Involvement of PPAR nuclear receptors in tissue injury and wound repair.

Tissue damage resulting from chemical, mechanical, and biological injury, or from interrupted blood flow and reperfusion, is often life threatening. The subsequent tissue response involves an intricate series of events including inflammation, oxidative stress, immune cell recruitment, and cell survival, proliferation, migration, and differentiation. In addition, fibrotic repair characterized by myofibroblast transdifferentiation and the deposition of ECM proteins is activated. Failure to initiate, maintain, or stop this repair program has dramatic consequences, such as cell death and associated tissue necrosis or carcinogenesis. In this sense, inflammation and oxidative stress, which are beneficial defense processes, can become harmful if they do not resolve in time. This repair program is largely based on rapid and specific changes in gene expression controlled by transcription factors that sense injury. PPARs are such factors and are activated by lipid mediators produced after wounding. Here we highlight advances in our understanding of PPAR action during tissue repair and discuss the potential for these nuclear receptors as therapeutic targets for tissue injury.

Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Collagen (NeuraGen®) nerve conduits and stem cells for peripheral nerve gap repair.

Collagen nerve guides are used clinically for peripheral nerve defects, but their use is generally limited to lesions up to 3 cm. In this study we combined collagen conduits with cells as an alternative strategy to support nerve regeneration over longer gaps. In vitro cell adherence to collagen conduits (NeuraGen(®) nerve guides) was assessed by scanning electron microscopy. For in vivo experiments, conduits were seeded with either Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC) or left empty (control group), conduits were used to bridge a 1cm gap in the rat sciatic nerve and after 2-weeks immunohistochemical analysis was performed to assess axonal regeneration and SC infiltration. The regenerative cells showed good adherence to the collagen walls. Primary SC showed significant improvement in distal stump sprouting. No significant differences in proximal regeneration distances were noticed among experimental groups. dMSC and dASC-loaded conduits showed a diffuse sprouting pattern, while SC-loaded showed an enhanced cone pattern and a typical sprouting along the conduits walls, suggesting an increased affinity for the collagen type I fibrillar structure. NeuraGen(®) guides showed high affinity of regenerative cells and could be used as efficient vehicle for cell delivery. However, surface modifications (e.g. with extracellular matrix molecule peptides) of NeuraGen(®) guides could be used in future tissue-engineering applications to better exploit the cell potential.

Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair.

OBJECTIVES/HYPOTHESIS: Facial nerve regeneration is limited in some clinical situations: in long grafts, by aged patients, and when the delay between nerve lesion and repair is prolonged. This deficient regeneration is due to the limited number of regenerating nerve fibers, their immaturity and the unresponsiveness of Schwann cells after a long period of denervation. This study proposes to apply glial cell line-derived neurotrophic factor (GDNF) on facial nerve grafts via nerve guidance channels to improve the regeneration. METHODS: Two situations were evaluated: immediate and delayed grafts (repair 7 months after the lesion). Each group contained three subgroups: a) graft without channel, b) graft with a channel without neurotrophic factor; and c) graft with a GDNF-releasing channel. A functional analysis was performed with clinical observation of facial nerve function, and nerve conduction study at 6 weeks. Histological analysis was performed with the count of number of myelinated fibers within the graft, and distally to the graft. Central evaluation was assessed with Fluoro-Ruby retrograde labeling and Nissl staining. RESULTS: This study showed that GDNF allowed an increase in the number and the maturation of nerve fibers, as well as the number of retrogradely labeled neurons in delayed anastomoses. On the contrary, after immediate repair, the regenerated nerves in the presence of GDNF showed inferior results compared to the other groups. CONCLUSIONS: GDNF is a potent neurotrophic factor to improve facial nerve regeneration in grafts performed several months after the nerve lesion. However, GDNF should not be used for immediate repair, as it possibly inhibits the nerve regeneration.

Suture repair of umbilical hernia during caesarean section: a case-control study.

PURPOSE: The objective of this study was to investigate the additional burdens in terms of pain, prolongation of surgery and morbidity which is added to elective caesarean section if umbilical hernia suture repair is performed simultaneously. Secondly, patient's satisfaction and hernia recurrence rate were assessed. METHODS: Consecutive women with symptomatic umbilical hernia undergoing internal or external suture repair during elective caesarean were included in this retrospective cohort-control study. Data on post-operative pain, duration of surgery and morbidity of a combined procedure were collected. These patients were matched 1:10 to women undergoing caesarean section only. Additionally, two subgroups were assessed separately: external and internal suture hernia repair. These subgroups were compared for patient's satisfaction, cosmesis, body image and recurrence rate. RESULTS: Fourteen patients with a mean age of 37 years were analysed. Internal suture repair (n = 7) prolonged caesarean section by 20 min (p = 0.001) and external suture repair (n = 7) by 34 min (p < 0.0001). Suture repair did not increase morphine use (0.38 ± 0.2 vs. 0.4 ± 02 mg/kg body weight), had no procedure-related morbidity and prolonged hospitalization by 0.5 days (p = 0.01). At a median follow-up of 37 (5-125) months, two recurrences in each surgical technique, internal and external suture repair, occurred (28 %). Body image and cosmesis score showed a higher level of functioning in internal suture repair (p = 0.02; p = 0.04). DISCUSSION: Despite a high recurrence rate, internal suture repair of a symptomatic umbilical hernia during elective caesarean section should be offered to women if requested. No additional morbidity or scar is added to caesarean section. Internal repair is faster, and cosmetic results are better, additional skin or fascia dissection is avoided, and it seems to be as effective as an external approach. Yet, women must be informed on the high recurrence rate.

Is the effect of inguinal field block with 0.5 % bupivacaine on postoperative pain after hernia repair enhanced by addition of ketorolac or S(+) ketamine ?

Résumé Malgré l'apparition de nouvelles techniques chirurgicales dites « sans tension », l'antalgie post-opératoire après cure de hernie inguinale reste un défi pour les anesthésiologistes. Récemment on a suggéré que l'addition de ketamine ou d'un anti-inflammatoire non-stéroïdien (AINS) à un anesthésique local pourrait améliorer et prolonger l'analgésie postopératoire. Le but de cette étude, à laquelle ont participé 36 patients ASA I-II, était d'évaluer si la coadministration de S(+) ketamine ou de ketorolac renforcerait les effets analgésiques de la bupivacaïne après cure ambulatoire de hernie inguinale sous anesthésie générale. L'analgésie a consisté en une infiltration de la plaie associé à un bloc inguinal avec soit 30 ml de bupivacaïne 0,5 % (n=12), soit 27 ml de bupivacaïne 0,5 % + 3 ml de S(+) ketamine (75 mg) (n=12), soit 28 ml de bupivacaïne 0,5 % + 2 ml de ketorolac (60 mg) (n=12). La prise orale d'antalgique en phase postopératoire était standardisée. L'intensité des douleurs a été évaluée au moyen d'une échelle visuelle analogique (EVA), d'un score verbal d'estimation et par algométrie de pression respectivement 2, 4, 6, 24 et 48 heures après l'intervention. Les trois groupes de patients ont présenté le score de douleur évalué par EVA le plus élevé à 24 heures, score significativement différent de ceux mesurés à 6 et 48 heures (P <0.05). A part une sensation de douleurs significativement moindre (score verbal d'estimation) dans le groupe ketorolac à 24 et 48 heures et seulement à 48 heures dans le groupe ketamine, il n'y avait aucune autre différence entre les groupes pour la durée de l'étude (48 heures) en ce qui concerne les scores de douleur, les seuils de douleur à la pression ou la prise postopératoire d'antalgiques « de secours ». En conclusion, l'addition de S(+) ketamine ou de ketorolac, n'améliore que marginalement l'effet analgésique de la bupivacaïne. Ceci peut-être mis en relation avec la technique de cure de hernie « sans tension » induisant un bas niveau de douleur postopératoire. Abstract Objective: The aim of the study was to assess whether coadministration of S(±) ketamine or ketorolac would enhance or prolong local analgesic effect of bupivacaine after inguinal hernia repair. Design: Prospective double-blind randomized study evaluating pain intensity after surgery under general anesthesia. Setting: Outpatient facilities of the University Hospital of Lausanne. Patient: Thirty-six ASA I-II outpatients scheduled for elective day-case inguinal herniorraphy. Intervention: Analgesia strategy consisted of a wound infiltration and an inguinal field block either with 30 mL bupivacairie (0.5%) or with the same volume of a mixture of 27 mL bupivacaine (0.5%) + 3 mL S(+) ketamine (75 mg) or a 28 mL bupivacaine (0.5%) + 2 ML ketorolac (60 mg). Postoperative analgesic regimen was standardized. Outcome Measures: Pain intensity was assessed with a Visual Analog Seale, a verbal rating score, and by pressure algometry 2, 4, 6, 24, and 48 hours after surgery. Results: The 3 groups of patients experienced the highest Visual Analog Scale pain score at 24 hours, which was different from those at 6 and 48 hours (P < 0.05). Apart from a significantly lower pain sensation (verbal rating score) in the ketorolac group at 24 and 48 hours and only at 48 hours with ketamine, there were no other differences in pain scores, pain pressure thresholds, or rescue analgesic consumption between groups throughout the 48-hour study period. Conclusion: The addition of S (+)-ketamine or ketorolac only minimally improves the analgesic effect of bupivacaine. This may be related to the tension-free hernia repair technique associated with low postoperative pain.

Peroxisome proliferator-activated receptors (PPARs) in skin health, repair and disease.

Peroxisome proliferator-activated receptors, PPARalpha, PPARbeta/delta and PPARgamma, are fatty acid activated transcription factors that belong to the nuclear hormone receptor family. While they are best known as transcriptional regulators of lipid and glucose metabolism, evidence has also accumulated for their importance in skin homeostasis. The three PPAR isotypes are expressed in rodent and human skin. Various cell culture and in vivo approaches suggest that PPARalpha contributes to fetal skin development, to epidermal barrier maturation and to sebocyte activity. PPARbeta/delta regulates sebocyte differentiation, promotes hair follicle growth and has pro-differentiating effects in keratinocytes in normal and inflammatory conditions. In contrast, the role of PPARgamma appears to be rather minor in keratinocytes, whereas its activity is required for sebaceous gland differentiation. Importantly, PPARalpha and beta/delta are instrumental in skin repair after an injury, each of them playing specific roles. Due to their collective diverse functions in skin biology, PPARs represent a major research target for the understanding and treatment of many skin diseases, such as benign epidermal tumors, papillomas, acne vulgaris and psoriasis.

Cardiac perforation after surgical repair of pectus excavatum.

Five days after surgical repair of pectus excavatum, this 7-year-old boy had a right-sided Kirschner wire protruding beneath the skin. The wire was repositioned blindly. Severe congestive heart failure developed. Surgical exploration showed a pierced right atrium, a torn septal leaflet of the tricuspid valve and noncoronary aortic cusp, and a large traumatic ventricular septal defect. The outcome and the indications and possible complications of surgery are discussed.

Living-engineered valves for transcatheter venous valve repair.

Background: Chronic venous insufficiency (CVI) represents a major global health problem with increasing prevalence and morbidity. CVI is due to an incompetence of the venous valves, which causes venous reflux and distal venous hypertension. Several studies have focused on the replacement of diseased venous valves using xeno- and allogenic transplants, so far with moderate success due to immunologic and thromboembolic complications. Autologous cell-derived tissue-engineered venous valves (TEVVs) based on fully biodegradable scaffolds could overcome these limitations by providing non-immunogenic, non-thrombogenic constructs with remodeling and growth potential. Methods: Tri- and bicuspid venous valves (n=27) based on polyglycolic acid-poly-4-hydroxybutyrate composite scaffolds, integrated into self-expandable nitinol stents, were engineered from autologous ovine bone-marrow-derived mesenchymal stem cells (BM-MSCs) and endothelialized. After in vitro conditioning in a (flow) pulse duplicator system, the TEVVs were crimped (n=18) and experimentally delivered (n=7). The effects of crimping on the tissue-engineered constructs were investigated using histology, immunohistochemistry, scanning electron microscopy, grating interferometry (GI), and planar fluorescence reflectance imaging. Results: The generated TEVVs showed layered tissue formation with increasing collagen and glycosaminoglycan levels dependent on the duration of in vitro conditioning. After crimping no effects were found on the MSC level in scanning electron microscopy analysis, GI, histology, and extracellular matrix analysis. However, substantial endothelial cell loss was detected after the crimping procedure, which could be reduced by increasing the static conditioning phase. Conclusions: Autologous living small-diameter TEVVs can be successfully fabricated from ovine BM-MSCs using a (flow) pulse duplicator conditioning approach. These constructs hold the potential to overcome the limitations of currently used non-autologous replacement materials and may open new therapeutic concepts for the treatment of CVI in the future.

Tissue specificity in DNA repair: lessons from trinucleotide repeat instability.

DNA must constantly be repaired to maintain genome stability. Although it is clear that DNA repair reactions depend on cell type and developmental stage, we know surprisingly little about the mechanisms that underlie this tissue specificity. This is due, in part, to the lack of adequate study systems. This review discusses recent progress toward understanding the mechanism leading to varying rates of instability at expanded trinucleotide repeats (TNRs) in different tissues. Although they are not DNA lesions, TNRs are hotspots for genome instability because normal DNA repair activities cause changes in repeat length. The rates of expansions and contractions are readily detectable and depend on cell identity, making TNR instability a particularly convenient model system. A better understanding of this type of genome instability will provide a foundation for studying tissue-specific DNA repair more generally, which has implications in cancer and other diseases caused by mutations in the caretakers of the genome.

In vitro and in vivo Repair Activities of Undifferentiated and Classically and Alternatively Activated Macrophages.

Objective: Macrophages play a critical role in wound repair. However, the specific role of the different macrophage subtypes in wound repair remains incompletely understood. The aim of this study was to compare the wound repair activities of undifferentiated macrophages (M0), classically activated macrophages (M1) and alternatively activated (M2) macrophages. Methods: The macrophage repair activities of intestinal wounds were evaluated using in vitro and in vivo models. Results: All three macrophage subtypes enhanced wound closure in vitro, with the M2 macrophages demonstrating greater repair activities than the M0 and M1 macrophages. Injection of M0 and M2 macrophages into mice with experimental dextran sodium sulfate-induced colitis significantly enhanced ulcer repair when compared to control mice. In contrast, injection of M1 macrophages did not affect ulcer repair. Conclusions: These results underscore the wound repair capacity of different macrophage subsets. Notably, wound repair activity is not restricted to M2 macrophages, as the current literature suggests. © 2014 S. Karger AG, Basel.

Cardiac function assessed by transesophageal echocardiography during pectus excavatum repair.

BACKGROUND: We assessed end-diastolic right ventricular (RV) dimensions and left ventricular (LV) ejection fraction by use of intraoperative transesophageal echocardiography before and after surgical correction of pectus excavatum in adults. METHODS: A prospective study was conducted including 17 patients undergoing surgical correction of pectus excavatum according to the technique of Ravitch-Shamberger between 1999 and 2004. Intraoperative transesophageal echocardiography was performed under general anesthesia before and after surgery to assess end-diastolic RV dimensions and LV ejection fraction. The end-diastolic RV diameter and area were measured in four-chamber and RV inflow-outflow view, and the RV volume was calculated from these data. The LV was assessed by transgastric short-axis view, and its ejection fraction was calculated by use of the Teichholz formula. RESULTS: The end-diastolic RV diameter, area, and volume all significantly increased after surgery (mean values +/- SD, respectively: 2.4 +/- 0.8 cm versus 3.0 +/- 0.9 cm, p < 0.001; 12.5 +/- 5.2 cm(2) versus 18.4 +/- 7.5 cm(2), p < 0.001; and 21.7 +/- 11.7 mL versus 40.8 +/- 23 mL, p < 0.001). The LV ejection fraction also significantly increased after surgery (58.4% +/- 15% versus 66.2% +/- 6%, p < 0.001). CONCLUSIONS: Surgical correction of pectus excavatum according to Ravitch-Shamberger technique results in a significant increase in end-diastolic RV dimensions and a significantly increased LV ejection fraction.