Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.     

Angiogenesis Is Induced and Wound Size Is Reduced by Electrical Stimulation in an Acute Wound Healing Model in Human Skin

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.     

Axl Tyrosine Kinase Protects against Tubulo-Interstitial Apoptosis and Progression of Renal Failure in a Murine Model of Chronic Kidney Disease and Hyperphosphataemia

Chronic kidney disease (CKD) is defined as the progressive loss of renal function often involving glomerular, tubulo-interstitial and vascular pathology. CKD is associated with vascular calcification; the extent of which predicts morbidity and mortality. However, the molecular regulation of these events and the progression of chronic kidney disease are not fully elucidated. To investigate the function of Axl receptor tyrosine kinase in CKD we performed a sub-total nephrectomy and fed high phosphate (1%) diet to Axl+/+ and Axl−/− mice. Plasma Gas6 (Axl'' ligand), renal Axl expression and downstream Akt signalling were all significantly up-regulated in Axl+/+ mice following renal mass reduction and high phosphate diet, compared to age-matched controls. Axl−/− mice had significantly enhanced uraemia, reduced bodyweight and significantly reduced survival following sub-total nephrectomy and high phosphate diet compared to Axl+/+ mice; only 45% of Axl−/− mice survived to 14 weeks post-surgery compared to 87% of Axl+/+ mice. Histological analysis of kidney remnants revealed no effect of loss of Axl on glomerular hypertrophy, calcification or renal sclerosis but identified significantly increased tubulo-interstitial apoptosis in Axl−/− mice. Vascular calcification was not induced in Axl+/+ or Axl−/− mice in the time frame we were able to examine. In conclusion, we identify the up-regulation of Gas6/Axl signalling as a protective mechanism which reduces tubulo-interstitial apoptosis and slows progression to end-stage renal failure in the murine nephrectomy and high phosphate diet model of CKD.     

基因治疗在慢性难愈创面的应用进展

众多疾病和创伤都会带来损伤创面的难愈问题,慢性难愈创面的长期存在不仅增加了局部感染的机会,而且大大影响了局部组织的结构和功能。基因治疗作为一种新的生物治疗方法,其相关理论和技术近来日臻完善,为慢性难愈创面的治疗提供了新的技术途径,有望带来突破性的治疗效果。     

Angiopoietin-2 Is Associated with Albuminuria and Microinflammation in Chronic Kidney Disease

Although cardiovascular disease (CVD) is the leading cause of mortality in patients with chronic kidney disease (CKD), the pathophysiology is not thoroughly understood. Given that elevated albuminuria or circulating angiopoietin-2 associates with CVD and mortality in CKD patients, we were intrigued by the relationship between albuminuria and angiopoietin-2. A total of 416 patients with CKD stages 3 to 5 were stratified by urine albumin-creatinine ratio as normoalbuminuria (<30 mg/g), microalbuminuria (30–300 mg/g), or macroalbuminuria (>300 mg/g). The levels of plasma angiopoietin-2 and vascular endothelial growth factor (VEGF) increased, and soluble Tie-2 decreased in the subgroups of albuminuria; whereas angiopoietin-1 did not change. Linear regression showed a positive correlation between urine albumin-creatinine ratio (ACR) and plasma angiopoietin-2 (correlation coefficient r = 0.301, 95% confidence interval 0.211–0.386, P<0.0001), but not between ACR and VEGF or soluble Tie-2. Multivariate linear regression analysis showed that plasma angiopoietin-2 was independently associated with ACR (P = 0.025). Furthermore, plasma angiopoietin-2 was positively correlated with high sensitive C-reactive protein (r = 0.114, 95% confidence interval 0.018–0.208, P = 0.020). In conclusion, plasma angiopoietin-2 was associated with albuminuria and markers of systemic microinflammation in CKD patients. Although previous evidence has shown that angiopoietin-2 destabilizes vasculature and induces inflammation in different scenarios, further study will be required to delineate the role of angiopoietin-2 in albuminuria and microinflammation in CKD patients.     

Transforming Growth Factor Beta 3 Is Required for Excisional Wound Repair In Vivo

Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms–TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.     

Trefoil Factor 1 Excretion Is Increased in Early Stages of Chronic Kidney Disease

Chronic kidney disease (CKD) is associated with high morbidity and mortality. In many patients CKD is diagnosed late during disease progression. Therefore, the implementation of potential biomarkers may facilitate the early identification of individuals at risk. Trefoil factor family (TFF) peptides promote restitution processes of mucous epithelia and are abundant in the urinary tract. We therefore sought to investigate the TFF peptide levels in patients suffering from CKD and their potential as biomarkers for CKD. We analysed TFF1 and TFF3 in serum and urine of 115 patients with CKD stages 1–5 without dialysis by ELISA. 20 healthy volunteers served as controls. Our results showed, that urinary TFF1 levels were significantly increased with the onset of CKD in stages 1–4 as compared to controls and declined during disease progression (p = 0.003, < 0.001, 0.005, and 0.007. median concentrations: 3.5 pg/mL in controls vs 165.2, 61.1, 17.2, and 15.8 pg/mL in CKD 1–4). TFF1 and TFF3 serum levels were significantly elevated in stages 3–5 as compared to controls (TFF1: p < 0.01; median concentrations: 12.1, 39.7, and 34.5 pg/mL in CKD 3–5. TFF3: p < 0.001; median concentrations: 7.1 ng/mL in controls vs 26.1, 52.8, and 78.8 ng/mL in CKD 3–5). TFF3 excretion was increased in stages 4 and 5 (p < 0.001; median urinary levels: 65.2 ng/mL in controls vs 231.5 and 382.6 ng/mL in CKD 4/5; fractional TFF3 excretion: 6.4 in controls vs 19.6 and 44.1 in CKD 4/5). ROC curve analyses showed, that monitoring TFF peptide levels can predict various CKD stages (AUC urinary/serum TFF > 0.8). In conclusion our results show increased levels of TFF1 and TFF3 in CKD patients with a pronounced elevation of urinary TFF1 in lower CKD stages. Furthermore, TFF1 and TFF3 seems to be differently regulated and show potential to predict various CKD stages, as shown by ROC curve analysis.     

KGF Phage Model Peptide Accelerates Cutaneous Wound Healing in a Diabetic Rat Model

International Journal of Peptide Research and Therapeutics - The authors have previously demonstrated that one phage-displayed keratinocyte growth factor (KGF) model peptide that can bind to...     

早期介入高压氧提高寒冷应激下小鼠存活率并促进创面愈合的研究

暴露于寒冷环境下的皮肤开放性伤口是一种高度危险的战场创伤,威胁在室外作业的人员健康。紧急治疗中,高压氧(hyperbaric oxygen, HBO)治疗已经证实能够安全有效地促进皮肤伤口愈合。然而,HBO治疗的最佳干预时间说法仍然并不统一。使用冷应激下的小鼠背部皮肤全层缺损创面模型,比较了HBO治疗的3种干预策略,即分别为创伤后0、24和48 h介入HBO。结果显示,创后立即实施高压氧治疗(0-hHBO组)降低死亡率的效果最佳,小鼠死亡率为33%,而对照组死亡率为100%,且0-hHBO组创面愈合率第5天已达到85%。进一步的血常规和组织免疫化学检测显示,0-hHBO治疗组改善了血液指标,并发挥了一定的抗凋亡作用,这种作用尤其在表皮干细胞中更为明显。因此,研究结果将为HBO的临床应用提供重要的实验数据和线索。     

Elevated Klotho Promoter Methylation Is Associated with Severity of Chronic Kidney Disease

Klotho (KL) expression is down-regulated in the renal tissues of chronic kidney disease (CKD) animal models and patients with end-stage renal disease. The putative role of KL promoter hypermethylation in the progression of CKD remains unclear. The present study aimed to determine renal and peripheral blood mononuclear cells (PBMC) levels of KL promoter methylation and analyze their relationship with clinical and histological severity in patients with CKD. Using bisulfite pyrosequencing, renal and PBMC levels of KL promoter methylation were quantified in 47 patients with CKD. 47 nephrectomy specimens of patients with renal cell carcinoma and 48 PBMC specimens of healthy volunteers were used as renal tissue and PBMC controls, respectively. Renal expression of KL protein was assayed by immunohistochemistry staining. Receiver operating characteristic (ROC) curve was used to identify the optimal cut-off value of PBMC KL promoter methylation level for renal KL promoter hypermethylation. Higher levels of KL promoter methylation were observed in renal tissue and PBMC in patients with CKD compared with controls (8.79±3.24 vs. 5.17±1.11%, P<0.001; 7.20±2.79 vs. 3.27±0.79%, P<0.001). In these patients, renal KL methylation level correlated inversely with renal KL immunostaining intensity (ρ=-0.794, P<0.001). Estimated glomerular filtration rate correlated inversely with renal and PBMC levels of KL promoter methylation (r=-0.829, P<0.001; r=-0.645, P<0.001), while tubulointerstistial fibrosis score correlated positively (ρ=0.826, P<0.001; ρ=0.755, P<0.001). PBMC KL promoter methylation level correlated positively with renal KL promoter methylation level in patients with CKD (r=0.787, P<0.001). In ROC curve, the area under curve was 0.964 (P<0.001) and the optimal cut-off value was 5.83% with a sensitivity of 93.8% and specificity of 86.7% to predict renal KL promoter hypermethylation. The degree of KL promoter methylation is associated with clinical and histological severity of CKD. PBMC KL promoter methylation level may act as a potential biomarker of renal KL promoter hypermethylation.     

Effects of Silver Nanoparticles on Burn Wound Healing in a Mouse Model

Biological Trace Element Research - Healing of injuries caused by exposure to heat has been discussed in many studies, although a few drugs have been shown to produce satisfactory results. In this...     

Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease

The effect of psychological stress on the gastrointestinal microbiota is widely recognized. Chronic psychological stress may be associated with increased disease activity in inflammatory bowel disease, but the relationships among psychological stress, the gastrointestinal microbiota, and the severity of colitis is not yet fully understood. Here, we examined the impact of 12-week repeated water-avoidance stress on the microbiota of two inbred strains of T cell receptor alpha chain gene knockout mouse (background, BALB/c and C57BL/6) by means of next-generation sequencing of bacterial 16S rRNA genes. In both mouse strains, knockout of the T cell receptor alpha chain gene caused a loss of gastrointestinal microbial diversity and stability. Chronic exposure to repeated water-avoidance stress markedly altered the composition of the colonic microbiota of C57BL/6 mice, but not of BALB/c mice. In C57BL/6 mice, the relative abundance of genus Clostridium, some members of which produce the toxin phospholipase C, was increased, which was weakly positively associated with colitis severity, suggesting that expansion of specific populations of indigenous pathogens may be involved in the exacerbation of colitis. However, we also found that colitis was not exacerbated in mice with a relatively diverse microbiota even if their colonic microbiota contained an expanded phospholipase C-producing Clostridium population. Exposure to chronic stress also altered the concentration of free immunoglobulin A in colonic contents, which may be related to both the loss of bacterial diversity in the colonic microbiota and the severity of the colitis exacerbation. Together, these results suggest that long-term exposure to psychological stress induces dysbiosis in the immunodeficient mouse in a strain-specific manner and also that alteration of microbial diversity, which may be related to an altered pattern of immunoglobulin secretion in the gastrointestinal tract, might play a crucial role in the development of chronic stress-induced colitis.     

Sodium-Glucose Linked Cotransporter-2 Inhibition Does Not Attenuate Disease Progression in the Rat Remnant Kidney Model of Chronic Kidney Disease

Pharmacological inhibition of the proximal tubular sodium-glucose linked cotransporter-2 (SGLT2) leads to glycosuria in both diabetic and non-diabetic settings. As a consequence of their ability to modulate tubuloglomerular feedback, SGLT2 inhibitors, like agents that block the renin-angiotensin system, reduce intraglomerular pressure and single nephron GFR, potentially affording renoprotection. To examine this further we administered the SGLT2 inhibitor, dapagliflozin, to 5/6 (subtotally) nephrectomised rats, a model of progressive chronic kidney disease (CKD) that like CKD in humans is characterised by single nephron hyperfiltration and intraglomerular hypertension and where angiotensin converting enzyme inhibitors and angiotensin receptor blockers are demonstrably beneficial. When compared with untreated rats, both sham surgery and 5/6 nephrectomised rats that had received dapagliflozin experienced substantial glycosuria. Nephrectomised rats developed hypertension, heavy proteinuria and declining GFR that was unaffected by the administration of dapagliflozin. Similarly, SGLT2 inhibition did not attenuate the extent of glomerulosclerosis, tubulointerstitial fibrosis or overexpression of the profibrotic cytokine, transforming growth factor-ß1 mRNA in the kidneys of 5/6 nephrectomised rats. While not precluding beneficial effects in the diabetic setting, these findings indicate that SGLT2 inhibition does not have renoprotective effects in this classical model of progressive non-diabetic CKD.