BACKGROUND
RESULTS
RESULTS
Nancy Yu1, Abdikarim Abdullahi MSc1, Saeid Amini Nik1 MD PhD, and Marc Jeschke1,2 MD PhD
1Sunny...
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NANCY SUNNYBROOK POSTER PRINT-1

Published on: Mar 3, 2016
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Transcripts - NANCY SUNNYBROOK POSTER PRINT-1

  • 1. BACKGROUND RESULTS RESULTS Nancy Yu1, Abdikarim Abdullahi MSc1, Saeid Amini Nik1 MD PhD, and Marc Jeschke1,2 MD PhD 1Sunnybrook Research Institute, Sunnybrook Hospital , 2University of Toronto Department of Surgery, Division of Plastic Surgery, Toronto, Canada ABSTRACT Conclusions REFERENCES RESULTS Discussion Purpose & Hypothesis Experimental Design Fig 1. Reduced pool of progenitor cells in the dermis of elderly patients compared with young. (A) Mortality rate among elderly vs young patients. (B) DAB stain showing decrease in the number of MSC in the skin of elderly human tissue. (C) Quantification and comparison of STRO1 + cells between young and old in dermis of burn skin. (D) DAB stain showing no significant difference between % of Ki67+ cells between young and elderly burnt human tissue. (E) Quantification of Ki67+ cells between young and old human tissue. Arrow indicate Ki67 +positive cells while arrowhead shows Ki67 negative cells. * = P Value <0.05 (n young=3, n old=4 for (C) and (E)) Fig. 5 Schematic showing β-Catenin and SCA1 expression in young vs old mice 2 weeks post burn. (A) Illustrate the trend seen in elderly mice: greater frequency of β-Catenin + cells (green cells) in the center of burn compared to young. Also, elderly has greater frequency of SCA1 (blue cells) in border of burn area compared with young counterparts. (B) Illustrate the trend observed in young mice: less frequency of β-Catenin in center of burn area compared to elderly. The younger population also has less SCA1 in border of burn area compared to elderly. •  One molecular candidate in the etiology of inadequate wound healing in aged populations is the Wnt family of proteins •  Wnt signaling pathway has a crucial role during skin healing through it’s regulation of stem cell renewal and differentiation •  Deregulation of this pathway has been linked to decreased healing post trauma injury Deficient progenitor pool in skin of elderly contributes to delayed skin healing post thermal injury A reduced progenitor pool in the elderly and subsequent de-regulation of the Wnt/ β-Catenin signalling pathway contributes to higher mortality rates post burn. Sample processing Human tissue was taken from patients at the RTBC immediately following skin graft surgery (with patient’s consent). A small piece of skin was extracted and sectioned into 5 micrometer thickness and mounted on slides. DAB staining Primary antibodies used were rabbit B-catenin, Rat Sca1, Rat STRO1 and Rabbit Ki67. Secondary antibodies used was from the MACH 3 Mouse HRP Polymer Detection. After coverslips were mounted with xylene based mounting medium, the slides were viewed under a light microscope and snapshots were taken under 10x and 40x magnification. Immunohistochemistry Immunohistochemical staining was performed on bone marrow cells harvested from mice 2 weeks post burn and double stained with Mouse BRDU and Rat SCA1. Western Blot Tissue samples were homogenized and lysate were subjected to SDS electrophoresis to measure for protein expression. Mice Model Five old mice (52 weeks) and five young mice (8 weeks) were kept in Sunnybrook’s animal facility for 2 weeks before burning. Prior to burning, all mice had saline injected under their skin, along the spine for protection. The anesthesia used was iso-flurane gas. All 10 mice were subject to 10s burning in 98 degrees Celsius water on their back. Animals were monitored for 14 days then killed. Skin, bone marrow, liver, blood and muscle were harvested. The skin was kept in formalin then moved to ethanol after 24 hours. RESULTS Fig. 3. Decreased MSC in dermis and bone marrow of mice 2 weeks post burn. (A) DAB stain showing decreased level of SCA1 in old at center of burn area. Arrows show SCA1+ cells and arrowhead show SCA1 negative cells (B) Quantification of SCA1+ cells in normal skin (n young=4, n old=3), border of burn area (n young=3, n old=2) and center of burn area ( n young=3, n old=2) (C) Immunofluroscent stain showing decreased level of BRDU+ and SCA1+ cells in old compared to young in bone marrow 2 weeks post burn. Arrows indicate BRDU+ cells and arrowheads show SCA1+ cells (D) Quantification of BRDU+ cells (n young=3, n old=2) (E) Quantification of SCA1+ cells (n young=2, n old=2) *=P value<0.05 •  There is a decreased stem cell pool in elderly mice and human which contributes to elderly’s slower rate of healing •  Decreased MSC in dermis and bone marrow of mice 2 weeks post burn •  Deficient healing in old mice in compare with young mice •  β-catenin activation augmented in the granulation tissue of old mice 2 weeks post thermal injury •  Reduced pool of progenitor cells in the dermis of elderly patients compared with young I would like to thank all members of Dr. Jeschke’s lab, with special mention to Cassandra Belo & Priscilla Yung. Fig 4. β-catenin activation augmented in the granulation tissue of old mice 2 weeks post thermal injury (A) DAB stain comparing β-catenin nuclear expression in mice 2 weeks post burn injury between different areas of a burn. (B) Quantification of β-catenin nuclear expression in human approximately 3-6 days post burn injury in normal skin (n young=4, n old=2), border of burn skin (n young=3, n old=2) and center of burn (n young=3, n old=2) (C) Shows western blot quantification of active β-catenin and non-active β-Catenin of mice tissue. (D) DAB stain comparing β-catenin nuclear expression in human 3-6 days post burn injury (E) Shows quantification of β-Catenin + cells from lower dermis (n young=3, n old=3) (F) Quantification of western blot on human tissue for β-Catenin expression. Arrowheads indicate β-catenin negative and arrows indicate β- catenin positive cells. *=P value < 0.05 Fig 2. Deficient healing in old mice in compare with young mice. (A) Trichrome stain showing decreased healing in old mice as measured by amount of cells in granulation tissue at border of burn area. (B) Quantification of cells in granulation tissue at border of burn area. (C) Degree of healing in old vs young mice as measured by amount of collagen deposition (D) Trichrome stain showing decreased healing in old mice vs young at center of burn area (E) Quantification of cells in granulation tissue at center of burn area (F) Degree of healing in old vs young mice as measured by amount of collagen deposition (G) DAB stain showing no significant difference between young and old mice that is % Ki67+ (H) Quantification of Ki67+ cells in normal (n young=4, n old=3), border of burn area (n young=3, n old=2), center of burn (n young=3, n old=2) (I) illustrate the percentage of Ki67 + cells of mice vs days post burn old young and elderly. Arrow indicate Ki67 +positive cells while arrowhead shows Ki67 negative cells. *=P value < 0.05, **= P value< 0.005 I.# C.# E.# A.# B.# C.# Burn trauma is not a single pathophysiological event but a devastating injury that causes structural and functional deficits in nearly every organ system1,2. Skin bing the most affected organ. After burn trauma to the skin, the wound healing response is activated (inflammation, regeneration and remodeling) to regenerate the missing parts of the afflicted area 3,4. However, when a wound is unable to heal effectively and remains unresolved, chronic inflammatory conditions like sepsis may occur 5. This has been observed more in elderly patients. Aging is perhaps one of the main factors that have been linked to inadequate wound healing 6. In fact, cutaneous wound repair has been shown to be delayed in aged individuals 7,8. Our preliminary data indicates that the skin of old mice and human burn tissue have increased activation of the Wnt/β-Catenin pathway in the center of the burn area; a vital signaling pathway important for stem cell renewal and differentiation. Moreover, our data also shows reduced number of mesenchyme stem cells (MSC) in the burnt skin of old mice and human compared to the young. Therefore, we propose that the reduced progenitor cell pool and disrupted Wnt/β-Catenin signaling pathway in the elderly are central for burn patient outcome, and the burn-induced changes and aging are associated with disrupted wound healing, resulting in increased morbidity and mortality. 1 Williams, F. N. et al. The leading causes of death after burn injury in a single pediatric burn center. Crit Care 13, R183, doi:cc8170 [pii] 10.1186/cc8170 (2009). 2 Keck, M., Herndon, D. H., Kamolz, L. P., Frey, M. & Jeschke, M. G. Pathophysiology of burns. Wien Med Wochenschr 159, 327-336, doi:10.1007/s10354-009-0651-2 (2009). 3 Li, J., Chen, J. & Kirsner, R. Pathophysiology of acute wound healing. Clin Dermatol 25, 9-18, doi:S0738-081X(06)00138-6 [pii] 10.1016/j.clindermatol.2006.09.007 (2007). 4 Gurtner, G. C., Werner, S., Barrandon, Y. & Longaker, M. T. Wound repair and regeneration. Nature 453, 314-321, doi:nature07039 [pii] 10.1038/nature07039 (2008). 5 Jeschke, M. G. et al. Burn size determines the inflammatory and hypermetabolic response. Crit Care 11, R90, doi:cc6102 [pii] 10.1186/cc6102 (2007). 6 Lau, K., Paus, R., Tiede, S., Day, P. & Bayat, A. Exploring the role of stem cells in cutaneous wound healing. Exp Dermatol 18, 921-933, doi:EXD942 [pii] 10.1111/j.1600-0625.2009.00942.x (2009). 7 Ashcroft, G. S., Horan, M. A. & Ferguson, M. W. The effects of ageing on wound healing: immunolocalisation of growth factors and their receptors in a murine incisional model. J Anat 190 ( Pt 3), 351-365 (1997). 8 Ashcroft, G. S., Horan, M. A. & Ferguson, M. W. Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model. J Invest Dermatol 108, 430-437, doi:S0022202X97801273 [pii] (1997). 9 McDonald, W. S. & Deitch, E. A. Hypertrophic skin grafts in burned patients: a prospective analysis of variables. J Trauma 27, 147-150 (1987). #####Young####################Old# Corner of Burn Burn Area Corner of Burn Normal Area EpidermisDermis Normal Area Normal Area Corner of Burn Burn Area Corner of Burn Normal Area EpidermisDermis STRO1 + STRO1 + STRO1 +STRO1 + β-Catenin + β-Catenin + β-Catenin + β-Catenin + Old Young Fig 5. Legend = β-Catenin + cells = SCA1 + cells

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