Autophagy protein ULK1 interacts with and regulates SARM1 during axonal injury.

Autophagy is a cellular catabolic pathway generally thought to be neuroprotective. However, autophagy and in particular its upstream regulator, the ULK1 kinase, can also promote axonal degeneration. We examined the role and the mechanisms of autophagy in axonal degeneration using a mouse model of contusive spinal cord injury (SCI). Consistent with activation of autophagy during axonal degeneration following SCI, autophagosome marker LC3, ULK1 kinase, and ULK1 target, phospho-ATG13, accumulated in the axonal bulbs and injured axons. SARM1, a TIR NADase with a pivotal role in axonal degeneration, colocalized with ULK1 within 1 h after SCI, suggesting possible interaction between autophagy and SARM1-mediated axonal degeneration. In our in vitro experiments, inhibition of autophagy, including Ulk1 knockdown and ULK1 inhibitor, attenuated neurite fragmentation and reduced accumulation of SARM1 puncta in neurites of primary cortical neurons subjected to glutamate excitotoxicity. Immunoprecipitation data demonstrated that ULK1 physically interacted with SARM1 in vitro and in vivo and that SAM domains of SARM1 were necessary for ULK1-SARM1 complex formation. Consistent with a role in regulation of axonal degeneration, in primary cortical neurons ULK1-SARM1 interaction increased upon neurite damage. Supporting a role for autophagy and ULK1 in regulation of SARM1 in axonal degeneration in vivo, axonal ULK1 activation and accumulation of SARM1 were both decreased after SCI in Becn1+/- autophagy hypomorph mice compared to wild-type (WT) controls. These findings suggest a regulatory crosstalk between autophagy and axonal degeneration pathways, which is mediated through ULK1-SARM1 interaction and contributes to the ability of SARM1 to accumulate in injured axons.

Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.

RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization.

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.

Etiology and pathophysiology of heart failure in people with HIV.

HIV-associated cardiomyopathy is a well-established sequela in people infected with HIV (PHIV). Despite significant advances in HIV management through the use of highly active anti-retroviral therapy (HAART), PHIV on HAART continue to have elevated risk of cardiomyopathy and heart failure, even when accounting for known cardiovascular risk factors. This review article will explore the proposed mechanisms by which chronic HIV infection induces cardiomyopathy and heart failure in the setting of HAART. Evaluation, work-up, and management of cardiomyopathy in PHIV will also be briefly discussed. The advent of HAART has altered the pathophysiology HIV-associated cardiomyopathy from a rapidly progressive cardiomyopathy, often with pericardial involvement, into a chronic process involving inflammation and persistent immune dysregulation. With the significant decrease in AIDS-related deaths, the prevalence of cardiomyopathy and the mortality associated with heart failure in PHIV have increased. Multiple immune-related and inflammatory mechanisms have been proposed, which may provide insight into evaluation and management of cardiomyopathy in PHIV.

Application of machine learning to determine top predictors of noncalcified coronary burden in psoriasis: An observational cohort study.

BACKGROUND: Psoriasis is associated with elevated risk of heart attack and increased accumulation of subclinical noncalcified coronary burden by coronary computed tomography angiography (CCTA). Machine learning algorithms have been shown to effectively analyze well-characterized data sets. OBJECTIVE: In this study, we used machine learning algorithms to determine the top predictors of noncalcified coronary burden by CCTA in psoriasis. METHODS: The analysis included 263 consecutive patients with 63 available variables from the Psoriasis Atherosclerosis Cardiometabolic Initiative. The random forest algorithm was used to determine the top predictors of noncalcified coronary burden by CCTA. We evaluated our results using linear regression models. RESULTS: Using the random forest algorithm, we found that the top 10 predictors of noncalcified coronary burden were body mass index, visceral adiposity, total adiposity, apolipoprotein A1, high-density lipoprotein, erythrocyte sedimentation rate, subcutaneous adiposity, small low-density lipoprotein particle, cholesterol efflux capacity and the absolute granulocyte count. Linear regression of noncalcified coronary burden yielded results consistent with our machine learning output. LIMITATION: We were unable to provide external validation and did not study cardiovascular events. CONCLUSION: Machine learning methods identified the top predictors of noncalcified coronary burden in psoriasis. These factors were related to obesity, dyslipidemia, and inflammation, showing that these are important targets when treating comorbidities in psoriasis.

Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.

Mapping a multiplexed zoo of mRNA expression.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Differences in skin blood flow oscillations between the plantar and dorsal foot in people with diabetes mellitus and peripheral neuropathy.

BACKGROUND: Understanding the differences in skin blood flow (SBF) on the plantar and dorsal foot in people with diabetes mellitus (DM) may help to assess the influence of diabetes and neuropathy on microvascular dysfunction and risks of diabetic foot ulcers in this population. However, there is no study comparing SBF oscillations between the plantar and dorsal foot in people with DM and peripheral neuropathy (PN). OBJECTIVE: The objective of this study was to compare SBF oscillations between the plantar and dorsal foot in people with DM and PN and investigate the underlying mechanisms responsible for the differences. METHODS: 18 people with Type 2 DM and PN and 8 healthy controls were recruited. Laser Doppler flowmetry (LDF) was used to measure SBF on the plantar and dorsal foot for 10 min when the subject was in the supine position. Wavelet analysis was used to quantify the relative amplitude of the characteristic frequency components of SBF oscillations. Sample entropy analysis was used to quantify the regularity degree of SBF oscillations. RESULTS: People with DM and PN had a higher SBF on the plantar foot compared to the dorsal foot. The relative wavelet amplitudes of metabolic and myogenic frequency components on the plantar foot were respectively higher and lower compared to the dorsal foot. Sample entropy analysis showed that SBF on the plantar foot had a higher degree of regularity compared to the dorsal foot. CONCLUSIONS: In people with DM and PN, higher SBF on the plantar foot is attributed to the metabolic and myogenic controls, and SBF on the plantar foot exhibits a higher degree of regularity compared to the dorsal foot. People with DM and PN also had higher plantar and dorsal SBF compared to the healthy controls. This study provides evidence to document differences in SBF of the plantar and dorsal foot in people with DM and PN.

Association of aortic vascular uptake of 18FDG by PET/CT and aortic wall thickness by MRI in psoriasis: a prospective observational study.

BACKGROUND: The contribution of inflammation to the incidence of cardiovascular disease (CVD) has been increasingly recognized in recent years. We investigated the relationship of aortic vascular uptake of 18F-FDG by PET/CT and aortic wall thickness (AWT) by MRI in psoriasis, a chronic inflammatory disease with increased incidence of CVD. One hundred sixty-five patients with plaque psoriasis participated in an ongoing longitudinal cohort study. Subclinical atherosclerosis was assessed as aortic uptake of 18F-FDG by PET/CT reported as target-to-background ratio (TBR) and AWT by MRI reported as maximal thickness. RESULTS: Patients with psoriasis were middle aged, predominantly male, and had mild CV risk by traditional risk factors. Psoriasis severity as measured by PASI score was a notable determinant of AWT (ρ = 0.20, p = 0.01). Moreover, aortic vascular uptake of 18F-FDG associated with AWT by MRI at baseline in unadjusted analysis (ß = 0.27 p = 0.001) and following adjustment for traditional cardiovascular risk factors, waist-to-hip ratio, and statin use (ß = 0.21 p = 0.01). Finally, following 1 year of psoriasis treatment, a decrease in aortic vascular uptake of 18F-FDG was associated with a reduction in AWT in fully adjusted models (ß = 0.33, p = 0.02). CONCLUSION: In conclusion, we demonstrate that psoriasis severity and aortic vascular uptake of 18F-FDG in the aorta were associated with AWT. Following treatment of psoriasis, a decrease in aortic vascular uptake of 18F-FDG was associated with a reduction in AWT at 1 year. These findings suggest that aortic vascular uptake of 18F-FDG is associated with early evidence of vascular disease assessed by aortic wall thickness. Prospective studies in larger populations including other inflammatory diseases are warranted.

Application of Non-invasive Imaging in Inflammatory Disease Conditions to Evaluate Subclinical Coronary Artery Disease.

PURPOSE OF REVIEW: Traditional risk models, such as the Framingham risk score, fail to capture the increased cardiovascular disease risk seen in patients with chronic inflammatory diseases. This review will cover imaging modalities and their emerging applications in assessing subclinical cardiovascular disease for both research and clinical care in patients with chronic inflammatory diseases. RECENT FINDINGS: Multiple imaging modalities have been studied to assess for subclinical cardiovascular disease via functional/physiologic, inflammatory, and anatomic assessment in patients with chronic inflammatory diseases. The use of imaging to evaluate subclinical cardiovascular disease in patients with chronic inflammatory diseases has the potential to capture early sub-clinical atherosclerosis, to improve risk stratification of future cardiovascular events, and to guide effective disease management.

Efficacy of Biophysical Energies on Healing of Diabetic Skin Wounds in Cell Studies and Animal Experimental Models: A Systematic Review.

We have systematically assessed published cell studies and animal experimental reports on the efficacy of selected biophysical energies (BPEs) in the treatment of diabetic foot ulcers. These BPEs include electrical stimulation (ES), pulsed electromagnetic field (PEMF), extracorporeal shockwave (ECSW), photo energies and ultrasound (US). Databases searched included CINAHL, MEDLINE and PubMed from 1966 to 2018. Studies reviewed include animal and cell studies on treatment with BPEs compared with sham, control or other BPEs. Information regarding the objective measures of tissue healing and data was extracted. Eighty-two studies were eventually selected for the critical appraisal: five on PEMF, four each on ES and ECSW, sixty-six for photo energies, and three about US. Based on the percentage of original wound size affected by the BPEs, both PEMF and low-level laser therapy (LLL) demonstrated a significant clinical benefit compared to the control or sham treatment, whereas the effect of US did not reveal a significance. Our results indicate potential benefits of selected BPEs in diabetic wound management. However, due to the heterogeneity of the current clinical trials, comprehensive studies using well-designed trials are warranted to confirm the results.

Constant Light Desynchronizes Olfactory versus Object and Visuospatial Recognition Memory Performance.

Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light (rLL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes Period1 and Period2 (Per1 and Per2), as well as the immediate-early gene Fos in the SCN, dorsal hippocampus, and olfactory bulb. Under rLL, rhythmic Per1 and Fos expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, Per1 and Fos expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under rLL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals.SIGNIFICANCE STATEMENT A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod).

Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.

Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

Programming biomolecular self-assembly pathways.

In nature, self-assembling and disassembling complexes of proteins and nucleic acids bound to a variety of ligands perform intricate and diverse dynamic functions. In contrast, attempts to rationally encode structure and function into synthetic amino acid and nucleic acid sequences have largely focused on engineering molecules that self-assemble into prescribed target structures, rather than on engineering transient system dynamics. To design systems that perform dynamic functions without human intervention, it is necessary to encode within the biopolymer sequences the reaction pathways by which self-assembly occurs. Nucleic acids show promise as a design medium for engineering dynamic functions, including catalytic hybridization, triggered self-assembly and molecular computation. Here, we program diverse molecular self-assembly and disassembly pathways using a 'reaction graph' abstraction to specify complementarity relationships between modular domains in a versatile DNA hairpin motif. Molecular programs are executed for a variety of dynamic functions: catalytic formation of branched junctions, autocatalytic duplex formation by a cross-catalytic circuit, nucleated dendritic growth of a binary molecular 'tree', and autonomous locomotion of a bipedal walker.

Prognostic Power of Quantitative Assessment of Functional Mitral Regurgitation and Myocardial Scar Quantification by Cardiac Magnetic Resonance.

BACKGROUND: The severity classification of functional mitral regurgitation (FMR) remains controversial despite adverse prognosis and rapidly evolving interventions. Furthermore, it is unclear if quantitative assessment with cardiac magnetic resonance can provide incremental risk stratification for patients with ischemic cardiomyopathy (ICM) or non-ICM (NICM) in terms of FMR and late gadolinium enhancement (LGE). We evaluated the impact of quantitative cardiac magnetic resonance parameters on event-free survival separately for ICM and NICM, to assess prognostic FMR thresholds and interactions with LGE quantification. METHODS: Patients (n=1414) undergoing cardiac magnetic resonance for cardiomyopathy (ejection fraction<50%) assessment from April 1, 2001 to December 31, 2017 were evaluated. The primary end point was all-cause death, heart transplant, or left ventricular assist device implantation during follow-up. Multivariable Cox analyses were conducted to determine the impact of FMR, LGE, and their interactions with event-free survival. RESULTS: There were 510 primary end points, 395/782 (50.5%) in ICM and 114/632 (18.0%) in NICM. Mitral regurgitation-fraction per 5% increase was independently associated with the primary end point, hazards ratios (95% CIs) of 1.04 (1.01-1.07; P=0.034) in ICM and 1.09 (1.02-1.16; P=0.011) in NICM. Optimal mitral regurgitation-fraction threshold for moderate and severe FMR were ≥20% and ≥35%, respectively, in both ICM and NICM, based on the prediction of the primary outcome. Similarly, optimal LGE thresholds were ≥5% in ICM and ≥2% in NICM. Mitral regurgitation-fraction×LGE emerged as a significant interaction for the primary end point in ICM (P=0.006), but not in NICM (P=0.971). CONCLUSIONS: Mitral regurgitation-fraction and LGE are key quantitative cardiac magnetic resonance biomarkers with differential associations with adverse outcomes in ICM and NICM. Optimal prognostic thresholds may provide important clinical risk prognostication and may further facilitate the ability to derive selection criteria to guide therapeutic decision-making.

Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice.

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.

Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice.

Elderly patients with traumatic brain injury (TBI) have greater mortality and poorer outcomes than younger individuals. The extent to which old age alters long-term recovery and chronic microglial activation after TBI is unknown, and evidence for therapeutic efficacy in aged mice is sorely lacking. The present study sought to identify potential inflammatory mechanisms underlying age-related outcomes late after TBI. Controlled cortical impact was used to induce moderate TBI in young and old male C57BL/6 mice. At 12 weeks post-injury, aged mice exhibited higher mortality, poorer functional outcomes, larger lesion volumes, and increased microglial activation. Transcriptomic analysis identified age- and TBI-specific gene changes consistent with a disease-associated microglial signature in the chronically injured brain, including those involved with complement, phagocytosis, and autophagy pathways. Dysregulation of phagocytic and autophagic function in microglia was accompanied by increased neuroinflammation in old mice. As proof-of-principle that these pathways have functional importance, we administered an autophagic enhancer, trehalose, in drinking water continuously for 8 weeks after TBI. Old mice treated with trehalose showed enhanced functional recovery and reduced microglial activation late after TBI compared to the sucrose control group. Our data indicate that microglia undergo chronic changes in autophagic regulation with both normal aging and TBI that are associated with poorer functional outcome. Enhancing autophagy may therefore be a promising clinical therapeutic strategy for TBI, especially in older patients.

Indentation Stiffness Measurement by an Optical Coherence Tomography-Based Air-Jet Indentation System Can Reflect Type I Collagen Abundance and Organisation in Diabetic Wounds.

There is a lack of quantitative and non-invasive clinical biomechanical assessment tools for diabetic foot ulcers. Our previous study reported that the indentation stiffness measured by an optical coherence tomography-based air-jet indentation system in a non-contact and non-invasive manner may reflect the tensile properties of diabetic wounds. As the tensile properties are known to be contributed by type I collagen, this study was aimed to establish the correlations between the indentation stiffness, and type I collagen abundance and organisation, in order to further justify and characterise the in vivo indentation stiffness measurement in diabetic wounds. In a male streptozotocin-induced diabetic rat model, indentation stiffness, and type I collagen abundance and organisation of excisional wounds were quantified and examined using the optical coherence tomography-based air-jet indentation system and picrosirius red polarised light microscopy, respectively, on post-wounding days 3, 5, 7, 10, 14, and 21. The results showed significant negative correlations between indentation stiffness at the wound centre, and the collagen abundance and organisation. The correlations between the indentation stiffness, as well as collagen abundance and organisation of diabetic wounds suggest that the optical coherence tomography-based air-jet indentation system can potentially be used to quantitatively and non-invasively monitor diabetic wound healing in clinical settings, clinical research or preclinical research.