Adipose cellular injection in the treatment of an intrasubstance Achilles tendon defect: a case report.

Our patient presented with a 1-year history of right sided Achilles tendon pain and weakness due to partial intrasubstance tear. The injury was refractory to conservative treatment, leading to a trial injection of microfragmented adipose tissue. Progressive healing and improved function were documented on physical exam and sonographically at subsequent follow-up appointments. About 4 weeks following the injection, the patient was able to return to his regular activity level. At the 6 month follow-up appointment, the patient continued to be pain free and had resumed all prior activities without limitations. This case highlights the potential microfragmented adipose tissue has as a regenerative treatment modality for the management of partial Achilles tendon tears.

Thoracic bone mineral density measured by quantitative computed tomography in patients undergoing spine surgery.

BACKGROUND CONTEXT: The thoracic spine is a common location for vertebral fractures as well as instrumentation failure after long spinal fusion procedures. The association between those complications and bone mineral density (BMD) are well recognized. Due to the overlying sternum and ribs in the thoracic spine, projectional BMD assessment tools such as dual energy x-ray absorptiometry (DXA) are limited to the lumbar spine. Quantitative computed tomography circumvents several shortcomings of DXA and allows for level-specific BMD measurements. Studies comprehensively quantifying BMD of the entire thoracic spine in patients undergoing spine surgery are limited. PURPOSE: The objective of this study was: (1) to assess the reliability of thoracic QCT measurements, (2) to determine possible level-specific BMD variation throughout the thoracic spine and (3) to assess the correlation between BMDs of the T1-T12 spinal levels. STUDY DESIGN/SETTING: Cross-sectional observation study. PATIENT SAMPLE: Patients undergoing spine surgery from 2016-2020 at a single, academic institution with available preoperative CT imaging of the thoracic spine were included in this study. OUTCOME MEASURES: The outcome measure was BMD measured by QCT. METHODS: Patients undergoing spine surgery from 2016-2020 at a single, academic institution with available preoperative CT imaging of the thoracic spine were included in this study. Subjects with previous instrumentation at any thoracic level, concurrent vertebral fractures, a Cobb angle of more than 20 degrees, or incomplete thoracic spine CT imaging were excluded. Asynchronous quantitative computed tomography (QCT) measurements of T1-T12 were performed. To assess inter- and intra-observer reliability, a validation study was performed on 120 vertebrae in 10 randomly selected patients. The interclass correlation coefficient (ICC) was calculated. A pairwise comparison of BMD was conducted and correlations between each thoracic level were evaluated. The statistical significance level was set at p<.05. RESULTS: 60 patients (men, 51.7%) met inclusion criteria. The study population was 90% Caucasian with a mean age of 62.2 years and a mean BMI of 30.2 kg/m2. The inter- and intra-observer reliability of the thoracic QCT measurements was excellent (ICC of 0.97 and 0.97, respectively). The trabecular BMD was highest in the upper thoracic spine and decreased in the caudal direction (T1 = 182.3 mg/cm3, T2 = 168.1 mg/cm3, T3 = 163.5 mg/cm3, T4 = 164.7 mg/cm3, T5 = 161.4 mg/cm3, T6 = 152.5 mg/cm3, T7 = 143.5 mg/cm3, T8 = 141.3 mg/cm3, T9 = 143.5 mg/cm3, T10 = 145.1 mg/cm3, T11 = 145.3 mg/cm3, T12 = 133.6 mg/cm3). The BMD of all thoracic levels cranial to T6 was statistically higher than the BMD of all levels caudal to T6 (p < .001). Nonetheless, significant correlations in BMD among all measured thoracic levels were observed, with a Pearson's correlation coefficient ranging from 0.74 to 0.97. CONCLUSIONS: There is significant regional BMD variation in the thoracic spine depending on spinal level. This BMD variation might contribute to several clinically relevant phenomena. First, vertebral fractures occur most commonly at the thoracolumbar junction including T12. In addition to mechanical reasons, these fractures might be partially attributed to thoracic BMD that is lowest at T12. Second, the optimal upper instrumented vertebra (UIV) for stopping long fusions to the sacrum and pelvis is controversial. The BMD of surgically relevant upper thoracic stopping points (T2-T4) was significantly higher than the BMD of lower thoracic stopping points (T10-T12). Besides stress concentration at the relatively mobile lower thoracic segments, the low BMD at these levels might contribute to previously suggested higher rates of junctional failures with short fusions.

Cholesterol regulates polymodal sensory transduction in Müller glia.

Over- and underexposure to cholesterol activates glia in neurodegenerative brain and retinal diseases but the molecular targets of cholesterol in glial cells are not known. Here, we report that disruption of unesterified membrane cholesterol content modulates the transduction of chemical, mechanical and temperature stimuli in mouse Müller cells. Activation of TRPV4 (transient receptor potential vanilloid type 4), a nonselective polymodal cation channel was studied following the removal or supplementation of cholesterol using the methyl-beta cyclodextrin (MßCD) delivery vehicle. Cholesterol extraction disrupted lipid rafts and caveolae without affecting TRPV4 trafficking or membrane localization protein. However, MßCD suppressed agonist (GSK1016790A)- and temperature-evoked elevations in [Ca2+ ]i , and suppressed transcellular propagation of Ca2+ waves. Lowering the free membrane cholesterol content markedly prolonged the time-course of the glial swelling response, whereas MßCD:cholesterol supplementation enhanced agonist- and temperature-induced Ca2+ signals and shortened the swelling response. Taken together, these data show that membrane cholesterol modulates polymodal transduction of agonists, swelling and temperature stimuli in retinal radial glia and suggest that dyslipidemic retinas might be associated with abnormal glial transduction of ambient sensory inputs.

Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency.

Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca2+-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca2+ stores through pharmacological and genetic suppression of ER Ca2+ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca2+-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP3R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP3R1 signaling contributes to Ca2+ homeostasis and cone survival. Consistent with the important contribution of organellar Ca2+ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca2+ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca2+ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.

TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye.

An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca(2+) influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca(2+)-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension.

Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels.

The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca(2+) signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca(2+)-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca(2+) release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca(2+) waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia. SIGNIFICANCE STATEMENT: Store-operated Ca(2+) signaling represents a major signaling pathway and source of cytosolic Ca(2+) in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory, and mechanosensory functions within the retina, is mediated through synergistic activation of transient receptor potential and Orai channels. The end-foot disproportionately expresses the depletion sensor stromal interacting molecule 1, which contains an extraordinarily high density of endoplasmic reticulum cisternae that shadow neuronal, astrocytic, vascular, and axonal structures; interface with mitochondria; but also originate store-operated Ca(2+) entry-induced transcellular Ca(2+) waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores, and contributes to astroglial function, regulation, and response to mechanical stress.

Moderate prenatal alcohol exposure reduces plasticity and alters NMDA receptor subunit composition in the dentate gyrus.

Although it is well documented that heavy consumption of alcohol during pregnancy impairs brain development, it remains controversial whether moderate consumption causes significant damage. Using a limited access, voluntary consumption paradigm, we recently demonstrated that moderate prenatal alcohol exposure (MPAE) is associated with dentate gyrus-dependent learning and memory deficits that are manifested in adulthood. Here, we identified a novel mechanism that may underlie this effect of MPAE. We found that MPAE mice exhibit deficits in NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) in the dentate gyrus. Further, using semiquantitative immunoblotting techniques, we found that the levels of GluN2B subunits were decreased in the synaptic membrane, while levels of C2'-containing GluN1 and GluN3A subunits were increased, in the dentate gyrus of MPAE mice. These data suggest that MPAE alters the subunit composition of synaptic NMDARs, leading to impaired NMDAR-dependent LTP in the dentate gyrus.