Measuring Parallelism to the Ground in Bipedal Stance Phase: Mechanical Versus Kinematic Alignment in Total Knee Arthroplasty.

Introduction The goal of total knee arthroplasty is to replace diseased cartilage and bone with an artificial implant to improve the patient's quality of life. The knee has historically been reconstructed to the patient's mechanical axis (MA). However, kinematically aligned techniques have been increasingly used. Kinematic alignment requires less soft-tissue resection and aligns the knee with what is anatomically natural to the patient, while there is concern that kinematically aligned knees will lead to earlier failure due to potential unequal weight distribution on the implant. The purpose of this study is to compare the parallelism from the floor of the joint-line cuts using kinematic and mechanical alignment and understand if the MA is a proper estimation of the tibial-ankle axis (TA). Methods A retrospective study was conducted by recruiting all high tibial osteotomy and distal femoral osteotomy recipients operated on by two surgeons in two MedStar Health hospitals from 01/2013 to 07/2020 with full-length films in preparation for restorative procedures. Baseline osteoarthritis was graded using the Kellgren-Lawrence classification system with all patients presenting as Grade 0. The TA and the joint-line orientations of the MA and kinematic axis (KA) were measured on 66 legs. The average distance from parallelism to the ground was compared between the MA and the KA and between the MA and the TA using a paired t-test. Results KA joint-line orientation (1.705° deviation) was more parallel to the floor in the bipedal stance phase than the MA (2.316° deviation, p=0.0156). The MA (2.316° deviation) was not a proper estimation of the TA (4.278° deviation, p=0.0001). Conclusion By utilizing the KA technique, the restoration of the natural joint line, as well as a joint that is more parallel to the floor in the stance phase compared to the MA, is achieved. The parallelism to the ground of the KA during the bipedal stance phase suggests an even load distribution across the knee. In addition, due to its similarity to the KA and anatomical significance in weight-bearing distribution, further investigation into the hip-to-calcaneal axis as an approximation of the joint line is warranted.

Interim 1-Year Radiographic and Clinical Outcomes Following Anterior Cervical Discectomy and Fusion Using Hydroxyapatite-Infused Polyetheretherketone Interbody Cages.

BACKGROUND: This is a multicenter observational registry analysis of 1-year radiographic and clinical outcomes following anterior cervical discectomy and fusion (ACDF) using hydroxyapatite (HA)-infused polyetheretherketone (PEEK) intervertebral cages. METHODS: Radiographic and clinical outcome data were collected preoperatively and at 6 weeks, 3 months, 6 months, and 12 months postoperatively. To assess fusion, dynamic flexion-extension radiographs were independently evaluated with a validated method. Clinical outcomes were assessed using the following disease-specific measures: Neck Disability Index (NDI) and visual analog scale (VAS) for neck, left arm, and right arm pain. Patient satisfaction was also evaluated. RESULTS: A total of 789 ACDF patients (men: 51.5%/women: 48.5%; mean body mass index: 29.9 kg/m2) were included at the time of analysis, and 1565 segments have been operated. Successful fusion was confirmed in 91.3% of all operated levels after 6 months and 92.2% after 12 months. Mean NDI scores improved significantly (P < 0.01) preoperatively (46.3, n = 771) to postoperatively (12 months: 25.2, n = 281). Consistently, mean VAS neck (preoperative: 64.2, n = 770; 12 months: 28.6, n = 278), VAS right arm (preoperative: 42.6, n = 766; 12 months: 20.4, n = 277), and VAS left arm (preoperative: 41.1, n = 768; 12 months: 20.8, n = 277) decreased significantly (P < 0.01). Patients reported high satisfaction rates after surgery with no significant changes in postoperative patient satisfaction between 6 weeks and 12 months (95.1%, n = 273). CONCLUSIONS: ACDF with HA-infused PEEK cages demonstrates promising radiographic and clinical outcomes, supporting the potential benefits of incorporating HA into PEEK cages to enhance fusion rates and improve patient outcomes. CLINICAL RELEVANCE: This study demonstrates a >90% fusion rate by level with reliable improvements in patient reported outcomes, along with a high rate of patient satisfaction, in a large patient cohort undergoing ACDF with HA-infused PEEK cages. LEVEL OF EVIDENCE: 2 .

Effects of donor-specific microvascular anatomy on hemodynamic perfusion in human choriocapillaris.

Evidence from histopathology and clinical imaging suggest that choroidal anatomy and hemodynamic perfusion are among the earliest changes in retinal diseases such as age-related macular degeneration (AMD). However, how inner choroidal anatomy affects hemodynamic perfusion is not well understood. Therefore, we sought to understand the influences of choroidal microvascular architecture on the spatial distribution of hemodynamic parameters in choriocapillaris from human donor eyes using image-based computational hemodynamic (ICH) simulations. We subjected image-based inner choroid reconstructions from eight human donor eyes to ICH simulation using a kinetic-based volumetric lattice Boltzmann method to compute hemodynamic distributions of velocity, pressure, and endothelial shear stress. Here, we demonstrate that anatomic parameters, including arteriolar and venular arrangements and intercapillary pillar density and distribution exert profound influences on inner choroidal hemodynamic characteristics. Reductions in capillary, arteriolar, and venular density not only reduce the overall blood velocity within choriocapillaris, but also substantially increase its spatial heterogeneity. These first-ever findings improve understanding of how choroidal anatomy affects hemodynamics and may contribute to pathogenesis of retinal diseases such as AMD.

Y chromosome proteins in female tissues.

Unreliable protein-based tools are impeding sex-based research.

Kamuvudine-9 Protects Retinal Structure and Function in a Novel Model of Experimental Rhegmatogenous Retinal Detachment.

Purpose: Rhegmatogenous retinal detachment (RRD) is a vision-threatening event that benefits from surgical intervention. While awaiting surgical reattachment, irreversible hypoxic and inflammatory damage to the retina often occurs. An interim therapy protecting photoreceptors could improve functional outcomes. We sought to determine whether Kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors (NRTIs) that inhibits inflammasome activation, and the NRTIs lamivudine (3TC) and azidothymidine (AZT) could protect the retina following RRD. Methods: RRD was induced in mice via subretinal injection (SRI) of 1% carboxymethylcellulose (CMC). To simulate outcomes following the clinical management of RRD, we determined the optimal conditions by which SRI of CMC induced spontaneous retinal reattachment (SRR) occurs over 10 days (RRD/SRR). K-9, 3TC, or AZT was administered via intraperitoneal injection. Inflammasome activation pathways were monitored by abundance of cleaved caspase-1, IL-18, and cleaved caspase-8, and photoreceptor death was assessed by TUNEL staining. Retinal function was assessed by full-field scotopic electroretinography. Results: RRD induced retinal inflammasome activation and photoreceptor death in mice. Systemic administration of K-9, 3TC, or AZT inhibited retinal inflammasome activation and photoreceptor death. In the RRD/SRR model, K-9 protected retinal electrical function during the time of RRD and induced an improvement following retinal reattachment. Conclusions: K-9 and NRTIs exhibit anti-inflammatory and neuroprotective activities in experimental RRD. Given its capacity to protect photoreceptor function during the period of RRD and enhance retinal function following reattachment, K-9 shows promise as a retinal neuroprotectant and warrants study in RRD. Further, this novel RRD/SRR model may facilitate experimental evaluation of functional outcomes relevant to RRD.

Reduction of human Alzheimer's disease risk and reversal of mouse model cognitive deficit with nucleoside analog use.

Innate immune signaling through the NLRP3 inflammasome has been implicated in the pathogenesis of Alzheimer's disease (AD), the most prevalent form of dementia. We previously demonstrated that nucleoside reverse transcriptase inhibitors (NRTIs), drugs approved to treat HIV and hepatitis B infections, also inhibit inflammasome activation. Here we report that in humans, NRTI exposure was associated with a significantly lower incidence of AD in two of the largest health insurance databases in the United States. Treatment of aged 5xFAD mice (a mouse model of amyloid-ß deposition that expresses five mutations found in familial AD) with Kamuvudine-9 (K-9), an NRTI-derivative with enhanced safety profile, reduced Aß deposition and reversed their cognitive deficit by improving their spatial memory and learning performance to that of young wild-type mice. These findings support the concept that inflammasome inhibition could benefit AD and provide a rationale for prospective clinical testing of NRTIs or K-9 in AD.

The Dean Effect: An Aortic Arch Flow Artifact Mimicking Dissection.

The unique hemodynamics of the aortic arch create conditions for potential formation of a flow-related artifact that mimics disease on CT angiographic images. The hemodynamic basis for this artifact can be explained by fluid mechanics incorporating a mathematical principle known as the Dean number. Therefore, in this review, the artifact is referred to as the Dean effect. It is important for radiologists and other clinicians to recognize this artifact when encountered. It is also helpful for the interpreting radiologist to have a basic understanding of the relevant hemodynamic principles. This review provides an example of the artifact, reviews the basic underlying hemodynamics, and presents methods of how to prevent this artifact and distinguish it from pathologic mimics in clinical practice. Keywords: CT Angiography, Vascular, Thorax, Aorta, Artifacts, Blood, Dissection, Hemodynamics/Flow Dynamics © RSNA, 2022.

Subretinal injection in mice to study retinal physiology and disease.

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a 'successful' SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with 'procedure success' defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7-14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. Conclusions: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain­containing protein 3, and apoptosis-associated speck-like protein­containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA­driven diseases.

A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD+; meanwhile, restoring NAD+ levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN (Crassostrea virginica and Nematostella vectensis), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

TMEM97 ablation aggravates oxidant-induced retinal degeneration.

The retinal pigment epithelium (RPE) is critical to the survival of the overlying photoreceptors. Subject to light exposure and active metabolism, the RPE and photoreceptors are particularly susceptible to oxidative damage that plays an important part in age-related macular degeneration (AMD). Recent meta-analyses identified TMEM97 as a new putative AMD risk locus, though it is yet to be functionally verified. The role of TMEM97 in the retina and RPE is not known. Here we investigated TMEM97 function using the sodium iodate model of oxidant-induced retinal degeneration in TMEM97 knockout (KO) mice. We found markedly increased reactive oxygen species (ROS) and loss of photoreceptos in TMEM97 KO mouse retinas relative to wild type (WT) controls. In vitro, sodium iodate treatment of CRISPR-mediated TMEM97 KO RPE cells resulted in diminished abundance of the master antioxidant transcription factor NRF2 and its target gene product SOD2, the mitochondrial superoxide dismutase, as well as elevated ROS and apoptosis markers. Moreover, TMEM97 KO affected proteins key to mitochondrial and lysosomal stability and impeded autophagy flux. These findings suggest that the absence of TMEM97 in RPE cells disturbs redox-balancing systems, thereby heightening oxidative stress. As TMEM97 is a druggable target, this study may inspire interest in basic and translational research in the context of retinal degeneration.

Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-ß induced retinal pigmented epithelium degeneration.

Nonfibrillar amyloid-ß oligomers (AßOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AßOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AßOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AßO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors-nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines-both inhibit AßOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

Repurposing anti-inflammasome NRTIs for improving insulin sensitivity and reducing type 2 diabetes development.

Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P < 0.0001; 95% prediction interval, 0.618 to 0.734). Meanwhile, an NRTI, lamivudine, improves insulin sensitivity and reduces inflammasome activation in diabetic and insulin resistance-induced human cells, as well as in mice fed with high-fat chow; mechanistically, inflammasome-activating short interspersed nuclear element (SINE) transcripts are elevated, whereas SINE-catabolizing DICER1 is reduced, in diabetic cells and mice. These data suggest the possibility of repurposing an approved class of drugs for prevention of diabetes.