Sex-specific effects of N-acetylcysteine in neonatal rats treated with hypothermia after severe hypoxia-ischemia

AbstractApproximately half of moderate to severely hypoxic-ischemic (HI) newborns do not respond to hypothermia, the only proven neuroprotective treatment. N-acetylcysteine (NAC), an antioxidant and glutathione precursor, shows promise for neuroprotection in combination with hypothermia, mitigating post-HI neuroinflammation due to oxidative stress. As mechanisms of HI injury and cell death differ in males and females, sex differences must be considered in translational research of neuroprotection. We assessed the potential toxicity and efficacy of NAC in combination with hypothermia, in male and female neonatal rats after severe HI injury. NAC 50mg/kg/d administered 1h after initiation of hypothermia significantly decreased iNOS expression and caspase 3 activation in the injured hemisphere versus hypothermia alone. However, only females treated with hypothermia +NAC 50mg/kg showed improvement in short-term infarct volumes compared with saline treated animals. Hypothermia alone had no effect in this severe model. When NAC was continued for 6 weeks, significant improvement in long-term neuromotor outcomes over hypothermia treatment alone was observed, controlling for sex. Antioxidants may provide insufficient neuroprotection after HI for neonatal males in the short term, while long-term therapy may benefit both sexes

Mutations in DCHS1 Cause Mitral Valve Prolapse

SUMMARY Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals1–3. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery4,5. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds) that segregates with MVP in the family. Morpholino knockdown of the zebrafish homolog dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 mRNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells, and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs as well as in Dchs1+/− mouse MVICs result in altered migration and cellular patterning, supporting these processes as etiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease

<i>Del1</i> Knockout Mice Developed More Severe Osteoarthritis Associated with Increased Susceptibility of Chondrocytes to Apoptosis

<div><p>Objective</p><p>We identified significant expression of the matricellular protein, DEL1, in hypertrophic and mature cartilage during development. We hypothesized that this tissue-specific expression indicated a biological role for DEL1 in cartilage biology.</p><p>Methods</p><p><i>Del1</i> KO and WT mice had cartilage thickness evaluated by histomorphometry. Additional mice underwent medial meniscectomy to induce osteoarthritis, and were assayed at 1 week for apoptosis by TUNEL staining and at 8 weeks for histology and OA scoring. <i>In vitro</i> proliferation and apoptosis assays were performed on primary chondrocytes.</p><p>Results</p><p>Deletion of the <i>Del1</i> gene led to decreased amounts of cartilage in the ears and knee joints in mice with otherwise normal skeletal morphology. Destabilization of the knee led to more severe OA compared to controls. <i>In vitro</i>, DEL1 blocked apoptosis in chondrocytes.</p><p>Conclusion</p><p>Osteoarthritis is among the most prevalent diseases worldwide and increasing in incidence as our population ages. Initiation begins with an injury resulting in the release of inflammatory mediators. Excessive production of inflammatory mediators results in apoptosis of chondrocytes. Because of the limited ability of chondrocytes to regenerate, articular cartilage deteriorates leading to the clinical symptoms including severe pain and decreased mobility. No treatments effectively block the progression of OA. We propose that direct modulation of chondrocyte apoptosis is a key variable in the etiology of OA, and therapies aimed at preventing this important step represent a new class of regenerative medicine targets.</p></div

<i>Del1</i> KO phenoptype.

<p>Appearance of the ear in WT (A) and KO (B) mice. Ears were harvested from 10-week old, male mice and stained (C) for measurement of auricular cartilage thickness (D, n = 12 WT and 18 KO). Width and length are measurements of the pinna. Photomicrographs shown are 40x magnification. Knees were harvested from 10-week old, male mice and stained (E) for measurement of tibial articular cartilage thickness (F, n = 4 for all groups). All values were normalized to tibial length. There was no difference in weight or tibial length between WT and KO mice. A bounding box at 200x magnification as shown was created and the area of cartilage within determined. Due to the variable thickness present within the ear and the undulating boundary between cartilage and bone in the knee, thickness was calculated by measuring the length and dividing into area. p value refers to difference between WT and KO mice. Picrosirius red staining of KO (G) and WT (H) ears and the medial surface of KO (I) and WT (J) knees. Representative sections are shown at 25x magnification.</p

Osteoarthritis susceptibility.

<p>(A) 25x magnification view of knee joints from WT and KO mice after sham operation or medial meniscectomy (MM). (B) Box and whiskers plot of histologic scoring of medial tibial and femoral surfaces for OA. *p = 0.0206 for tibia, p = 0.0003 for femur, n = 18 WT and 17 KO. Representative photomicrographs of TUNEL staining of articular surfaces at 1 week after knee destabilization in the injured (C), and sham operated (D) knees. Apoptotic cells seen in the same area of the articular cartilage were counted at 200x magnification as shown and quantified (E). *p<0.001 for femur and p<0.00001 for tibia, n = 5 WT and 6 KO.</p

Biomechanical testing of cartilage.

<p>Articular surfaces were tested to measure (A) stiffness, (B) elasticity, and (C) resistance to penetration. Numerical values are shown (D) and statistical significance calculated with Student’s t test with p<0.05 seen to be significant, n = 10 WT and 10 KO.</p