Christophe Céleste

Comparative study on microvascular occlusion and apoptosis in body and limb wounds in the horse

Wound repair in horse limbs is often complicated by exuberant granulation tissue, a condition characterized by excessive fibroplasia and scarring and that resembles hypertrophic scars and keloids in man. The aim of this study was to compare microvascular occlusion and apoptosis in wounds of the limb with those of the body, which heal normally. Five 6.25 cm2 wounds were created on both forelimbs and on the body of six horses. One limb was bandaged to stimulate excessive fibroplasia. Weekly biopsies were evaluated histologically and immunohistochemically for mutant p53 protein by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling to localize and quantify apoptosis, and by electron microscopy to measure microvessel luminal diameters. Histologic examination revealed protracted inflammation as well as slowed epithelialization and deficient fibroblast orientation in limb wounds, particularly those with excessive fibroplasia. Microvessels were occluded significantly more often in limb wounds, and the balance of apoptotic signals was altered against apoptosis in the former, although this could not be confirmed quantitatively. Data suggest that microvascular occlusion and a dysregulated apoptotic process may be involved in the excessive accumulation of extracellular matrix within limb wounds. This might provide a basis for the development of targeted therapies to prevent and treat excessive fibroplasia and extensive scarring in horses.

Regional differences in wound oxygenation during normal healing in an equine model of cutaneous fibroproliferative disorder

Wound repair in horse limbs is often complicated by the development of exuberant granulation tissue (EGT) and excessive scarring while body wounds tend to repair uneventfully. EGT resembles the human keloid. While the events leading to keloid formation are not fully elucidated, tissue hypoxia has been proposed as a major contributing factor. The objective of this study was to investigate tissue oxygen saturation in healing full‐thickness wounds created on the horse limb and body, using near‐infrared spectroscopy. Spectroscopic reflectance data were collected from both anatomic sites at specific times following wounding. The oxygen saturation values of limb wounds were significantly inferior to those of body wounds during the early period of healing, indicating a temporary, relative state of hypoxia in the former during the inflammatory phase of repair. Horses present a weak, persistent inflammatory response to wounding, especially at the limb level. The relative hypoxia present acutely in limb wounds of horses may promote a feeble yet prolonged inflammatory response, which could interfere with and retard the subsequent phases of healing. Ongoing low‐grade inflammation in horse wounds is accompanied by up‐regulation of various inflammatory and profibrotic mediators, which might ultimately promote the development of fibroproliferative disorders such as EGT.

Skin temperature during cutaneous wound healing in an equine model of cutaneous fibroproliferative disorder: kinetics and anatomic-site differences

OBJECTIVE To map skin temperature kinetics, and by extension skin blood flow throughout normal or abnormal repair of full-thickness cutaneous wounds created on the horse body and limb, using infrared thermography. STUDY DESIGN Experimental. ANIMALS Standardbreds (n = 6), aged 3-4 years. METHODS Three cutaneous wounds were created on the dorsolateral surface of each metacarpus and on the lateral thoracic wall. Thoracic skin wounds and those on 1 randomly chosen forelimb healed by second intention without a bandage, whereas contralateral limb wounds were bandaged to induce formation of exuberant granulation tissue (EGT). Thermal data were collected from all planned wound sites before the surgical procedure (baseline), and at 24, 48, 96 hours, 1, 2, and 4 weeks after wounding. Data were analyzed using repeated measures ANOVA and a priori contrasts submitted to Bonferroni sequential correction. Level of significance was P < .05. RESULTS Cutaneous wound temperature (CWT) increased temporally from preoperative period to week 1 postwounding, independently of anatomic location (P < .0001). CWT of limb wounds was significantly less than that of body wounds throughout healing (P < .01). CWT of limb wounds managed with bandages and developing EGT was significantly less than that of unbandaged limb wounds, which did not develop EGT (P ≤ .01). CONCLUSIONS CWT varied with anatomic location and throughout healing. CWT of wounds developing EGT was significantly less than that of wounds without EGT.

Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1.

BACKGROUND Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. OBJECTIVE The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or limb respond differently to hypoxia. METHODS EDFs were isolated and cultured from skin from body or limb under normoxic or hypoxic conditions for up to 7days. RESULTS Hypoxia significantly stimulated EDF proliferation, but had no effect on cell survival. The hypoxia-mimetic agent CoCl(2) up-regulated COL1A1 expression and down-regulated MMP2 expression, suggesting an increase in ECM synthesis and a decrease in turnover. Both regulatory effects were inhibited by the addition of echinomycin, indicating that they are mediated by the transcriptional regulatory activity of HIF. No differences were observed between EDFs originating from body or limb for any effect of hypoxia or CoCl(2), suggesting that EGT development does not depend on intrinsic properties of limb fibroblasts. CONCLUSIONS We conclude that hypoxia regulates ECM remodeling via HIF1 in EDFs, and that this may be an important determinant in the pathogenesis of equine EGT.

Constitutive expression of hypoxia‐inducible factor‐1 α in keratinocytes during the repair of skin wounds in horses

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia‐inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin‐dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia‐independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.

β-Catenin Stabilization in Gonadotropes Impairs FSH Synthesis in Male Mice In Vivo

Although classically considered a WNT signaling intermediary, β-catenin (CTNNB1) can also mediate GnRH induction of gonadotropin β-subunit (Fshb and Lhb) transcription in the murine gonadotrope-like cell line LβT2. Here, we assessed CTNNB1s role in gonadotropin synthesis in vivo. We used a Cre/lox approach to introduce both gain- and loss-of-function mutations in the murine Ctnnb1 gene in gonadotrope cells. Gonadotropin production and fertility were normal in Ctnnb1 knockout mice. Similarly, females harboring a deletion of exon 3 of Ctnnb1, which stabilizes the resulting CTNNB1 protein, showed normal fertility and gonadotropin synthesis. Interestingly, males with the activating CTNNB1-Δexon 3 mutation exhibited 50% reductions in FSH synthesis and secretion, without a corresponding change in LH. This selective regulation of FSH suggested an alteration in the activin/inhibin/follistatin system. Indeed, CTNNB1-Δexon 3 males showed a 60% increase in serum inhibin B levels, and in culture, their pituitaries exhibited a greater sensitivity to exogenous inhibin than controls. At the same time, pituitary, but not testicular, follistatin (Fst) expression was increased significantly in these mice. Castration normalized FSH levels in CTNNB1-Δexon 3 males to those seen in castrated controls. Paradoxically, pituitaries from CTNNB1-Δexon 3 males exhibited greater basal and activin-stimulated FSH synthesis in vitro. Similarly, CTNNB1-Δexon 3 overexpression potentiated activin A-induced murine Fshb promoter activity in LβT2 cells. Together, these results indicate that CTNNB1 is dispensable for gonadotropin synthesis in vivo. However, sustained CTNNB1 signaling potentiates activin-induced Fshb expression in gonadotropes, but this effect is overcome in vivo by enhanced inhibin feedback sensitivity and Fst expression.

mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice

ABSTRACT The mammalian target of rapamycin (Mtor) gene encodes a serine/threonine kinase that acts as a master regulator of processes as diverse as cell growth, protein synthesis, cytoskeleton reorganization, and cell survival. In the testis, physiological roles for Mtor have been proposed in perinatal Sertoli cell proliferation and blood–testis barrier (BTB) remodeling during spermatogenesis, but no in vivo studies of Mtor function have been reported. Here, we used a conditional knockout approach to target Mtor in Sertoli cells. The resulting Mtorflox/flox; Amhr2cre/+ mice were characterized by progressive, adult-onset testicular atrophy associated with disorganization of the seminiferous epithelium, loss of Sertoli cell polarity, increased germ cell apoptosis, premature release of germ cells, decreased epididymal sperm counts, increased sperm abnormalities, and infertility. Histopathologic analysis and quantification of the expression of stage-specific markers showed a specific loss of pachytene spermatocytes and spermatids. Although the BTB and the ectoplasmic specializations did not appear to be altered in Mtorflox/flox;Amhr2cre/+ mice, a dramatic redistribution of gap junction alpha-1 (GJA1) was detected in their Sertoli cells. Phosphorylation of GJA1 at Ser373, which is associated with its internalization, was increased in the testes of Mtorflox/flox; Amhr2cre/+ mice, as was the expression and phosphorylation of AKT, which phosphorylates GJA1 at this site. Together, these results indicate that Mtor expression in Sertoli cells is required for the maintenance of spermatogenesis and the progression of germ cell development through the pachytene spermatocyte stage. One mechanism of mTOR action may be to regulate gap junction dynamics by inhibiting AKT, thereby decreasing GJA1 phosphorylation and internalization. mTOR regulates gap junction alpha-1 protein distribution in Sertoli cells and is necessary for progression through the pachytene spermatocyte stage.

Electrosurgical Tenoscopic Desmotomy of the Accessory Ligament of the Superficial Digital Flexor Muscle (Proximal Check Ligament) in Horses

OBJECTIVE To report a tenoscopic technique using monopolar electrosurgery to transect the accessory ligament of superficial digital flexor muscle (AL-SDFM) and outcome in 33 horses. STUDY DESIGN Case series. ANIMALS Horses (n=33). METHODS Medical files and surgery video recordings of horses that had AL-SDFM desmotomy performed by tenoscopy with monopolar electrosurgical electrodes were reviewed. RESULTS Of 33 horses, 24 were Standardbred racehorses with surgery performed bilaterally for superficial digital flexor tendonitis and 9 horses had flexural deformity. Severe (n=6) and mild (6) intrathecal hemorrhage was the most common intraoperative complication. Large intrathecal vessels including the nutrient artery were successfully electrocoagulated and AL-SDFM transection was completed. Clear/serosanguinous drainage from skin incisions was observed for 4.3±3.3 days (mean, SD). Protracted wound drainage for >4 days occurred in 10 horses, principally in the group treated for flexural deformities (P=.01). CONCLUSIONS Sixty-four AL-SDFM were transected under tenoscopic observation using monopolar electrodes. Electrocoagulation of large intrathecal vessels, including the nutrient artery, was possible in all cases and allowed completion of desmotomy. Postoperative wound care was similar to routine tenoscopy in most (70%) horses. Aseptic protracted wound drainage was observed in 30% of horses (principally those with flexural deformity), and led to a prolonged hospitalization.Objective: To report a tenoscopic technique using monopolar electrosurgery to transect the accessory ligament of superficial digital flexor muscle (AL-SDFM) and outcome in 33 horses. Study Design: Case series. Animals: Horses (n=33). Methods: Medical files and surgery video recordings of horses that had AL-SDFM desmotomy performed by tenoscopy with monopolar electrosurgical electrodes were reviewed. Results: Of 33 horses, 24 were Standardbred racehorses with surgery performed bilaterally for superficial digital flexor tendonitis and 9 horses had flexural deformity. Severe (n=6) and mild (6) intrathecal hemorrhage was the most common intraoperative complication. Large intrathecal vessels including the nutrient artery were successfully electrocoagulated and AL-SDFM transection was completed. Clear/serosanguinous drainage from skin incisions was observed for 4.3±3.3 days (mean, SD). Protracted wound drainage for >4 days occurred in 10 horses, principally in the group treated for flexural deformities (P=.01). Conclusions: Sixty-four AL-SDFM were transected under tenoscopic observation using monopolar electrodes. Electrocoagulation of large intrathecal vessels, including the nutrient artery, was possible in all cases and allowed completion of desmotomy. Postoperative wound care was similar to routine tenoscopy in most (70%) horses. Aseptic protracted wound drainage was observed in 30% of horses (principally those with flexural deformity), and led to a prolonged hospitalization.

A hypoxia response element in the Vegfa promoter is required for basal Vegfa expression in skin and for optimal granulation tissue formation during wound healing in mice

Hypoxia in skin wounds is thought to contribute to healing through the induction of hypoxia inducible factor-1 (HIF-1). Although HIF-1 can regulate the expression of vascular endothelial growth factor A (Vegfa), whether hypoxia and HIF-1 are required to induce Vegfa expression in the context of wound healing is unknown. To test this hypothesis, we evaluated Vegfa expression and wound healing in mutant mice that lack a functional HIF-1 binding site in the Vegfa promoter. Full-thickness excisional wounds were made using a biopsy punch, left to heal by second intention, and granulation tissue isolated on a time course during healing. mRNA levels of Vegfa and its target genes platelet-derived growth factors B (Pdgfb) and stromal cell-derived factor-1 (Sdf1) were measured by RT-qPCR, and HIF-1alpha and VEGFA protein levels measured by immunoblotting. Lower levels of Vegfa, Pdgf1 and Sdf1 mRNA were found in intact skin of mutant mice relative to wild-type controls (n = 6 mice/genotype), whereas levels in granulation tissue during wound healing were unaltered. VEGFA protein levels were also lower in intact skin of the mutant versus the wild-type mice. Decreased Vegfa mRNA levels in skin of mutant mice could not be attributed to decreased HIF-1alpha protein expression, and were therefore a consequence of the loss of HIF-1 responsiveness of the Vegfa promoter. Comparative histologic analyses of healing wounds in mutant and wild-type mice (n = 8 mice/genotype) revealed significant defects in granulation tissue in the mutant mice, both in terms of quantity and capillary density, although epithelialization and healing rates were unaltered. We conclude that HIF-1 is not a major regulator of Vegfa expression during wound healing; rather, it serves to maintain basal levels of expression of Vegfa and its target genes in intact skin, which are required for optimal granulation tissue formation in response to wounding.