Ivan Kováč

Open Wound Healing In Vivo: Monitoring Binding and Presence of Adhesion/Growth-Regulatory Galectins in Rat Skin during the Course of Complete Re-Epithelialization

Galectins are a family of carbohydrate-binding proteins that modulate inflammation and immunity. This functional versatility prompted us to perform a histochemical study of their occurrence during wound healing using rat skin as an in vivo model. Wound healing is a dynamic process that exhibits three basic phases: inflammation, proliferation, and maturation. In this study antibodies against keratins-10 and -14, wide-spectrum cytokeratin, vimentin, and fibronectin, and non-cross-reactive antibodies to galectins-1, -2, and -3 were applied to frozen sections of skin specimens two days (inflammatory phase), seven days (proliferation phase), and twenty-one days (maturation phase) after wounding. The presence of binding sites for galectins-1, -2, -3, and -7 as a measure for assessing changes in reactivity was determined using labeled proteins as probes. Our study detected a series of alterations in galectin parameters during the different phases of wound healing. Presence of galectin-1, for example, increased during the early phase of healing, whereas galectin-3 rapidly decreased in newly formed granulation tissue. In addition, nuclear reactivity of epidermal cells for galectin-2 occurred seven days post-trauma. The dynamic regulation of galectins during re-epithelialization intimates a role of these proteins in skin wound healing, most notably for galectin-1 increasing during the early phases and galectin-3 then slightly increasing during later phases of healing. Such changes may identify a potential target for the development of novel drugs to aid in wound repair and patients’ care.

The Effect of Equal Daily Dose Achieved by Different Power Densities of Low-Level Laser Therapy at 635 and 670 nm on Wound Tensile Strength in Rats: A Short Report

OBJECTIVE The aim of our study was to compare the effects of different power densities of LLLT at 635 and 670 nm achieving a daily dose of 5 J/cm(2) on wound tensile strength (TS) in rats. BACKGROUND DATA Optimal parameters of low-level laser therapy (LLLT) are still unknown. MATERIALS AND METHODS Under general anesthesia, one full-thickness skin incision was performed on the back of each rat (n = 40) and immediately closed using an intradermal running suture. Rats were separated into five groups depending on treatment parameters: (1) sham irradiated control group (SIC); (2) 635 nm laser-treated group at 4 mW/cm(2) (L-635/4); (3) 635 nm laser-treated group at 15 mW/cm(2) (L-635/15); (4) 670 nm laser-treated group at 4 mW/cm(2) (L-670/4); and (5) 670 nm laser-treated group at 15 mW/cm(2) (L-670/15). The total daily dose was 5 J/cm(2). Seven days after surgery each wound was removed for wound TS measurement. RESULTS The lowest wound TS results were measured in the SIC rats (10.5 +/- 2.8 g/mm(2)). Higher wound TS results were measured in group L-670/15 (11.5 +/- 2.5 g/mm(2)) and group L-635/4 (11.7 +/- 4.3 g/mm(2)) rats, while significantly higher results were found in group L-670/4 (15.8 +/- 4.4 g/mm(2)) and group L-635/15 (15.9 +/- 4.8 g/mm(2)). The differences were significant between certain groups (p < 0.01: SIC vs. L-635/15, SIC vs. L-670/4; p < 0.05: L-635/4 vs. L-635/15, L-635/4 vs. L-670/4, L-635/15 vs. L-670/15, L-670/4 vs. L-670/15). CONCLUSION Both red lasers significantly increased wound TS at selected parameters. Whereas the 635 nm laser significantly improved wound healing by using the higher power density, the 670 nm laser improved healing using a lower power density.

Low-Level Laser Therapy with 810 nm Wavelength Improves Skin Wound Healing in Rats with Streptozotocin-Induced Diabetes

OBJECTIVE The aim of present study was to evaluate whether low-level laser therapy (LLLT) can reverse the impaired wound healing process in diabetic rats. BACKGROUND DATA Impaired wound healing in diabetic patients represents a major health problem. Recent studies have indicated that LLLT may improve wound healing in diabetic rats, but the optimal treatment parameters are still unknown. MATERIALS AND METHODS Male Sprague-Dawley rats (n=21) were randomly divided into three groups: a healthy control group, a diabetic sham-treated group, and a diabetic LLLT-treated group. Diabetes mellitus was then induced by streptozotocin administration to the two diabetic groups. One 4 cm long full thickness skin incision and one full thickness circular excision (diameter=4 mm) were performed on the back of each rat. An infrared 810 nm laser with an output of 30 mW, a power density of 30 mW/cm(2), and a spot size of 1 cm(2) was used to irradiate each wound for 30 sec (daily dose of 0.9 J/cm(2)/wound/day). RESULTS In diabetic rats, the histology of LLLT-treated excisions revealed a similar healing response to that in nondiabetic controls, with significantly more mature granulation tissue than in the sham-treated diabetic control group. LLLT reduced the loss of tensile strength, and increased the incision wound stiffness significantly compared with sham-irradiated rats, but this did not achieve the same level as in the nondiabetic controls. CONCLUSIONS Our study demonstrates that infrared LLLT can improve wound healing in diabetic rats. Nevertheless, further research needs to be performed to evaluate the exact underlying mechanism and to further optimize LLLT parameters for clinical use.

Agrimonia eupatoria L. and Cynara cardunculus L. Water Infusions: Phenolic Profile and Comparison of Antioxidant Activities

Reactive oxygen species (ROS) are highly considered in the ethiopathogenesis of different pathological conditions because they may cause significant damage to cells and tissues. In this paper, we focused on potential antioxidant properties of two medical plants such as the Agrimonia eupatoria L. and Cynara cardunculus L. Both plants have previously been studied for their pharmacological activities, especially as hepatoprotective and hypoglycemic activities. It has been suggested, that their effects are related to the antioxidant properties of polyphenols, which are dominant compounds of the plants’ extracts. In the present study HPLC-MS analysis of water infusion was performed allowing the identification of several phenolic constituents. Furthermore, antioxidant effects of the two extracts were compared showing higher effects for agrimony extract compared to artichoke. Thus, agrimony was selected for the in vivo study using the skin flap viability model. In conclusion, our results provide evidence that the A. eupatoria extract may be a valuable source of polyphenols to be studied for the future development of supplements useful in the prevention of diseases linked to oxidative stress.

Pharmacological activation of estrogen receptors-α and -β differentially modulates keratinocyte differentiation with functional impact on wound healing

Estrogen deprivation is considered responsible for many age-related processes, including poor wound healing. Guided by previous observations that estradiol accelerates re-epithelialization through estrogen receptor (ER)-β, in the present study, we examined whether selective ER agonists [4,4′,4″-(4-propyl [1H] pyrazole-1,3,5-triyl)-trisphenol (PPT), ER-α agonist; 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), ER-β agonist] affect the expression of basic proliferation and differentiation markers (Ki-67, keratin-10, -14 and -19, galectin-1 and Sox-2) of keratinocytes using HaCaT cells. In parallel, ovariectomized rats were treated daily with an ER modulator, and wound tissue was removed 21 days after wounding and routinely processed for basic histological analysis. Our results revealed that the HaCaT keratinocytes expressed both ER-α and -β, and thus are well-suited for studying the effects of ER agonists on epidermal regeneration. The activation of ER-α produced a protein expression pattern similar to that observed in the control culture, with a moderate expression of Ki-67 being observed. However, the activation of ER-β led to an increase in cell proliferation and keratin-19 expression, as well as a decrease in galectin-1 expression. Fittingly, in rat wounds treated with the ER-β agonist (DPN), epidermal regeneration was accelerated. In the present study, we provide information on the mechanisms through which estrogens affect the expression patterns of selected markers, thus modulating keratinocyte proliferation and differentiation; in addition, we demonstrate that the pharmacological activation of ER-α and -β has a direct impact on wound healing.

Plantago lanceolata L. water extract induces transition of fibroblasts into myofibroblasts and increases tensile strength of healing skin wounds.

Although the exact underlying mechanisms are still unknown, Plantago lanceolata L. (PL) water extracts are frequently used to stimulate wound healing and to drain abscesses. Therefore, in this experimental study the effect of PL water extract on skin wound healing was studied in Sprague‐Dawley rats.

Genistein Improves Skin Flap Viability in Rats: A Preliminary In Vivo and In Vitro Investigation

Selective estrogen receptor modulators (SERMs) have been developed to achieve beneficial effects of estrogens while minimizing their side effects. In this context, we decided to evaluate the protective effect of genistein, a natural SERM, on skin flap viability in rats and in a series of in vitro experiments on endothelial cells (migration, proliferation, antioxidant properties, and gene expression profiling following genistein treatment). Our results showed that administration of genistein increased skin flap viability, but importantly, the difference is only significant when treatment is started 3 days prior the flap surgery. Based on our in vitro experiments, it may be hypothesized that the underlying mechanism may rather by mediated by increasing SOD activity and Bcl-2 expression. The gene expression profiling further revealed 9 up-regulated genes (angiogenesis/inflammation promoting: CTGF, CXCL5, IL-6, ITGB3, MMP-14, and VEGF-A; angiogenesis inhibiting: COL18A1, TIMP-2, and TIMP-3). In conclusion, we observed a protective effect of genistein on skin flap viability which could be potentially applied in plastic surgery to women undergoing a reconstructive and/or plastic intervention. Nevertheless, further research is needed to explain the exact underlying mechanism and to find the optimal treatment protocol.