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Andressa A Magalhaes,1 Camila MF Gama,1 Raquel M Gonçalves,1 Liana CL Portugal,2 Isabel A David,1 Fernanda Serpeloni,3 Liana Wernersbach Pinto,3 Simone G Assis,3,4 Joviana Q Avanci,3 Eliane Volchan,5 Ivan Figueira,6 Liliane MP Vilete,6 Mariana P Luz,6 William Berger,6 Fatima S Erthal,5 Mauro V Mendlowicz,7 Izabela Mocaiber,8 Mirtes G Pereira,1 Leticia de Oliveira1 1Biomedical Institute, Fluminense Federal University, Niterói, RJ, 24210-130, Brazil; 2Department of Physiological Sciences, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20550-170, Brazil; 3Department of Violence and Health Studies, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, 21040-900, Brazil; 4The Neurology Postgraduate Program, Hospital Gaffrée and Guinle, Unirio, RJ, 20270-901, Brazil; 5Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil; 6Psychiatry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, 22290-140, Brazil; 7Department of Psychiatry and Mental Health, Fluminense Federal University, Niterói, RJ, 24030-215, Brazil; 8Institute of Humanities and Health, Federal Fluminense University, Rio das Ostras, RJ, 28890-000, BrazilCorrespondence: Leticia de OliveiraBiomedical Institute, Fluminense Federal University, 101 Hernani Mello, Niterói, RJ, 24210-130, BrazilTel +55 21 2629-2446Email oliveira_leticia@id.uff.brPurpose: Growing evidence suggests that peritraumatic tonic immobility, an involuntary defensive response that involves extreme physical immobility and the perceived inability to escape, is a significant predictor of post-traumatic stress disorder (PTSD) symptomatology. However, this issue has not been specifically addressed in adolescents. Here, we investigated whether tonic immobility response experienced during the worst childhood or adolescent trauma is associated with PTSD symptom severity in a non-clinical student sample.Methods: The sample was composed of students in 9th grade who were attending public and private schools. Symptoms of post-traumatic stress and tonic immobility were assessed using questionnaires. We performed bivariate and multivariate negative binomial regressions to examine whether tonic immobility was associated with PTSD symptomatology after controlling for confounders (peritraumatic dissociation, peritraumatic panic reactions, gender, age and time since trauma).Results: We found an association between tonic immobility and PTSD symptom severity, even after controlling for confounders. Therefore, tonic immobility is associated with PTSD symptoms in trauma-exposed adolescents.Conclusion: These findings highlight tonic immobility as a possible risk factor that could be used to provide direction for more targeted trauma interventions for individuals, particularly those at risk for developing PTSD. Therefore, it contributes to preventing and reducing the psychiatric burden in adolescence and later in life.Keyswords: post-traumatic stress disorder, adolescence, youth, peritraumatic reaction, psychometry, trauma

Purpose: Formation of terminal complement complex (TCC), a downstream complement system activation product inducing inflammatory processes and cell lysis, has been identified in degenerated discs. However, it remains unclear which molecular factors regulate complement activation during disc degeneration (DD). This study investigated a possible involvement of the pro-inflammatory cytokine interleukin-1β (IL-1β) and the lysosomal protease cathepsin D (CTSD)., Methods: Disc biopsies were collected from patients suffering from DD (n = 43) and adolescent idiopathic scoliosis (AIS, n = 13). Standardized tissue punches and isolated cells from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were stimulated with 5% human serum (HS) alone or in combination with IL-1β, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed., Results: In DD tissue cultures, IL-1β stimulation decreased the percentage of TCC + cells in AF and EP (P < 0.05), whereas CTSD stimulation significantly increased TCC deposition in NP (P < 0.01) and zymosan in EP (P < 0.05). Overall, the expression of CD46, CD55 and CD59 significantly increased in all isolated cells during culture (P < 0.05). Moreover, cellular TCC deposition was HS concentration dependent but unaffected by IL-1β, CTSD or zymosan., Conclusion: These results suggest a functional relevance of IL-1β and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1β may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology., (© 2021. The Author(s).)

Background: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions., Aims: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models., Materials & Methods: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability., Results: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 μm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories., Conclusion: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Purpose: The complement system is a crucial part of innate immunity. Recent work demonstrated an unexpected contribution to tissue homeostasis and degeneration. This study investigated for the first time, in human disc tissues, the deposition profile of the complement activation product terminal complement complex (TCC), an inflammatory trigger and inducer of cell lysis, and its inhibitor CD59, and their correlation with the degree of disc degeneration (DD)., Methods: Disc biopsies were collected from patients diagnosed with DD (n = 39, age 63 ± 12) and adolescent idiopathic scoliosis (AIS, n = 10, age 17 ± 4) and compared with discs from healthy Young (n = 11, age 7 ± 7) and Elder (n = 10, age 65 ± 15) donors. Immunohistochemical detection of TCC and CD59 in nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) was correlated with age, Pfirrmann grade and Modic changes., Results: Higher percentage of TCC+ cells was detected in the NP and EP of DD compared to Elder (P < 0.05), and in the EP of Young versus Elder (P < 0.001). In DD, TCC deposition was positively correlated with Pfirrmann grade, but not with Modic changes, whereas for Young donors, a negative correlation was found with age, indicating TCC's involvement not only in DD, but also in early stages of skeletal development. Higher CD59 positivity was found in AIS and DD groups compared to Young (P < 0.05), and it was negatively correlated with the age of the patients., Conclusion: TCC deposition positively correlated with the degree of disc degeneration. A functional relevance of TCC may exist in DD, representing a potential target for new therapeutics.

Purpose: The pathomechanism of annulus fibrosus (AF) failure is still unknown. We hypothesise that mechanical overload and an inflammatory microenvironment contribute to AF structural weakening. Therefore, the objective of this study was to investigate the influence of these factors on the AF, particularly the translamellar bridging network (TLBN) which connects the AF lamellae., Methods: A bovine AF organ culture (AF-OC) model of standardised AF rings was used to study the individual and combined effects of cyclic tensile strain (CTS) and IL-1β (1 ng/mL) culture medium supplementation. AF-OCs were analysed for PGE 2 production (ELISA) and deposition of IL-6, COX-2, fibrillin, and MMP3 in the tissue (immunohistochemistry, IHC). The mechanical strength of the TLBN was evaluated using a peel test to measure the strength required to separate an AF segment along a lamellar bound., Results: The combination of CTS + IL-1β led to a significant increase in PGE 2 production compared to Control (p < 0.01). IHC evaluations showed that the CTS + IL-1β group exhibited higher production of COX-2 and MMP3 within the TLBN regions compared to the adjacent lamellae and a significant increase in IL-6 ratio compared to Control (p < 0.05). A significant decrease in the annular peel strength was observed in the CTS + IL1β group compared to Control (p < 0.05)., Conclusion: Our findings suggest that CTS and IL-1β act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN, leading to a weakening of the tissue. This standardised model enables the investigation of AF/TLBN structure-function relationship and is a platform to test AF-focused therapeutics. These slides can be retrieved under Electronic Supplementary Material.

Study Design: Ex vivo experimental study., Objective: To investigate the effect of proinflammatory/degenerative intervertebral disc (IVD) microenvironment on the regenerative and immunomodulatory behavior of mesenchymal stem/stromal cells (MSCs), using an ex vivo model from bovine origin., Summary of Background Data: Low back pain is a cause of disability worldwide, most frequently associated with IVD degeneration and inflammation, and characterized by increased levels of inflammatory mediators, often disregarded. MSC-based therapies to low back pain have been advocated, but the involvement of inflammation in IVD remodeling mechanism, promoted by MSCs has not yet been explored., Methods: Bovine IVD organ cultures of nucleus pulposus punches were stimulated with needle puncture and culture medium supplementation with 10 ng/mL of interleukin (IL)-1β, to induce a proinflammatory/degenerative environment, as previously established. Human bone marrow-derived MSCs were cultured on top of transwells, placed above nucleus pulposus punches, for up to 16 days. MSCs were analyzed by screening cell viability/apoptosis, metabolic activity, migration, and inflammatory cytokines production in response to the proinflammatory environment. IVD extracellular matrix (ECM) remodeling, gene expression profile of IVD cells, and inflammatory cytokine profile in the presence of MSCs in basal versus proinflammatory conditions were also evaluated., Results: Proinflammatory/degenerative IVD conditions did not affect MSCs viability, but promoted cell migration, while increasing IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 and reducing transforming growth factor-β1 production by MSCs. MSCs did not stimulate ECM production (namely type II collagen or aggrecan) in neither basal nor inflammatory conditions, instead MSCs downregulated bovine proinflammatory IL-6, IL-8, and TNF-α gene expression levels in IL-1β-stimulated IVDs., Conclusion: The present study provides evidence for an immunomodulatory paracrine effect of MSCs in degenerated IVD without an apparent effect in ECM remodeling, and suggest an MSCs mechanism-of-action dependent on a cytokine feedback loop., Level of Evidence: 5.

Changes in drought around the globe are among the most daunting potential effects of climate change. However, changes in droughts are often not well distinguished from changes in aridity levels. As drought constitutes conditions of aridity, the projected declines in mean precipitation tend to override changes in drought. This results in projections of more dire changes in drought than ever. The overestimate of changes can be attributed to the use of 'static' normal precipitation in the derivation of drought events. The failure in distinguishing drought from aridity is a conceptual problem of concern, particularly to drought policymakers. Given that the key objective of drought policies is to determine drought conditions, which are rare and so protracted that they are beyond the scope of normal risk management, for interventions. The main objective of this Case Study of Brazil is to demonstrate the differences between projections of changes in drought based on 'static' and '30-year dynamic' precipitation normal conditions. First we demonstrate that the 'static' based projections suggest 4-fold changes in the probability of drought-year occurrences against changes by the dynamic normal precipitation. The 'static-normal mean precipitation' based projections tend to be monotonically increasing in magnitude, and were arguably considered unrealistic. Based on the '30-year dynamic' normal precipitation conditions, the 13-member GCM ensemble median projection estimates of changes for 2050 under rcp4.5 1 and rcp8.5 2 suggest: (i) Significant differences between changes associated with rcp4.5 and rcp8.5, and are more noticeable for droughts at long than short timescales in the 2070; (ii) Overall, the results demonstrate more realistic projections of changes in drought characteristics over Brazil than previous projections based on 'static' normal precipitation conditions. However, the uncertainty of response of droughts to climate change in CMIP5 simulations is still large, regardless of GCMs selection and translation processes undertaken., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For Brazil, a country frequented by droughts and whose rural inhabitants largely depend on groundwater, reliance on isotope for its monitoring, though accurate, is expensive and limited in spatial coverage. We exploit total water storage (TWS) derived from Gravity Recovery and Climate Experiment (GRACE) satellites to analyse spatial-temporal groundwater changes in relation to geological characteristics. Large-scale groundwater changes are estimated using GRACE-derived TWS and altimetry observations in addition to GLDAS and WGHM model outputs. Additionally, TRMM precipitation data are used to infer impacts of climate variability on groundwater fluctuations. The results indicate that climate variability mainly controls groundwater change trends while geological properties control change rates, spatial distribution, and storage capacity. Granular rocks in the Amazon and Guarani aquifers are found to influence larger storage capability, higher permeability (>10 -4 m/s) and faster response to rainfall (1 to 3months' lag) compared to fractured rocks (permeability <10 -7 m/s and lags > 3months) found only in Bambui aquifer. Groundwater in the Amazon region is found to rely not only on precipitation but also on inflow from other regions. Areas beyond the northern and southern Amazon basin depict a 'dam-like' pattern, with high inflow and slow outflow rates (recharge slope > 0.75, discharge slope < 0.45). This is due to two impermeable rock layer-like 'walls' (permeability <10 -8 m/s) along the northern and southern Alter do Chão aquifer that help retain groundwater. The largest groundwater storage capacity in Brazil is the Amazon aquifer (with annual amplitudes of > 30cm). Amazon's groundwater declined between 2002 and 2008 due to below normal precipitation (wet seasons lasted for about 36 to 47% of the time). The Guarani aquifer and adjacent coastline areas rank second in terms of storage capacity, while the northeast and southeast coastal regions indicate the smallest storage capacity due to lack of rainfall (annual average is rainfall <10cm)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To provide information useful in policy formulation and management of drought impacts in Brazil, in this study, a sequence of drought events based on monthly rainfall of 1901-2013 on ~25 km x 25 km grid are derived at 4 timescales that include short-timescales (3-month and 6-month) and medium to long-timescales (12-month and 24-month). Subsequently, probability of drought occurrences, intensity, duration and areal-extent are calculated. The probabilities of occurrence of severe and extreme droughts at short-timescales are 1 in 12 and 1 in 66 years, respectively, all over the country. At medium to long-timescales, the probability of severe droughts is about 1 in 20 years in northern Brazil, and 1 in 10 years in the south. The probabilities of extreme droughts are 1 in 9 and 1 in 12 years over northern Brazil and in the south, respectively. In general, no evidence of significant (α =0.05) trend is detected in drought frequency, intensity, and duration over the last 11 decades (since 1901) at all the 4 timescales. The drought areal-extent show increasing trends of 3.4%/decade over Brazil for both 3-month and 6-month timescales. However, the trend increases for the 12-month and 24-month timescales are relatively smaller, i.e., 2.4%/decade and 0.5%/decade, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Intervertebral disc (IVD) degeneration is one of the most common causes of low back pain (LBP), the leading disorder in terms of years lived with disability. Inflammation can play a role in LPB, while impairs IVD regeneration. In spite of this, different inflammatory targets have been purposed in the context of IVD regeneration. Anti-inflammatory nanoparticles (NPs) of Chitosan and Poly-(γ-glutamic acid) with a non-steroidal anti-inflammatory drug, diclofenac (Df), were previously shown to counteract a pro-inflammatory response of human macrophages. Here, the effect of intradiscal injection of Df-NPs in degenerated IVD was evaluated. For that, Df-NPs were injected in a bovine IVD organ culture in pro-inflammatory/degenerative conditions, upon stimulation with needle-puncture and interleukin (IL)-1β. Df-NPs were internalized by IVD cells, down-regulating IL-6, IL-8, MMP1 and MMP3, and decreasing PGE2 production, compared with IL-1β-stimulated IVD punches. Interestingly, at the same time, Df-NPs promoted an up-regulation of extracellular matrix (ECM) proteins, namely collagen type II and aggrecan. Allover, this study suggests that IVD treatment with Df-NPs not only reduces inflammation, but also delays and/or decreases ECM degradation, opening perspectives to new intradiscal therapies for IVD degeneration, based on the modulation of inflammation., Statement of Significance: Degeneration of the IVD is an age-related progressive process considered to be the major cause of spine disorders. The pro-inflammatory environment and biomechanics of the degenerated IVD is a challenge for regenerative therapies. The novelty of this work is the intradiscal injection of an anti-inflammatory therapy based on Chitosan (Ch)/Poly-(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) with an anti-inflammatory drug (diclofenac, Df), previously developed by us. This drug delivery system was tested in a pro-inflammatory/degenerative intervertebral disc ex vivo model. The main findings support the success of an anti-inflammatory therapy for degenerated IVD that not only reduces inflammation but also promotes native IVD matrix production., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The conventional techniques used to extract natural products have many disadvantages, and alternative methods have been used, such as supercritical fluid extraction (SFE-CO2). We compared the anti-Mycobacterium tuberculosis activity and cytotoxicity of extracts and major pure compounds were obtained from the leaves of Calophyllum brasiliense by SFE-CO2, maceration and Soxhlet. Anti-M tuberculosis activity was evaluated by resazurin microtiter assay plate and cytotoxicity assay was performed using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The (-) mammea A/BB, (-) mammea B/BB, mammea B/BB cyclo D, ponnalide, mammea A/BA cyclo D, and amentoflavone were identified as the majority compounds. SFE-CO2, especially at 313 K and 10.92 MPa showed better yield for (-) mammea A/BB. Anti-M. tuberculosis activity (62.5 μg/mL) and cytotoxicity (Selectivity Index = 0.320-0.576) were similar for the three extracts. Mammea B/BB cyclo D had a minimum inhibitory concentration (MIC) of 125 μg/mL, and ponnalide and mammea A/BA cyclo D had MICs > 250 μg/mL. The pure compounds isolated showed low Selectivity Index (< 0.09). SFE-CO2 may be more promising than conventional methods for the extraction of compound (-) mammea A/BB, which presented the best anti-M. tuberculosis activity in our previous study. This is important for current industrial requirements to obtain extracts from medicinal plants using clean technologies.

Patients with biliary dyskinesia have symptoms consistent with biliary colic and an abnormal gallbladder ejection fraction (GEF) in the absence of cholelithiasis. Cholecystokinin hepatobiliary scan quantifies gallbladder function and may assist in selecting patients with acalculous biliary pain who would benefit from cholecystectomy. Seventy-eight patients with an abnormal GEF (< 35%) on cholecystokinin hepatobiliary scan without cholelithiasis were studied retrospectively. Patients were divided into groups based on diagnosis and treatment. In Group I, the patients who underwent cholecystectomy, 80 per cent (35 of 44) had complete symptomatic resolution whereas the remaining 20 per cent (9 of 44) had symptomatic improvement. Pathology reports demonstrated chronic cholecystitis in 95 per cent of specimens. Group II were patients with symptoms attributable to biliary dyskinesia, but did not undergo cholecystectomy. Persistence of symptoms was noted in 75 per cent (18 of 24) of patients whereas 25 per cent (6 of 24) had symptomatic resolution without any treatment. Group III consisted of patients with an abnormal ejection fraction who had improvement of symptoms after treatment for an alternative diagnosis (n = 10). These findings suggest that an abnormal ejection fraction does not always indicate gallbladder disease. Alternative diagnoses must be investigated and treated. Patients with persistent biliary type symptoms in combination with an abnormal GEF in the absence of other attributable causes can expect a favorable response to cholecystectomy.