Superior Capsule Reconstruction With Fascia Lata Allograft Has Initial Stiffness and Ultimate Load Comparable to the Native Shoulder Superior Capsule: A Cadaveric Biomechanical Study.

PURPOSE: The purpose of this study was to compare the biomechanical characteristics of a fascia lata superior capsule reconstruction (FL-SCR) to the native superior capsule. METHODS: The native superior capsule of 8 cadaveric shoulders was tested with cyclic loading from 10 to 50 N for 30 cycles in 20° of glenohumeral abduction followed by load to failure at 60 mm/min. Following native superior capsule testing, FL-SCR was performed, which was tested as described for the native capsule. Paired t test was used for statistical analyses with P < .05 for significance. RESULTS: The stiffness for cycle 1 to 50 N was significantly higher for the native superior capsule compared to the FL-SCR (P = .001). By cycle 30, the stiffness between the two was not statistically different (P = .734). During load to failure, the initial stiffness to 2 mm for the FL-SCR and the native superior capsule was not statistically different (P = .262). The linear stiffness and yield load of the native superior capsule were significantly greater than that of the FL-SCR (94.5 vs 28.0 N/mm, P = .013; 386.9 vs 123.8 N, P = .029). There was no significant difference in ultimate load between the native superior capsule and the FL-SCR (444.9 vs 369.0 N, P = .413). CONCLUSIONS: FL-SCR has initial stiffness and ultimate load similar to the native superior capsule. CLINICAL RELEVANCE: The biomechanical properties of FL allograft make it an appealing option as a graft choice for superior capsule reconstruction.

Anterior Cable Reconstruction of the Superior Capsule Using Semitendinosus Allograft for Large Rotator Cuff Defects Limits Superior Migration and Subacromial Contact Without Inhibiting Range of Motion: A Biomechanical Analysis.

PURPOSE: To biomechanically assess translation, contact pressures, and range of motion for anterior cable reconstruction (ACR) using hamstring allograft for large to massive rotator cuff tears. METHODS: Eight cadaveric shoulders (mean age, 68 years) were tested with a custom testing system. Range of motion (ROM), superior translation of the humeral head, and subacromial contact pressure were measured at 0°, 30°, 60°, and 90° of external rotation (ER) with 0°, 20°, and 40° of glenohumeral abduction. Three conditions were tested: intact, stage III tear (supraspinatus + anterior half of infraspinatus), and stage III tear + allograft ACR (involving 2 supraglenoid anchors for semitendinosus tendon allograft fixation. Allograft ACR included loop-around fixation using 3 side-to-side sutures and an anchor at the articular margin to restore capsular anatomy along the anterior edge of the cuff defect. RESULTS: ACR with allograft for stage III tears showed significantly higher total ROM compared with intact at all angles (P ≤ .028). Augmentation significantly decreased superior translation for stage III tears at 0°, 30°, and 60° ER for both 0° and 20° abduction, and at 0° and 30° ER for 40° abduction (P ≤ .043). Augmentation for stage III tears significantly reduced overall subacromial contact pressure at 30° ER with 0° and 40° abduction, and at 60° ER with 0° and 20° abduction (P ≤ .016). CONCLUSION: Anterior cable reconstruction using cord-like allograft semitendinosus tendon can biomechanically improve superior migration and subacromial contact pressure (primarily in the lower combined abduction and rotation positions), without limiting range of motion for large rotator cuff tendon defects or tears. CLINICAL RELEVANCE: In patients with superior glenohumeral instability, using hamstring allograft for ACR may improve rotator cuff tendon defect longevity by providing basic static ligamentous support to the dynamic tendon while helping to limit superior migration, without restricting glenohumeral kinematics.

The hippocampal extracellular matrix regulates pain and memory after injury.

Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.

Over-expression of astrocytic ET-1 attenuates neuropathic pain by inhibition of ERK1/2 and Akt(s) via activation of ETA receptor.

A differential role of endothelin-1 (ET-1) in pain processing has recently been suggested. However, the function of central ET-1 in neuropathic pain (NP) has not been fully elucidated to date. We report here the action of endogenous central ET-1 in sciatic nerve ligation-induced NP (SNL-NP) in a transgenic animal model that over-expresses ET-1 in the astrocytes (GET-1 mice). We hypothesized that the over-expression of astrocytic ET-1 would exert anti-allodynic and anti-hyperalgesic effects in NP, as demonstrated by mechanical threshold and plantar withdrawal latency using the von Frey filament and heat stimuli. In our animal model, GET-1 mice showed an increase in the withdrawal threshold and latency in response to the mechanical and thermal stimuli, respectively, in pain behavior tests after SNL. ET-1 and endothelin type A receptor (ETA-R) levels were increased significantly in L4-L6 segments of the spinal cord (ipsilateral to SNL) of GET-1 mice at 7 and 21days after surgery. Moreover, intrathecal administration of a specific ETA-R antagonist, BQ-123, attenuated the anti-allodynic and anti-hyperalgesic phenotype in GET-1 mice. The effects of BQ-123 on the mRNA expression of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase B/serine protein kinase (Akt(s)) were assessed in the ipsilateral L4-L6 segments harvested 30min after BQ-123 administration on day 7 after surgery. Phosphorylation of ERK1/2 and Akt(s) in the ipsilateral spinal cord of GET-1 mice was reduced following SNL, whereas no reduction was observed after intrathecal injection of BQ-123. In conclusion, our results showed that the xover-expression of astrocytic ET-1 reduced SNL-induced allodynia and hyperalgesia by inhibiting the activation of ERK1/2 and Akt(s) via the ETA-R-mediated pathway.

Propofol ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction via heme oxygenase-1/signal transducer and activator of transcription 3 signaling pathway in rats.

OBJECTIVES: Heme oxygenase-1 is inducible in cardiomyocytes in response to stimuli such as oxidative stress and plays critical roles in combating cardiac hypertrophy and injury. Signal transducer and activator of transcription 3 plays a pivotal role in heme oxygenase-1-mediated protection against liver and lung injuries under oxidative stress. We hypothesized that propofol, an anesthetic with antioxidant capacity, may attenuate hyperglycemia-induced oxidative stress in cardiomyocytes via enhancing heme oxygenase-1 activation and ameliorate hyperglycemia-induced cardiac hypertrophy and apoptosis via heme oxygenase-1/signal transducer and activator of transcription 3 signaling and improve cardiac function in diabetes. DESIGN: Treatment study. SETTING: Research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: In vivo and in vitro treatments. MEASUREMENTS AND MAIN RESULTS: At 8 weeks of streptozotocin-induced type 1 diabetes in rats, myocardial 15-F2t-isoprostane was significantly increased, accompanied by cardiomyocyte hypertrophy and apoptosis and impaired left ventricular function that was coincident with reduced heme oxygenase-1 activity and signal transducer and activator of transcription 3 activation despite an increase in heme oxygenase-1 protein expression as compared to control. Propofol infusion (900 µg/kg/min) for 45 minutes significantly improved cardiac function with concomitantly enhanced heme oxygenase-1 activity and signal transducer and activator of transcription activation. Similar to the changes seen in diabetic rat hearts, high glucose (25 mmol/L) exposure for 48 hours led to cardiomyocyte hypertrophy and apoptosis, both in primary cultured neonatal rat cardiomyocytes and in H9c2 cells compared to normal glucose (5.5 mmol/L). Hypertrophy was accompanied by increased reactive oxygen species and malondialdehyde production and caspase-3 activity. Propofol, similar to the heme oxygenase-1 inducer cobalt protoporphyrin, significantly increased cardiomyocyte heme oxygenase-1 and p-signal transducer and activator of transcription protein expression and heme oxygenase-1 activity and attenuated high-glucose-mediated cardiomyocyte hypertrophy and apoptosis and reduced reactive oxygen species and malondialdehyde production (p < 0.05). These protective effects of propofol were abolished by heme oxygenase-1 inhibition with zinc protoporphyrin and by heme oxygenase-1 or signal transducer and activator of transcription 3 gene knockdown. CONCLUSIONS: Heme oxygenase-1/signal transducer and activator of transcription 3 signaling plays a critical role in propofol-mediated amelioration of hyperglycemia-induced cardiomyocyte hypertrophy and apoptosis, whereby propofol improves cardiac function in diabetic rats.

Evaluation of Tibiofemoral Contact Mechanics After a Novel Hybrid Procedure for Femoral Osteochondral Defect Repairs With a Subchondral Implant and Dermal Matrix.

Background: There is a lack of procedures that adequately address the subchondral bone structure and function for reconstructing osteochondral defects in the femoral condyles. Purpose: To biomechanically evaluate the tibiofemoral joint contact characteristics before and after reconstruction of femoral condylar osteochondral defects using a novel hybrid reconstructive procedure, which was hypothesized to restore the contact characteristics to the intact condition. Study Design: Controlled laboratory study. Methods: Tibiofemoral contact areas, contact forces, and mean contact pressures were measured in 8 cadaveric knees (mean age 52 ± 11 years; 6 women, 2 men) using a custom testing system and pressure mapping sensors. Five conditions were tested for each condyle: intact, 8-mm defect, 8-mm repair, 10-mm defect, and 10-mm repair. Medial femoral condylar defects were evaluated at 30° of knee flexion and lateral condylar defects were evaluated at 60° of knee flexion, with compressive loads of 50, 100, and 150 N. The defects were reconstructed with a titanium fenestrated threaded implant countersunk in the subchondral bone and an acellular dermal matrix allograft. Repeated-measures analysis of variance with Bonferroni correction for multiple comparisons was used to compare the results between the 5 testing conditions at each load. Results: Medial condylar defects significantly increased mean contact pressure on the lateral side (P < .042), which was restored to the intact levels with repair. The lateral condylar defect decreased the mean contact pressure laterally while increasing the mean pressure medially. The lateral and medial mean contact pressures were restored to intact levels with the 8-mm lateral condylar defect repair. The medial mean contact pressure was restored to intact levels with the 10-mm lateral condylar defect repair. The lateral mean contact pressure decreased compared with the intact state with the lateral condylar 10-mm defect repair. Conclusion: Tibiofemoral joint contact pressure was restored to the intact condition after reconstruction of osteochondral defects with dermal allograft matrix and subchondral implants for the repair of both 8- and 10-mm lateral condylar defects as well as 8-mm medial condylar defects but not completely for 10-mm medial condylar defects. Clinical Relevance: The novel hybrid procedure for osteochondral defect repair restored tibiofemoral joint contact characteristics to normal in a cadaveric model.

Does Reusable Instrumentation for Four-Anchor Rotator Cuff Repair Offer Decreased Waste Disposal Costs and Lower Waste-Related Carbon Emissions?

INTRODUCTION: Orthopaedic surgery is culpable, in part, for the excessive carbon emissions in health care partly due to the utilization of disposable instrumentation in most procedures, such as rotator cuff repair (RCR). To address growing concerns about hospital waste, some have considered replacing disposable instrumentation with reusable instrumentation. The purpose of this study was to estimate the cost and carbon footprint of waste disposal of RCR kits that use disposable instrumentation compared with reusable instrumentation. METHODS: The mass of the necessary materials and their packaging to complete a four-anchor RCR from four medical device companies that use disposable instrumentation and one that uses reusable instrumentation were recorded. Using the cost of medical waste disposal at our institution ($0.14 per kilogram) and reported values from the literature for carbon emissions produced from the low-temperature incineration of noninfectious waste (249 kgCO 2 e/t) and infectious waste (569 kgCO 2 e/t), we estimated the waste management cost and carbon footprint of waste disposal produced per RCR kit. RESULTS: The disposable systems of four commercial medical device companies had 783%, 570%, 1,051%, and 478%, respectively, greater mass and waste costs when compared with the reusable system. The cost of waste disposal for the reusable instrumentation system costs on average $0.14 less than the disposable instrumentation systems. The estimated contribution to the overall carbon footprint produced from the disposal of a RCR kit that uses reusable instrumentation was on average 0.37 kg CO2e less than the disposable instrumentation systems. CONCLUSION: According to our analysis, reusable instrumentation in four-anchor RCR leads to decreased waste and waste disposal costs and lower carbon emissions from waste disposal. Additional research should be done to assess the net benefit reusable systems may have on hospitals and the effect this may have on a long-term decrease in carbon footprint. LEVEL OF EVIDENCE: Level II.

Use of allograft bone matrix in clinical orthopedics.

Clinical orthopedics continuously aims to improve methods for bone formation. Clinical applications where bone formation is necessary include critical long bone defects in orthopedic trauma or tumor patients. Though some biomaterials combined with autologous stem cells significantly improve bone repair, critical-size damages are still challenged with the suitable implantation of biomaterials and donor cell survival. Extracellular matrix (ECM) is the fundamental structure in tissues that can nest and nourish resident cells as well as support specific functions of the tissue type. ECM also plays a role in cell signaling to promote bone growth, healing and turnover. In the last decade, the use of bone-derived ECMs or ECM-similar biomaterials have been widely investigated, including decellularized and demineralized bone ECM. In this article, we reviewed the current productions and applications of decellularized and demineralized bone matrices. We also introduce the current study of whole limb decellularization and recellularization.

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The advances of blood clots used as biomaterials in regenerative medicine.

The physiologic process of blood clot formation is well understood and occurs naturally in the setting of tissue injury to achieve hemostasis and begin the process of wound healing. While the investigation of blood clots as a biomaterial is still in the early stages, there has been some research with similar biomaterials made of the components of blood clots that support the innovative idea of using an autologous blood clot as a scaffold or delivery method for therapeutic agents. Here, we review the physiology of blood clots in wound healing and how using blood clots as a biomaterial and delivery system can potentially promote wound healing, provide targeted therapeutic agent delivery and use it as an innovative tool in regenerative medicine.

Wounds and tissue injuries are unfortunately still common, and research is very active in trying to discover ways to augment the healing process. Various biomaterials have been developed and studied to analyze their therapeutic potential in promoting regeneration of different tissues in the human body. The formation of blood clots is a natural process that occurs in response to tissue injury, and there has been recent work to investigate the utility of blood clots as therapeutic adjuncts. The practicality of obtaining blood clots, along with the feasibility of integrating therapeutic agents into them, make blood clots an attractive tool in regenerative medicine to be explored. This review article synthesizes current research on blood clot biology, physiology and potential therapeutic utility.

Radiocapitellar Contact Characteristics After Osteochondral Defect Repair Using a Novel Hybrid Reconstructive Procedure.

Background: Many procedures to reconstruct osteochondral defects of the elbow radiocapitellar (RC) joint lack versatility or durability or do not directly address the subchondral bone structure and function. Purpose/Hypothesis: To biomechanically characterize the RC joint contact area, force, pressure, and peak pressure before and after reconstruction of osteochondral defects using a novel hybrid reconstructive procedure. It was hypothesized that the procedure would restore the contact characteristics to the intact condition. Study Design: Controlled laboratory study. Methods: A total of 10 cadaveric elbows (mean age 67 ± 2.7 years) were dissected to isolate the humerus and radial head. RC contact area, contact force, mean contact pressure, and peak contact pressure were measured with the elbow at 45° of flexion and neutral forearm rotation at compressive loads of 25, 50, and 75 N. Osteochondral defects 8 and 11 mm in diameter were created at the center of the capitellum; the defects were then reconstructed with a titanium fenestrated threaded implant, countersunk in the subchondral bone, with an acellular dermal matrix allograft sutured in place on top of the implant. Five conditions (intact, 8-mm defect, 8-mm repair, 11-mm defect, and 11-mm repair) were tested and results were compared using repeated-measures analysis of variance. Results: Both 8- and 11-mm defects significantly increased RC mean contact pressure at all compressive loads (P ≤ .008) and significantly increased peak contact pressure at compressive loads of 50 and 75 N (P < .002) compared with the intact condition. Repair of the 8-mm defect significantly decreased RC mean contact pressure at 25- and 50-N loads (P ≤ .009) and significantly decreased peak contact pressure at 50- and 75-N loads (P ≤ .035) compared with the defect condition. Repair of the 11-mm defect decreased mean contact pressure significantly at all compressive loads (P ≤ .001) and peak contact pressure at 50- and 75-N loads (P < .044) compared with the defect condition. Conclusion: RC joint contact pressure was restored to intact conditions while avoiding increased peak contact pressure or edge loading after repairing osteochondral defects related to osteochondrosis with a novel hybrid reconstruction technique. Clinical Relevance: This hybrid procedure that addresses the entire osteochondral unit may provide a new treatment option for osteochondral defects.

Exacerbated VEGF up-regulation accompanies diabetes-aggravated hemorrhage in mice after experimental cerebral ischemia and delayed reperfusion.

Reperfusion therapy is the preferred treatment for ischemic stroke, but is hindered by its short treatment window, especially in patients with diabetes whose reperfusion after prolonged ischemia is often accompanied by exacerbated hemorrhage. The mechanisms underlying exacerbated hemorrhage are not fully understood. This study aimed to identify this mechanism by inducing prolonged 2-hour transient intraluminal middle cerebral artery occlusion in diabetic Ins2Akita/+ mice to mimic patients with diabetes undergoing delayed mechanical thrombectomy. The results showed that at as early as 2 hours after reperfusion, Ins2Akita/+ mice exhibited rapid development of neurological deficits, increased infarct and hemorrhagic transformation, together with exacerbated down-regulation of tight-junction protein ZO-1 and up-regulation of blood-brain barrier-disrupting matrix metallopeptidase 2 and matrix metallopeptidase 9 when compared with normoglycemic Ins2+/+ mice. This indicated that diabetes led to the rapid compromise of vessel integrity immediately after reperfusion, and consequently earlier death and further aggravation of hemorrhagic transformation 22 hours after reperfusion. This observation was associated with earlier and stronger up-regulation of pro-angiogenic vascular endothelial growth factor (VEGF) and its downstream phospho-Erk1/2 at 2 hours after reperfusion, which was suggestive of premature angiogenesis induced by early VEGF up-regulation, resulting in rapid vessel disintegration in diabetic stroke. Endoplasmic reticulum stress-related pro-apoptotic C/EBP homologous protein was overexpressed in challenged Ins2Akita/+ mice, which suggests that the exacerbated VEGF up-regulation may be caused by overwhelming endoplasmic reticulum stress under diabetic conditions. In conclusion, the results mimicked complications in patients with diabetes undergoing delayed mechanical thrombectomy, and diabetes-induced accelerated VEGF up-regulation is likely to underlie exacerbated hemorrhagic transformation. Thus, suppression of the VEGF pathway could be a potential approach to allow reperfusion therapy in patients with diabetic stroke beyond the current treatment window. Experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong [CULATR 3834-15 (approval date January 5, 2016); 3977-16 (approval date April 13, 2016); and 4666-18 (approval date March 29, 2018)].

Effects of augmented somatosensory input using vibratory insoles to improve walking in individuals with chronic post-stroke hemiparesis.

BACKGROUND: Stroke survivors suffer from hemiparesis and somatosensory impairments, which adversely impact walking performance, placing them at higher risks for trips and falls. Post-stroke, somatosensory deficits are commonly observed as impaired interpretation of afferent input and increased threshold. Diminishing or augmenting somatosensory inputs via various techniques have been demonstrated to be able to modify static and dynamic balance, postural and locomotor control in non-neurologically impaired as well as neurologically impaired individuals. RESEARCH QUESTION: We sought to investigate whether enhancing somatosensory input using vibratory insoles, can improve post-stroke gait. We hypothesized that with augmentation of somatosensory input at the soles via vibratory insoles would improve post-stroke gait via increased propulsive forces, decreased braking forces and increased ankle angle movements in the paretic legs of individuals with chronic post-stroke hemiparesis. METHODS: Fifteen individuals with chronic post-stroke hemiparesis and 15 age-similar non-neurologically impaired controls participated in this cross-sectional study. Enhanced somatosensory stimulation was delivered using a pair of tactor-embedded insoles, providing suprathreshold vibratory stimulation to the bottom of the feet. Participants walked over an instrumented treadmill with self-selected speeds, under 5 conditions: no insole in shoe (NT), insoles in shoe with no vibration (BOFF), vibration under both feet (BON), vibration under one foot only (ION, CON). Kinetics and kinematics during walking were recorded and analyzed offline. RESULTS: Suprathreshold vibratory stimulations did not alter gait kinetics under any stimulation conditions. We observed increased paretic ankle dorsiflexions in the paretic legs, when vibratory stimuli were applied unilaterally. SIGNIFICANCE: Vibratory stimulations applied at suprathreshold intensity to the bottom of the feet to augment somatosensory feedback can potentially be used as a low-cost solution to address the inadequate toe clearance during walking in people post-stroke, which is an important goal in post-stroke rehabilitation.

Retinoid X Receptor Activation During Adipogenesis of Female Mesenchymal Stem Cells Programs a Dysfunctional Adipocyte.

Early life exposure to endocrine-disrupting chemicals (EDCs) is an emerging risk factor for the development of obesity and diabetes later in life. We previously showed that prenatal exposure to the EDC tributyltin (TBT) results in increased adiposity in the offspring. These effects linger into adulthood and are propagated through successive generations. TBT activates two nuclear receptors, the peroxisome proliferator-activated receptor (PPAR) γ and its heterodimeric partner retinoid X receptor (RXR), that promote adipogenesis in vivo and in vitro. We recently employed a mesenchymal stem cell (MSC) model to show that TBT promotes adipose lineage commitment by activating RXR, not PPARγ. This led us to consider the functional consequences of PPARγ vs RXR activation in developing adipocytes. We used a transcriptomal approach to characterize genome-wide differences in MSCs differentiated with the PPARγ agonist rosiglitazone (ROSI) or TBT. Pathway analysis suggested functional deficits in TBT-treated cells. We then compared adipocytes differentiated with ROSI, TBT, or a pure RXR agonist IRX4204 (4204). Our data show that RXR activators ("rexinoids," 4204 and TBT) attenuate glucose uptake, blunt expression of the antidiabetic hormone adiponectin, and fail to downregulate proinflammatory and profibrotic transcripts, as does ROSI. Finally, 4204 and TBT treatment results in an inability to induce markers of adipocyte browning, in part due to sustained interferon signaling. Taken together, these data implicate rexinoids in the development of dysfunctional white adipose tissue that could potentially exacerbate obesity and/or diabetes risk in vivo. These data warrant further screening and characterization of EDCs that activate RXR.

Deletion of aldose reductase leads to protection against cerebral ischemic injury.

Previously, we reported that transgenic mice overexpressing endothelin-1 in astrocytes showed more severe neurological deficits and increased infarct after transient focal ischemia. In those studies, we also observed increased level of aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, which has been implicated in osmotic and oxidative stress. To further understand the involvement of the polyol pathway, the mice with deletion of enzymes in the polyol pathway, AR, and sorbitol dehydrogenase (SD), which is the second enzyme in this pathway, were challenged with similar cerebral ischemic injury. Deletion of AR-protected animals from severe neurological deficits and large infarct, whereas similar protection was not observed in mice with SD deficiency. Most interestingly, AR(-/-) brains showed lowered expression of transferrin and transferrin receptor with less iron deposition and nitrotyrosine accumulation. The protection against oxidative stress in AR(-/-) brain was also associated with less poly(adenosine diphosphate-ribose) polymerase (PARP) and caspase-3 activation. Pharmacological inhibition of AR by Fidarestat also protected animals against cerebral ischemic injury. These findings are the first to show that AR contributes to iron- and transferrin-related oxidative stress associated with cerebral ischemic injury, suggesting that inhibition of AR but not SD may have therapeutic potential against cerebral ischemic injury.

Retinoid X Receptor Activation Alters the Chromatin Landscape To Commit Mesenchymal Stem Cells to the Adipose Lineage.

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

Identification of KRT16 as a target of an autoantibody response in complex regional pain syndrome.

OBJECTIVE: Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens in the skin of the affected limb. METHODS: A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets validated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of selected protein targets, we quantified the binding of IgM in control and fracture mice sera, as well as in control and CRPS human sera, to the recombinant protein. RESULTS: We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after fracture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane localization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracture mice and CRPS patients showed increased autoantibody binding to recombinant kRT16 protein. CONCLUSIONS: Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condition and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology.

Longitudinal Assessments of Normal and Perilesional Tissues in Focal Brain Ischemia and Partial Optic Nerve Injury with Manganese-enhanced MRI.

Although manganese (Mn) can enhance brain tissues for improving magnetic resonance imaging (MRI) assessments, the underlying neural mechanisms of Mn detection remain unclear. In this study, we used Mn-enhanced MRI to test the hypothesis that different Mn entry routes and spatiotemporal Mn distributions can reflect different mechanisms of neural circuitry and neurodegeneration in normal and injured brains. Upon systemic administration, exogenous Mn exhibited varying transport rates and continuous redistribution across healthy rodent brain nuclei over a 2-week timeframe, whereas in rodents following photothrombotic cortical injury, transient middle cerebral artery occlusion, or neonatal hypoxic-ischemic brain injury, Mn preferentially accumulated in perilesional tissues expressing gliosis or oxidative stress within days. Intravitreal Mn administration to healthy rodents not only allowed tracing of primary visual pathways, but also enhanced the hippocampus and medial amygdala within a day, whereas partial transection of the optic nerve led to MRI detection of degrading anterograde Mn transport at the primary injury site and the perilesional tissues secondarily over 6 weeks. Taken together, our results indicate the different Mn transport dynamics across widespread projections in normal and diseased brains. Particularly, perilesional brain tissues may attract abnormal Mn accumulation and gradually reduce anterograde Mn transport via specific Mn entry routes.

Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.

Targeted Overexpression of Astrocytic Endothelin-1 Attenuates Neuropathic Pain by Upregulating Spinal Excitatory Amino Acid Transporter-2.

We previously demonstrated that endogenous endothelin-1 (ET-1) inhibits pathological pain in a transgenic mouse model with astrocyte-specific ET-1 overexpression (GET-1 mice); however, the underlying mechanism is unclear. ET-1 regulates excitatory amino acid transporter-2 (EAAT-2), a predominant subtype of glutamate transporters that plays critical role in pain modulation in spinal astrocytes. We hypothesized that astrocytic ET-1 overexpression alleviates neuropathic pain through modulating EAAT-2. GET-1 or nontransgenic (NTg) mice either received sham operation or sciatic nerve ligation (SNL) with or without ceftriaxone (CEF, an EAAT-2 inducer, for 4 days before termination). In GET-1 mice, mRNA and protein expressions of EAAT-2, but not EAAT-1, were upregulated associated with reduced SNL-induced neuropathic pain. Despite that SNL induced a significant reduction of EAAT-2 mRNA expression in both genotypes of mice, post-SNL EAAT-2 mRNA expression was higher in GET-1 mice than that in NTg mice. EAAT-2 induction by CEF reduced SNL-induced neuropathic pain in both NTg and GET-1 mice. In cultured rat astrocytic cell line, overexpression of ET-1 mRNA expression also elevated EAAT-2 mRNA expression, which was reversed by ET receptor antagonists. In conclusion, overexpressed astrocytic ET-1 suppressed neuropathic pain by upregulating spinal EAAT-2 expression via ET receptors.

Selective astrocytic endothelin-1 overexpression contributes to dementia associated with ischemic stroke by exaggerating astrocyte-derived amyloid secretion.

Endothelin-1 (ET-1) is synthesized by endothelial cells and astrocytes in stroke and in brains of Alzheimer's disease patients. Our transgenic mice with ET-1 overexpression in the endothelial cells (TET-1) showed more severe blood-brain barrier (BBB) breakdown, neuronal apoptosis, and glial reactivity after 2-hour transient middle cerebral artery occlusion (tMCAO) with 22-hour reperfusion and more severe cognitive deficits after 30 minutes tMCAO with 5 months reperfusion. However, the role of astrocytic ET-1 in contributing to poststroke cognitive deficits after tMCAO is largely unknown. Therefore, GET-1 mice were challenged with tMCAO to determine its effect on neurologic and cognitive deficit. The GET-1 mice transiently displayed a sensorimotor deficit after reperfusion that recovered shortly, then more severe deficit in spatial learning and memory was observed at 3 months after ischemia compared with that of the controls. Upregulation of TNF-α, cleaved caspase-3, and Thioflavin-S-positive aggregates was observed in the ipsilateral hemispheres of the GET-1 brains as early as 3 days after ischemia. In an in vitro study, ET-1 overexpressing astrocytic cells showed amyloid secretion after hypoxia/ischemia insult, which activated endothelin A (ETA) and endothelin B (ETB) receptors in a PI3K/AKT-dependent manner, suggesting role of astrocytic ET-1 in dementia associated with stroke by astrocyte-derived amyloid production.