Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar ...formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury.
Fourteen adult male Sprague Dawley rats were divided into four groups. Groups 1 (n = 4) and 2 (n = 2) were used to investigate the anatomy and histology of the soft palate, respectively. Group 3 (n = 6) was used for surgical wounding of the soft palate, and group 4 (n = 2) was used as unwounded control group. The wounds (1 mm) were evaluated by (immuno)histochemistry (AZAN staining, Pax7, MyoD, MyoG, MyHC, and ASMA) after 7 days.
The present study shows that the anatomy and histology of the soft palate muscles of the rat is largely comparable with that in humans. All wounds showed clinical evidence of healing after 7 days. AZAN staining demonstrated extensive collagen deposition in the wound area, and initial regeneration of muscle fibers and salivary glands. Proliferating and differentiating satellite cells were identified in the wound area by antibody staining.
This model is the first, suitable for studying muscle regeneration in the rat soft palate, and allows the development of novel adjuvant strategies to promote muscle regeneration after cleft palate surgery.
Skin wounds may lead to scar formation and impaired functionality. Remote ischemic preconditioning (RIPC) can induce the anti-inflammatory enzyme heme oxygenase-1 (HO-1) and protect against tissue ...injury. We aim to improve cutaneous wound repair by RIPC treatment via induction of HO-1. RIPC was applied to HO-1-
transgenic mice and HO-1 promoter activity and mRNA expression in skin and several other organs were determined in real-time. In parallel, RIPC was applied directly or 24h prior to excisional wounding in mice to investigate the early and late protective effects of RIPC on cutaneous wound repair, respectively. HO-1 promoter activity was significantly induced on the dorsal side and locally in the kidneys following RIPC treatment. Next, we investigated the origin of this RIPC-induced HO-1 promoter activity and demonstrated increased mRNA in the ligated muscle, heart and kidneys, but not in the skin. RIPC did not change HO-1 mRNA and protein levels in the wound 7 days after cutaneous injury. Both early and late RIPC did not accelerate wound closure nor affect collagen deposition. RIPC induces HO-1 expression in several organs, but not the skin, and did not improve excisional wound repair, suggesting that the skin is insensitive to RIPC-mediated protection.
•Impaired function of the soft palate is the main problem for cleft palate patients.•We developed a real-time wireless system for EMG of the soft palate in rats.•Reduced muscle function was shown ...after surgical wounding of the soft palate.•This system will help us to develop strategies to improve soft palate function in patients.
The aim of this study was to analyze the function of the palatal muscles in vivo by real-time wireless electromyography in rats. The effects of palatal wounding were also analyzed.
Microelectrodes were implanted six rats; in the masseter muscle (two-rats) for comparison, in the unwounded soft palate (two-rats) and the soft palate that received a surgical wound (two-rats). Two weeks after implantation, a wound was made in the soft palate using a 1 mm biopsy-punch. Electromyographic measurements and video-recordings were taken weekly to monitor train-duration and peak-amplitude during eating, grooming and drinking.
The train-duration of the masseter muscle during eating was 0.49 ± 0.11 s (rat-1) and 0.56 ± 0.09 s (rat-2), which was higher than during grooming. In the unwounded soft palate the train-duration during eating was 0.63 ± 0.12 s (rat-1) and 0.69 ± 0.069 s (rat-2), which was higher than during grooming and drinking. The peak-amplitude for eating in the normal soft palate before surgery was 0.31 ± 0.001 mV (rat-1) and 0.33 ± 0.02 mV (rat-2). This decreased to 0.23 ± 0.03 mV and 0.25 ± 0.11 mV respectively, after surgery. For drinking the peak-amplitude was 0.30 ± 0.01 mV (rat-1) and 0.39 ± 0.01 mV (rat-2) before surgery, which decreased to 0.23 ± 0.09 mV and 0.20 ± 0.14 mV respectively, after surgery.
The reduced peak-amplitude suggests impaired soft palate function after wounding. This is the first study into the in vivo function of the soft palate after surgical wounding. This model will contribute to develop strategies to improve soft palate function in patients.
Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its ...cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14+ monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14+ monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation.
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•HO-1 has immunomodulatory effects via its specific functions in mononuclear cells.•Reports of HO-1 regulation by lipopolysaccharide (LPS) in mononuclear cells are contradictory.•HO-1 is downregulated by LPS in primary human mononuclear cells in vitro and in vivo.•Bach1 mediates the LPS-dependent regulation of HO-1 in these cells.•HO-1 regulation in different animal and cell culture models needs careful evaluation.
Aims
Preclinical results suggest therapeutic potential of mild hyperbilirubinemia in T2DM and cardiovascular disease. Translational data are limited, because an appropriate bilirubin formulation for ...parenteral human use is lacking. Considering its use in both clinical practice and medical research in the past, we explored the feasibility to reintroduce parenteral bilirubin for translational experiments.
Methods
We developed a preparation method in accordance with good manufacturing practice and evaluated the parenteral applicability in healthy volunteers (n = 8). Explorative pharmacokinetic and safety data were compared to the results from a literature study on the former parenteral use of bilirubin. Bilirubin was administered intra‐arterially to raise the local plasma concentration in the forearm vascular bed (n = 4) and intravenously to raise the systemic plasma concentration (n = 4). Finally, pharmacokinetic characteristics were studied following a single bolus infusion (n = 3).
Results
During parenteral application, no side effects occurred. Adverse events mentioned during the two‐week observation period were in general mild and self‐limiting. Three more significant adverse events (appendicitis, asymptomatic cardiac arrhythmia and atopic eczema) were judged unrelated by independent physicians.
A dose–concentration relationship appeared sufficiently predictable for both intra‐arterial and intravenous administration. In line with existing knowledge, bilirubin pharmacokinetics could be described best according to a two‐compartment model with a volume of distribution of 9.9 (±2.0) l and a total plasma clearance of 36 (±16) ml per minute.
Conclusions
Supported by previous reports, our data suggest that it is both feasible and safe to perform translational experiments with parenteral albumin bound bilirubin.
Remote ischaemic preconditioning (RIPC) is a strategy to protect a target organ against ischaemia-reperfusion injury (IRI) by inducing short-term ischaemia/reperfusion (I/R) in a remote organ. RIPC ...of the kidney by temporary limb occlusion would be a safe, inexpensive and noninvasive method to prevent renal damage in, e.g., transplantation and aortic surgery. We investigated whether brief hind limb occlusion can protect against renal IRI and whether this protection is adenosine dependent.
Rats underwent either no RIPC, unilateral RIPC or bilateral RIPC. The preconditioning stimulus was either continuous (12'/12' I/R) or fractionated (three times 4'/4' I/R). After the last reperfusion period, we induced 25' ischaemia in the right kidney.
After 24 h of reperfusion, renal function was improved by 30-60% in both bilateral RIPC groups and in the fractionated unilateral group. Renal tubule damage and kidney injury molecule-1 expression were reduced in three of four RIPC groups. Treatment with the adenosine receptor blocker 8-(p-sulfophenyl)theophylline had no effect on fractionated or continuous RIPC.
Brief hind limb ischaemia induces protection against renal IRI, which makes this a promising strategy to prevent renal IRI in a clinical setting. Bilateral RIPC was more effective than unilateral RIPC, and this protection occurs via an adenosine-independent mechanism.
ABSTRACT
Muscle repair is a crucial component of palatoplasty but little is known about muscle regeneration after cleft palate repair. We hypothesized that the formation of new myofibers is hampered ...by collagen accumulation after experimental injury of the soft palate of rats. One‐millimeter excisional defects were made in the soft palates of 32 rats. The wound area was evaluated after 3, 7, 28, and 56 days using azocarmine G and aniline blue to stain for collagen and immunohistochemistry to identify myofibroblasts and to monitor skeletal muscle differentiation. To evaluate age effects, 16 unwounded animals were evaluated at 3 and 56 days. Staining was quantified by image analysis, and one‐way ANOVA was used for the statistical analysis. At day 56, the area percentage of collagen‐rich tissue was higher in the injured palatal muscles (46.7 ± 6.9%) than in nonwounded controls (15.9 ± 1.0%, p < 0.05). Myofibroblasts were present in the injured muscles at days 3 and 7 only. The numbers of proliferating and differentiating myoblasts within the wound area were greater at day 7 (p < 0.05), but only a few new myofibers had formed by 56 days. No age effects were found. The results indicate that surgical wounding of the soft palate results in muscle fibrosis. Although activated satellite cells migrated into the wound area, no new myofibers formed. Thus, regeneration and function of the soft palate muscles after injury may be improved by regenerative medicine approaches.
Fibrosis is characterized by the formation of fibrous connective tissue in response to primary injury. As a result, an affected organ may lose part of its functionality due to chronic, organ-specific ...tissue damage. Since fibrosis is a leading cause of death worldwide, targeting fibrotic diseases with antifibrotic hydrogels can be a lifesaving therapeutic strategy. This study developed a novel hybrid antifibrotic hydrogel by combining the synthetic polyisocyanide (PIC) with hyaluronic acid (HA). Gels of PIC are highly tailorable, thermosensitive, and strongly biomimetic in architecture and mechanical properties, whereas HA is known to promote non-fibrotic fetal wound healing and inhibits inflammatory signaling. The developed HA-PIC hybrids were biocompatible with physical properties comparable to those of the PIC gels. The antifibrotic nature of the gels was assessed by 3D cultures of human foreskin fibroblasts in the presence (or absence as control) of TGFβ1 that promotes differentiation into myofibroblasts, a critical step in fibrosis. Proliferation and macroscopic contraction assays and studies on the formation of stress fibers and characteristic fibrosis markers all indicate a strong antifibrotic nature of HA-PIC hydrogel. We showed that these effects originate from both the lightly crosslinked architecture and the presence of HA itself. The hybrid displaying both these effects shows the strongest antifibrotic nature and is a promising candidate for use as in vivo treatment for skin fibrosis.
Reliable in vitro models closely resembling native tissue are urgently needed for disease modeling and drug screening applications. Recently, conductive biomaterials have received increasing ...attention in the development of in vitro models as they permit exogenous electrical signals to guide cells toward a desired cellular response. Interestingly, they have demonstrated that they promote cellular proliferation and adhesion even without external electrical stimulation. This paper describes the development of a conductive, fully synthetic hydrogel based on hybrids of the peptide-modified polyisocyanide (PIC-RGD) and the relatively conductive poly(aniline-co-N-(4-sulfophenyl)aniline) (PASA) and its suitability as the in vitro matrix. We demonstrate that incorporating PASA enhances the PIC-RGD hydrogel’s electroactive nature without significantly altering the fibrous architecture and nonlinear mechanics of the PIC-RGD network. The biocompatibility of our model was assessed through phenotyping cultured human foreskin fibroblasts (HFF) and murine C2C12 myoblasts. Immunofluorescence analysis revealed that PIC–PASA hydrogels inhibit the fibrotic behavior of HFFs while promoting myogenesis in C2C12 cells without electrical stimulation. The composite PIC–PASA hydrogel can actively change the cell fate of different cell types, providing an attractive tool to improve skin and muscle repair.
Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth ...factor (TGF)-β1 play a key role in these processes. The persistence of (myo)fibroblasts and their excessive ECM production and continuous wound contraction have been linked to pathological scarring. The identification of compounds reducing myofibroblast survival and function may thus offer promising therapeutic strategies to optimize impaired wound healing. The plant-derived polyphenol curcumin has shown promising results as a wound healing therapeutic in vivo; however, the exact mechanism is still unclear. In vitro, curcumin induces apoptosis in various cell types via a reactive oxygen species (ROS)-dependent mechanism. Here we treated human dermal fibroblasts with TGF-β1 to induce myofibroblast differentiation, and compared the responses of fibroblasts and myofibroblasts to 25µM curcumin. Curcumin induced caspase-independent apoptosis in both fibroblasts and myofibroblasts in a ROS-dependent manner. Oxidative stress leads to the induction of several antioxidant systems to regain cellular homeostasis. We detected stress-induced induction of heme oxygenase (HO)-1 in fibroblasts but not in myofibroblasts following curcumin exposure. Instead, myofibroblasts expressed higher levels of heat shock protein (HSP)72 compared to fibroblasts in response to curcumin, suggesting that TGF-β1 treatment alters the stress-responses of the cells. However, we did not detect any differences in curcumin toxicity between the two populations. The differential stress responses in fibroblasts and myofibroblasts may open new therapeutic approaches to reduce myofibroblasts and scarring.
•Curcumin induces apoptosis in fibroblasts and myofibroblasts.•TGF-β1 treatment induces myofibroblast differentiation in foreskin fibroblasts.•Curcumin reduces pro-fibrotic gene expression in myofibroblasts.•Curcumin induces HO-1 in fibroblasts but not in myofibroblasts.•Curcumin induces higher levels of Hsp72 in myofibroblasts than in fibroblasts.
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