant increase of cell stiffness and induced an altered organization of the cytoskeleton, which has been shown to reinforce cell stiffness. Furthermore, IDH1R132H expression decreased the expression of vimentin, an important component of the cytoskeleton and regulator of the cell stiffness. The results emphasize the important role of mutant IDH1 in treatment of patients with diffuse gliomas especially in response to radiation. Hence, detection of the genetic status of IDH1 before therapy massively expands the utility of immunohistochemistry to accurately distinguish patients with a less aggressive and radiosensitive IDH1-mutant diffuse glioma suitable for radiotherapy from those with a more aggressive IDH1-wildtype diffuse glioma who might benefit from an individually intensified therapy comprising radiotherapy and alternative medical treatments.

Fragility Fractures of the Pelvis Classification: A Multicenter Assessment of the Intra-Rater and Inter-Rater Reliabilities and Percentage of Agreement.

BACKGROUND: The fragility fractures of the pelvis (FFP) classification was established to address the specific fracture morphology and dynamic instability in the elderly. Although this system is frequently used, data on the intra-rater and inter-rater reliabilities are lacking. METHODS: Six experienced and 6 inexperienced surgeons and 1 surgeon trained by the originator of the FFP classification ("gold standard") each used the FFP classification 3 times to grade the computed tomography (CT) scans of 60 patients from 6 hospitals. We assessed intra-rater and inter-rater reliabilities using Fleiss kappa statistics and the percentage of agreement using the "gold standard," the submitting hospital, and the majority vote as references. RESULTS: The intra-rater reliability for the FFP classification was mainly moderate, with a mean Fleiss kappa coefficient (and 95% confidence interval) of 0.46 (0.40 to 0.50) for the complete classification (i.e., both the main-group FFP ratings [I through III] and the subgroup ratings [a, b, and c]) and 0.60 (0.53 to 0.65) for the main group only. The inter-rater reliability was substantial for the main group classification (0.61 [0.54 to 0.66]) and moderate for the complete classification (0.53 [0.48 to 0.58]). The percentage of agreement was 68% to 80%. The lowest agreement was found for FFP II and III. CONCLUSIONS: The FFP classification displayed moderate and substantial intra-rater and inter-rater reliabilities. CLINICAL RELEVANCE: With moderate to substantial intra-rater and inter-rater reliabilities, the FFP classification forms a solid basis for future clinical investigations. The differentiation of FFP II from FFP III should be evaluated thoroughly, as the initial treatment changes from nonoperative for II to operative for III.