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Skin regeneration with conical and hair follicle structure of deep second-degree scalding injuries via combined expression of the EPO receptor and beta common receptor by local subcutaneous injection of nanosized rhEPO.

Bader A, Ebert S, Giri S, Kremer M, Liu S, Nerlich A, Günter CI, Smith DU, Machens HG - Int J Nanomedicine (2012)

Bottom Line: Our aim was to determine if joint expression of EPOR and βCR is a prerequisite for the tissue-protective effect of rhEPO.In addition, the quality of regenerated skin was improved.These results highlight the potential role of rhEPO in the improved treatment of burns patients, which might be crucial for the development of innovative new therapy regimes.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Stem Cell Biology and Cell Techniques, Centre for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, Leipzig, Germany. augustinus.bader@bbz.uni-leipzig.de

ABSTRACT

Background: Acceleration of skin regeneration is still an unsolved problem in the clinical treatment of patients suffering from deep burns and scalds. Although erythropoietin (EPO) has a protective role in a wide range of organs and cells during ischemia and after trauma, it has been recently discovered that EPO is not tissue-protective in the common β subunit receptor (βCR) knockout mouse. The protective capacity of EPO in tissue is mediated via a heteroreceptor complex comprising both the erythropoietin receptor (EPOR) and βCR. However, proof of coexpression of these heterogenic receptors in regenerating skin after burns is still lacking.

Methods: To understand the role of nanosized recombinant human erythropoietin (rhEPO) in wound healing, we investigated the effects of subcutaneous injections of EPO on skin regeneration after deep second-degree scalding injuries. Our aim was to determine if joint expression of EPOR and βCR is a prerequisite for the tissue-protective effect of rhEPO. The efficiency in wound regeneration in a skin scalding injury mouse model was examined. A deep second-degree dermal scald injury was produced on the backs of 20 female Balb/c mice which were subsequently randomized to four experimental groups, two of which received daily subcutaneous injections of rhEPO. At days 7 and 14, the mice were sacrificed and the effects of rhEPO were analyzed with respect to grade of re-epithelialization (wound closure) and stage of epidermal maturation. This was investigated using different histological parameters of epithelial covering, such as depth of the epidermal layer, epidermal stratification, and presence of conical and hair follicle structures.

Results: Expression of EPOR, βCR, and growth hormone receptor at the mRNA and protein levels was demonstrated with reverse transcriptase polymerase chain reaction and Western blot analysis. After rhEPO treatment, the rate of re-epithelialization of the scalding injury was increased and the time to final wound closure was reduced. In addition, the quality of regenerated skin was improved. In this investigation, for the first time, we demonstrated coexpression of EPOR and βCR at the RNA and protein levels in vivo using a deep second-degree scalding injury mouse model. These results highlight the potential role of rhEPO in the improved treatment of burns patients, which might be crucial for the development of innovative new therapy regimes.

Conclusion: Local injection of nanosized rhEPO directly to the injury site rather than systemic administration for deep second-degree scalding injuries achieved complete skin regeneration with conical and hair follicle structure via combined expression of EPOR and βCR.

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Representative hematoxylin and eosin stainings of scalded skin tissue samples from mice on day 7 after injury and continuous application of rhEPO or vehicle by subcutaneous injection. (A) Control group without EPO (vehicle only), mouse 1 (a–f); (B) EPO group, mouse 2 (a–f) on day 14; (C) control group, mouse 5 (a–f); (D) EPO group, mouse 8 (a–f). Magnification 40× or 100×, scale bar is 500 μm or 200 μm, respectively. In order to obtain a three-dimensional view of the wound area, each 100 μm thickness comparative slides were subjected to qualitative and quantitative evaluation. All evaluations were performed on hematoxylin and eosin routine stainings. The following histological parameters were examined and scored: wound closure in the form of assessment of remaining wound area and re-epithelialization (presence or absence of epithelial covering, degree of epithelial covering relative to number of epithelial cell layers as monolayer or multilayer designed, presence of conical structures). All criteria used for histological scoring of wound healing are summarized in Table 1. In order to render the observations comparable, sections every 100–200 μm in depth were evaluated at low-power (40×) and high-power magnification (100×).Notes: a–f: from a to b; b to c: c to d; d to e; e to f is a 100–200 μm distance of the cryosection.
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f3-ijn-7-1227: Representative hematoxylin and eosin stainings of scalded skin tissue samples from mice on day 7 after injury and continuous application of rhEPO or vehicle by subcutaneous injection. (A) Control group without EPO (vehicle only), mouse 1 (a–f); (B) EPO group, mouse 2 (a–f) on day 14; (C) control group, mouse 5 (a–f); (D) EPO group, mouse 8 (a–f). Magnification 40× or 100×, scale bar is 500 μm or 200 μm, respectively. In order to obtain a three-dimensional view of the wound area, each 100 μm thickness comparative slides were subjected to qualitative and quantitative evaluation. All evaluations were performed on hematoxylin and eosin routine stainings. The following histological parameters were examined and scored: wound closure in the form of assessment of remaining wound area and re-epithelialization (presence or absence of epithelial covering, degree of epithelial covering relative to number of epithelial cell layers as monolayer or multilayer designed, presence of conical structures). All criteria used for histological scoring of wound healing are summarized in Table 1. In order to render the observations comparable, sections every 100–200 μm in depth were evaluated at low-power (40×) and high-power magnification (100×).Notes: a–f: from a to b; b to c: c to d; d to e; e to f is a 100–200 μm distance of the cryosection.

Mentions: The stainings were analyzed by light microscopy and images were taken on an Olympus IX 51 microscope (Olympus, Tokyo, Japan) using a CC12 camera and imaging software of the Olympus cell* family (cell* Imaging Software for Life Sciences Microscopy, Soft Imaging System GMbH, Münster, Germany). The mean values of all sections were allocated to the relevant score values for all individual specimens and all relevant groups, and used for statistical analysis. The scoring data served as a reference for comparison of both experimental groups. The histological scoring system ranged between 0 and 4 for wound closure and 0 and 2 for each of the three partial scores of epithelial covering (Table 1). The score values for epithelial covering were summarized (up to 6) and are represented graphically (Figure 2). The specimens were evaluated in parallel by two independent researchers, including one pathologist, in a blinded manner (Figure 3).


Skin regeneration with conical and hair follicle structure of deep second-degree scalding injuries via combined expression of the EPO receptor and beta common receptor by local subcutaneous injection of nanosized rhEPO.

Bader A, Ebert S, Giri S, Kremer M, Liu S, Nerlich A, Günter CI, Smith DU, Machens HG - Int J Nanomedicine (2012)

Representative hematoxylin and eosin stainings of scalded skin tissue samples from mice on day 7 after injury and continuous application of rhEPO or vehicle by subcutaneous injection. (A) Control group without EPO (vehicle only), mouse 1 (a–f); (B) EPO group, mouse 2 (a–f) on day 14; (C) control group, mouse 5 (a–f); (D) EPO group, mouse 8 (a–f). Magnification 40× or 100×, scale bar is 500 μm or 200 μm, respectively. In order to obtain a three-dimensional view of the wound area, each 100 μm thickness comparative slides were subjected to qualitative and quantitative evaluation. All evaluations were performed on hematoxylin and eosin routine stainings. The following histological parameters were examined and scored: wound closure in the form of assessment of remaining wound area and re-epithelialization (presence or absence of epithelial covering, degree of epithelial covering relative to number of epithelial cell layers as monolayer or multilayer designed, presence of conical structures). All criteria used for histological scoring of wound healing are summarized in Table 1. In order to render the observations comparable, sections every 100–200 μm in depth were evaluated at low-power (40×) and high-power magnification (100×).Notes: a–f: from a to b; b to c: c to d; d to e; e to f is a 100–200 μm distance of the cryosection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3298388&req=5

f3-ijn-7-1227: Representative hematoxylin and eosin stainings of scalded skin tissue samples from mice on day 7 after injury and continuous application of rhEPO or vehicle by subcutaneous injection. (A) Control group without EPO (vehicle only), mouse 1 (a–f); (B) EPO group, mouse 2 (a–f) on day 14; (C) control group, mouse 5 (a–f); (D) EPO group, mouse 8 (a–f). Magnification 40× or 100×, scale bar is 500 μm or 200 μm, respectively. In order to obtain a three-dimensional view of the wound area, each 100 μm thickness comparative slides were subjected to qualitative and quantitative evaluation. All evaluations were performed on hematoxylin and eosin routine stainings. The following histological parameters were examined and scored: wound closure in the form of assessment of remaining wound area and re-epithelialization (presence or absence of epithelial covering, degree of epithelial covering relative to number of epithelial cell layers as monolayer or multilayer designed, presence of conical structures). All criteria used for histological scoring of wound healing are summarized in Table 1. In order to render the observations comparable, sections every 100–200 μm in depth were evaluated at low-power (40×) and high-power magnification (100×).Notes: a–f: from a to b; b to c: c to d; d to e; e to f is a 100–200 μm distance of the cryosection.
Mentions: The stainings were analyzed by light microscopy and images were taken on an Olympus IX 51 microscope (Olympus, Tokyo, Japan) using a CC12 camera and imaging software of the Olympus cell* family (cell* Imaging Software for Life Sciences Microscopy, Soft Imaging System GMbH, Münster, Germany). The mean values of all sections were allocated to the relevant score values for all individual specimens and all relevant groups, and used for statistical analysis. The scoring data served as a reference for comparison of both experimental groups. The histological scoring system ranged between 0 and 4 for wound closure and 0 and 2 for each of the three partial scores of epithelial covering (Table 1). The score values for epithelial covering were summarized (up to 6) and are represented graphically (Figure 2). The specimens were evaluated in parallel by two independent researchers, including one pathologist, in a blinded manner (Figure 3).

Bottom Line: Our aim was to determine if joint expression of EPOR and βCR is a prerequisite for the tissue-protective effect of rhEPO.In addition, the quality of regenerated skin was improved.These results highlight the potential role of rhEPO in the improved treatment of burns patients, which might be crucial for the development of innovative new therapy regimes.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Stem Cell Biology and Cell Techniques, Centre for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, Leipzig, Germany. augustinus.bader@bbz.uni-leipzig.de

ABSTRACT

Background: Acceleration of skin regeneration is still an unsolved problem in the clinical treatment of patients suffering from deep burns and scalds. Although erythropoietin (EPO) has a protective role in a wide range of organs and cells during ischemia and after trauma, it has been recently discovered that EPO is not tissue-protective in the common β subunit receptor (βCR) knockout mouse. The protective capacity of EPO in tissue is mediated via a heteroreceptor complex comprising both the erythropoietin receptor (EPOR) and βCR. However, proof of coexpression of these heterogenic receptors in regenerating skin after burns is still lacking.

Methods: To understand the role of nanosized recombinant human erythropoietin (rhEPO) in wound healing, we investigated the effects of subcutaneous injections of EPO on skin regeneration after deep second-degree scalding injuries. Our aim was to determine if joint expression of EPOR and βCR is a prerequisite for the tissue-protective effect of rhEPO. The efficiency in wound regeneration in a skin scalding injury mouse model was examined. A deep second-degree dermal scald injury was produced on the backs of 20 female Balb/c mice which were subsequently randomized to four experimental groups, two of which received daily subcutaneous injections of rhEPO. At days 7 and 14, the mice were sacrificed and the effects of rhEPO were analyzed with respect to grade of re-epithelialization (wound closure) and stage of epidermal maturation. This was investigated using different histological parameters of epithelial covering, such as depth of the epidermal layer, epidermal stratification, and presence of conical and hair follicle structures.

Results: Expression of EPOR, βCR, and growth hormone receptor at the mRNA and protein levels was demonstrated with reverse transcriptase polymerase chain reaction and Western blot analysis. After rhEPO treatment, the rate of re-epithelialization of the scalding injury was increased and the time to final wound closure was reduced. In addition, the quality of regenerated skin was improved. In this investigation, for the first time, we demonstrated coexpression of EPOR and βCR at the RNA and protein levels in vivo using a deep second-degree scalding injury mouse model. These results highlight the potential role of rhEPO in the improved treatment of burns patients, which might be crucial for the development of innovative new therapy regimes.

Conclusion: Local injection of nanosized rhEPO directly to the injury site rather than systemic administration for deep second-degree scalding injuries achieved complete skin regeneration with conical and hair follicle structure via combined expression of EPOR and βCR.

Show MeSH
Related in: MedlinePlus