United States

Background
Diabetic retinopathy (DR) is one of the leading complications of diabetes mellitus (DM) and is a progressive neovascular disease that causes pathological changes in neuronal, vascular, and immune systems. Inflammation and inflammasome activation have been associated with multiple neurovascular conditions and are implicated in the pathogenesis of diabetic retinopathy. NLRP3 is a well-studied inflammasome made up of the constituents NLRP3, Asc, and Caspase-1. This inflammasome has been demonstrated to be activated by a multitude of danger signals including elevated glucose and free fatty acids, which are present in diabetes mellitus. However, there remains uncertainty regarding the role of the NLRP3 inflammasome in DR development.

Methods
Diabetes was induced using the streptozotocin (STZ) model in C57BL/6J wild type (WT) male mice (N=40) and knock out (KO) male mice lacking certain NLRP3 inflammasome constituents (Caspase-1 KO (N=7) and ASC KO (N=9)). Vehicle control was injected for control (CTL) mice of each genotype. After one and three months of hyperglycemia, retinal neurodegeneration was evaluated using scotopic electroretinography (ERG) and bilateral eyes were harvested for immunoblotting to evaluate the neuroretina for inflammasome activity. 

Results
Immunoblotting of whole retinal lysates showed minimal increase of inflammasome activation in WT DM mice compared to WT CTL mice. However, on ERG evaluation at three months, there was a 23.1% decrease in a-wave and a 32.3% decrease in b-wave between DM and CTL WT mice. For both strains of KO mice, there was a lesser decrease of 10.8% and 21.8% in a- and b-waves respectively (Caspase-1 KO), and of 5% and 18.3% in a- and b-waves respectively (ASC KO) between DM and CTL mice.

Conclusion
Our results suggest that while NLRP3 inflammasome activation may not be evident on protein analysis, the decrease in readings on ERG evaluation may be indicative of a reduction in neuroretinal function by three months of DR. Additionally, the reduced loss of electrical function in both Caspase-1 KO and ASC KO mice may represent a partial rescue of neuroretinal function when components of the NLRP3 inflammasome are absent. This finding helps to establish a direct connection between NLRP3 inflammasome constituents to the pathological processes of DR. 


AMA Research Challenge 2022

NLRP3 inflammasome activation in diabetic retinopathy affects neuroretinal function.

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Charles Lopresto, DO

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poster

Background
Sig1R is a pluripotent modulator of cell survival, the activation of which by the high-affinity, high-specificity ligand pentazocine ((+)-PTZ), has marked retinal neuroprotective effects in the RhoP23H/+ model of adRP compared to non-treated mutants. These findings were obtained in mice administered (+)-PTZ beginning at postnatal day 14 (P14); whether prenatal administration of (+)-PTZ would afford retinal protection was not known and was investigated in the current study. 

Methods
RhoP23H/+ mice were administered (+)-PTZ 3X/week beginning at either (a) P14 (“post”-RhoP23H/+) or (b) RhoP23H/+ mouse dams received (+)-PTZ during adolescence, throughout pregnancy, and pups were treated beginning at P14 (“pre”-RhoP23H/+). Pups from both groups were administered (+)-PTZ through P180 at which time rod and cone photoreceptor function was evaluated by scotopic and photopic electroretinography (ERG), visual acuity by OptoMotry (OMR), in vivo retinal architecture in dorsal and ventral retina by optical coherence tomography (OCT). Retinal structure was examined post-mortem by light microscopy; the thickness of the outer nuclear layer (ONL) of rod/cone nuclei was quantified. Data were analyzed by 2-way ANOVA.

Results
ERG: Scotopic and photopic highest flash intensity b-wave amplitudes were significantly impaired in pre-RhoP23H/+ mice (123.9±13.7μV; 44.3±5.5μV, respectively) at P180 compared to age-matched post-RhoP23H/+ mice (203.4±12.4μV; 70.9±4.5μV) p <0.0001 and 0.0003, respectively. OMR: Acuity in pre-RhoP23H/+ mice was significantly reduced (0.344±0.013 c/d) compared to post-RhoP23H/+ (0.387±0.003c/d) p <0.0001. OCT: ONL thickness was significantly greater in pre-RhoP23H/+ (15.56±0.29μm) versus post-RhoP23H/+(14.3±0.22μm) along the dorsal retina (p = 0.0155). Microscopy: ONL thickness along the dorsal retina in pre-RhoP23H/+ was significantly less than in post-RhoP23H/+ (24.37±3.19 v. 35.15±1.52μm) (p <0.0001).

Conclusion
We anticipated that prenatal plus postnatal treatment with Sig1R would yield a more beneficial response than postnatal treatment alone. However, in our study design, mice in the prenatal Sig1R activation cohort had diminished ERG responses, reduced visual acuity and decreased ONL thickness by histologic examination, although OCT revealed thicker ONL in the dorsal retina. This pilot study raises compelling questions about pre- versus post-natal Sig1R activation and its effects on retinal development. Sig1R is gaining attention in the field of neurodegenerative diseases requiring careful analysis of optimal administration of its ligands.

Prenatal activation of Sigma 1 Receptor (Sig1R) yields intriguing consequences on photoreceptor (PRC) function in a murine model of autosomal dominant Retinitis Pigmentosa (adRP)

Background
Diabetic retinopathy (DR) is a serious diabetes complication that affects millions worldwide. Current therapies are invasive and only slow late-stage progression, thus the development of less invasive, novel therapies that attenuate progression are paramount. In the diabetic milieu, inducible nitric oxide synthase (iNOS) expression is upregulated in retinal macrophages/microglia, which increases production of inflammatory cytokines and reactive oxygen species, worsening ischemia, blood-retinal-barrier (BRB) compromise, and disease progression. Arginase 1 (Arg1) is a ureohydrolase that competes with iNOS for their common substrate, L-arginine. Recent studies revealed a neuroprotective role of Arg1 in murine models of retinal ischemia/reperfusion injury and ischemic stroke and demonstrated the beneficial effects of Arg1 administration in these models. As DR also involves ischemia and inflammation, we hypothesized that exogenous Arg1 would be similarly beneficial through iNOS substrate depletion, resulting in decreased retinal inflammation and oxidative stress.

Methods
Using the obese, type 2 diabetic db/db mouse model that well-approximates human DR pathophysiology, the visual function, BRB, retinal inflammation, and oxidative stress were characterized in these mice aged 16-24-weeks and compared to lean, littermate controls. Visual function was determined by electroretinography (ERG) responses, visual acuity, and contrast threshold measurements. Retinas were collected for immunofluorescence or western blot analyses for iNOS, inflammatory cytokines (IL-1β, TNF-α), oxidative stress (3-nitrotyrosine, 4-hydroxynonenal), and BRB assessment, determined by both retinal albumin extravasation and co-labeling for ZO-1 (tight junction protein) and CD31 (endothelial cell-specific protein). To assess Arg1 therapeutic potential, recombinant Arg1 conjugated to polyethylene glycol (PEG-Arg1), for improved half-life, or a PEG-5000 vehicle control was administered intraperitoneally (25mg/kg) to 16-24-week-old db/db mice thrice weekly for 2 weeks and the same experiments were performed.

Results
Compared to lean littermate controls, db/db mice exhibited significant visual dysfunction, BRB compromise, and elevated retinal levels of iNOS, IL-1β, TNF-α, 3-nitrotyrosine, and 4-hydroxynonenal. PEG-Arg1 treatment significantly ameliorated visual function impairment and BRB compromise in these mice and markedly reduced iNOS levels, retinal inflammation, and oxidative stress compared to vehicle-treated db/db mice. 

Conclusion
The retinal pathology of db/db mice significantly improved with PEG-Arg1 therapy. Though further investigation is necessary, these results suggest that systemic PEG-Arg1 administration has potential as a novel DR therapeutic.

Systemic Administration of Pegylated Arginase-1 Attenuates Progression of Diabetic Retinopathy in the db/db Mouse

Background
Superior Capsule Reconstruction (SCR) allows for the repair of irreparable rotator cuff tears in the younger active patient population. While the use of various graft materials has been described, the choice of using an autograft versus an allograft currently remains unclear. This study aimed to biomechanically investigate SCR with the use of a long head of the biceps tendon (LHBT) autograft and human dermal (HD) allograft in regard to functional abduction force and superior translation of the humeral head. 

Methods
8 fresh-frozen cadaveric shoulder specimens were tested in 5 conditions: (1) intact, (2) complete supraspinatus tear, (3) LHBT autograft, (4) LHBT autograft with side-to-side suturing, and (5) HD allograft with side-to-side suturing. Functional abduction force, superior translation of the humeral head, translational range of motion, and rotational range of motion were tested at 0º, 30º, 60º, and 90º of abduction within each condition. Data was analyzed using analysis of variance with post-hoc Tukey testing for pairwise comparison, with a significance value set at 0.05.

Results
Functional abduction force in the LHBT, LHBT+ suture, and HD+ suture conditions was significantly increased compared to the supraspinatus tear condition at abduction angles of 30º (p=0.011, 0.001, and 0.017 respectively), 60º (p=0.004, 0.001, and 0.002 respectively), and 90º (p=0.013, 0.001, and 0.038 respectively). Additionally, superior translation of the humeral head in the LHBT, LHBT+ suture, and HD+ suture conditions was significantly decreased compared to the tear condition at abduction angles of 30º (p=0.03, 0.049, 0.03 respectively) and 60º (p=0.02, 0.04, 0.03 respectively). All 3 reconstructive techniques were statistically identical to the intact rotator cuff condition in regard to translational and rotational range of motion.

Conclusion
SCR with LHBT autograft without side-to-side suturing, LHBT with posterior side-to-side suturing, and HD allograft with posterior side-to-side suturing all equivalently restore functional abduction force and decrease superior translation of the humeral head after a complete supraspinatus tear. Additionally, all three reconstructive techniques display no deficits in both translational and rotational range of motion, resembling the intact rotator cuff state.


Orthopaedic Surgery - Biceps Autograft versus Dermal Allograft for Superior Capsule Reconstruction after Rotator Cuff Tear - Orthopaedic Surgery

Orthopaedic Surgery - Changes to Excursion of Rotator Cuff Muscles during Abduction after Superior Capsular Reconstruction (SCR) and Reverse Total Shoulder Arthroplasty (rTSA) - Orthopaedic Surgery

Background
Volumetric muscle loss (VML) is a traumatic or surgical orthopaedic injury characterized by a significant loss of skeletal muscle mass and function. Clinically there is no treatment that targets skeletal muscle after VML, and lifelong functional impairments lead to musculoskeletal deformity and comorbidities. While mechanisms driving chronic lack of regeneration remain unclear, pathologic fibrotic deposition and loss of myogenic signaling after injury are thought to be involved. One near-term solution to improve function following VML is to repurpose FDA-approved drugs. This study’s objective was to evaluate effectiveness of an anti-fibrotic, nintedanib, and a hypertrophy stimulant, formoterol combined with leucine, to improve muscle function and regeneration after VML. Muscle function after VML is the key clinical outcome; evaluation of torque thus serves as the main measure of success.

Methods
Adult (12-week-old) male C57BL/6 mice (n=29) underwent unilateral VML surgery removing ~15% of the posterior muscle compartment (gastrocnemius, soleus, plantaris) or remained injury-naïve controls. VML-injured mice were randomized to nintedanib (6mg/kg/day), formoterol (3mg/kg/day) and leucine (24g/kg/day), or no treatment. Six weeks post-VML activity and whole-body metabolism were evaluated. Maximal in vivo muscle torque was evaluated 8 weeks post-VML, and tissue was collected terminally. Data were evaluated using a one-way ANOVA and Tukey post-hoc, with significance at p<0.05.

Results
Twenty-four-hour metabolic rate was ~17% and 8% higher in the formoterol+leucine group (16.5kcal/hour) compared to injury naïve and VML alone, respectively (p<0.001); despite no ambulation or respiratory exchange ratio differences (p≥0.069). However, formoterol+leucine treatment increased lipid oxidation (p=0.005). After 8-weeks, gastrocnemius mass:body mass was reduced for VML alone, but both treatments ameliorated this difference (p=0.004). Maximal in vivo torque was greatest in injury naïve mice (609.6mN•m/kg) and ~41% and 49% lower in VML untreated and nintedanib groups, respectively, while formoterol+leucine mitigated this decline (p=0.012).

Conclusion
Understanding the effects of repurposed FDA-approved medications on functional recovery after VML can establish translational options. Formoterol+leucine treatment improved torque of the posterior muscle compartment and muscle mass to a level not statistically different from injury-naïve mice, supporting its role in promoting functional recovery after VML. This strategy warrants further research to consider applications for VML-injured patients.

Orthopaedic Surgery - Drug Therapies after Volumetric Muscle Loss Injury: Recovery of Function and Muscle Mass with Formoterol - Orthopaedic Surgery

Orthopaedic Surgery - Effects of different 3D-printed titanium surfaces on cell behaviors in vitro - Orthopaedic Surgery

Orthopaedic Surgery - Fibro-adipogenic Progenitors (FAPs) in Heart, Diaphragm and Muscle in a Novel PDGFRa-reporter Duchenne’s Muscular Dystrophy (DMD) Mouse Model - Orthopaedic Surgery

Background:
There is a strong desire in orthopaedics to have soft tissue, particularly tendon, grow into metallic implants. Extensor mechanism and abductor reconstructions in total joint arthroplasty are problematic, and current methods for repair are associated with substantial complications and failure rates. With the introduction of three-dimensional (3D) printed porous metal implants, we hypothesized that tendons could directly attach to the implants. However, the effects of the porous metal structure on tissue growth and penetration into the pores are unknown. In order to improve outcomes after tendon injury, a better understanding of the optimal conditions to promote healing and prevent fibrosis is needed. Using a rat model, we investigated the effect of pore size on tendon repair fixation using 3D printed titanium implants.

Methods:
There were three experimental groups of eight Sprague Dawley rats (n = 24) plus control (n = 3). Implants had defined pore sizes of 400μm (n = 8), 700μm (n = 8), and 1000μm (n = 8). A defect was created in the Achilles tendon and the implant positioned between cut ends and secured with suture. Specimens were harvested at twelve weeks. Half the specimens underwent mechanical testing to assess tensile load to failure. The remaining specimens were fixed and processed for hard tissue histological analysis.

Results:
The average load to failure was 72.6N for controls (SD 10.04), 29.95N for 400μm (SD 17.95), 55.08N for 700μm (SD 13.47), and 63.08N for 1000μm (SD 1.87). The load to failure was generally better in the larger pore sizes. The 700m and 1000m specimens performed similarly, while the 400m showed significant differences vs control (p = 0.039), vs 1000μm (p = 0.010), and approached significance vs 700μm (p = 0.066). There was increasing ingrowth as pore sized increased. Histology showed fibrous tendon tissue within and around the implants, with collagen fibers organized in bundles.

Conclusion:
Tendon repair utilizing implants with 700μm and 1000μm pores exhibited similar load to failure as controls. Using a defined pore structure at the attachment points of tendons to implants may allow predictable tendon ingrowth onto/into an implant at the time of revision arthroplasty.

Orthopaedic Surgery - Influence of Porosities of Titanium Implants on Tensile Properties in a Rat Tendon Repair Model - Orthopaedic Surgery

Background
Postoperative complications are a concern for surgeons due to worse outcomes and increased cost. It has been shown that social determinants of health disparities (SDHD) are associated with higher rates of postoperative complications. Understanding the role SDHD plays in patient’s health can help prepare providers to optimize patients’ conditions prior to surgery and improve postoperative care. The purpose of this study was to compare the postoperative complication rates between patients undergoing total shoulder arthroplasty (TSA) with a past medical history of SDHD with a control group without SDHD diagnoses.
Methods
A retrospective cohort analysis was conducted using a national insurance claims database. Using International Classification of Diseases (ICD) and Current Procedural Terminology (CPT) codes patients who underwent primary TSA with at least two years of follow-up in the database were identified. The SDHD and control groups were matched and analyzed using multivariable analysis for 90-day medical complications and 2-year surgical complications.
Results
A total of 120,367 patients underwent TSA between 2010-2019: 110,822 patients in control group and 9,394 in SDHD group. SDHD group had significantly longer length of stay (mean [SD]; Control = 1.99 days [1.39] vs SDHD = 3.84 days [18.32]; p<0.001) along with significantly higher odds ratios for 90-day medical complications including heart failure (OR: 3.71; p<0.001), cerebrovascular accident (OR: 2.48; p<0.001), renal failure (OR: 2.91; p<0.001), death (OR: 3.32; p<0.001), deep vein thrombosis (OR: 3.16; p<0.001), pulmonary embolism (OR: 1.95; p<0.001), pneumonia (OR: 2.84; p<0.001), sepsis (OR: 4.94; p<0.001), and urinary tract infection (OR: 2.60; p<0.001). Additionally, the SDHD group had a significantly higher odds ratio for revision surgery within 2-years following TSA (OR: 1.45; p<0.001).
Conclusion
This study highlights the negative impact of SDHD on patient outcomes following TSA including a higher rate of postoperative complications within 90-days and revision surgery within 2-years. Additionally, patients with a history of SDHD had a significantly longer length of hospital stay than patients without SDHD. Quantifying the risk of complications and differences in length of stay for TSA patients with a history of SDHD are important in determining value-based payment models and risk stratification to optimize patient care.

Orthopaedic Surgery - Social Determinants of Health Disparities Impact Postoperative Complications in Patients Undergoing Total Shoulder Arthroplasty - Orthopaedic Surgery

Background
Neurofibromatosis type 2 (NF2) is an autosomal dominant tumor predisposition syndrome resulting from mutations in the NF2 gene. NF2 patients are prone to developing schwannomas, which are benign tumors originating from Schwann cells of the peripheral nervous system. Though the hallmark of NF2 is the development of bilateral vestibular schwannomas, schwannomas can occur on any cranial or peripheral nerve. Facial nerve schwannoma (FNS) is the most common primary neoplasm of the facial nerve, although it is uncommon overall. Due to the rarity of FNS, animal models are imperative for therapeutic testing of pharmaceutical compounds. The Postn-Cre;Nf2flox/flox mouse model is known to develop cranial nerve schwannomas, including FNS. However, the nature of these tumors has not been robustly characterized.

Methods
Postn-Cre;Nf2flox/flox and control littermates were euthanized at 12 months of age. A novel method was employed for dissection of the intratemporal facial nerve. Whole heads or isolated facial nerves were formalin-fixed and paraffin embedded. Sections were stained by hematoxylin and eosin or immunohistochemistry. Schwann cell density and tumorlets were quantified in geniculate ganglia and peri-geniculate facial nerves. Comparative statistical analysis was performed using Student’s t-test (GraphPad Prism).

Results
In this study, we report the first thorough histopathological analysis of FNS development in the Postn-Cre;Nf2flox/flox mouse model. Schwann cell density is increased in mutant facial nerves and geniculate ganglia compared to control (p<0.001). Tumorlets are present only in Postn- Cre;Nf2flox/flox mutant mouse ganglia (p<0.01). Neuron density is decreased in mutant geniculate ganglia (p<0.001). Merlin, S100, SOX10, GFAP, FABP7, Neurofilament, and Ki-67 immunostaining confirmed a Schwann cell phenotype.

Conclusion
The Postn-Cre;Nf2flox/flox mouse model demonstrates consistent and predictable development of facial nerve schwannoma. While this model has been described to develop cranial nerve schwannomas, our study establishes the first quantifiable readout of tumor burden through measurement of Schwann cell and tumorlet density. These findings provide a novel endpoint for preclinical testing of targeted therapies for neurofibromatosis type 2-associated cranial nerve schwannomas in the Postn-Cre;Nf2flox/flox mouse model.

Next from AMA Research Challenge 2022

Prenatal activation of Sigma 1 Receptor (Sig1R) yields intriguing consequences on photoreceptor (PRC) function in a murine model of autosomal dominant Retinitis Pigmentosa (adRP)

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