The Effect of Thrombocytopenia on Dermal Wound Healing Anna M. Szpaderska, Eric I. Egozi, Richard L. Gamelli, Luisa A. DiPietro  Journal of Investigative Dermatology  Volume 120, Issue 6, Pages 1130-1137 (June 2003) DOI: 10.1016/S0022-202X(18)32320-0 Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Induction of thrombocytopenia in mice. (A) Mice were rendered transiently thrombocytopenic by a single injection of antiplatelet antiserum 24 h prior to wound placement. Platelet counts were performed on each experimental day. Recovery to normal platelet levels occurred by day 3. n = 6. (B) Mice were subjected to sustained thrombocytopenia by injection of antiplatelet antiserum 24 h prior to wound placement and then once daily thereafter. Platelet counts were performed on each experimental day. n = 6. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Electron micrographs of wounds from control (A, B, D) and thrombocytopenic (C, E) mice at either 30 min (A, B, C) or 2 h (D, E) after injury. (A) (B) At 30 min, control wounds exhibit large numbers of degranulated platelets (black arrow) and polymorphonuclear neutrophils (white arrow). (Q Wounds from thrombocytopenic mice at 30 min show polymorphonuclear neutrophils (white arrow) but no platelets. (D) At 2 h after injury, control wounds show a large number of degranulated platelets in the subcutaneous tissue (black arrows) surrounded by edema and red blood cell ghosts (white arrow with ball). (E) Two-hour-old wounds from thrombocytopenic mice exhibited a large amount of edema in the subcutaneous tissue (black arrow with ball) but no platelets. Scale bars (lower left corner of each panel): (A), (Q, (£) 2.5 um; (B) 0.6 urn; (D) 1.1 μrn. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Comparison of inflammatory cell content of wounds of control and thrombocytopenic mice. n = 6. (A) Neutrophil content of day 1 wounds as measured by MPO level. No significant difference was observed. (B) Macrophage content of wounds at days 1, 2, and 3 after injury as determined by immunohistochemical quantitation. *p<0.05. (Q Tcell content of wounds at days 7,10, and 15 after injury. **p<0.005. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Wound healing in mice with transient thrombocytopenia. (/I) Reepithelialization of wounds. The time course of reepithelialization was nearly identical in thrombocytopenic and control mice. Both groups exhibited complete reepithelialization by day 7. n = 6. (B) Collagen content of wounds. Hydroxyproline, as an indicator of collagen content of wounds, was examined at days 5, 7, 10, and 14 after injury. The collagen content of the wounds of transiently thrombocytopenic mice showed no difference from that of control mice, n = 5. (Q Wound angiogenesis. No significant difference in wound capillary density was observed between the transiently thrombocytopenic and control mice, n = 6. (D) Granulation tissue. No significant difference in the granulation tissue was observed between the transiently thrombocytopenic and control mice, n = 6. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Wound healing in mice with sustained thrombocytopenia. (A) Mice with sustained thrombocytopenia exhibited normal rates of reepithelialization. Both groups exhibited complete reepithelialization by day 7, and the rates of reepithelialization were similar, n = 6. (B) The collagen content of the wounds of mice with sustained thrombocytopenia showed no difference from that of control mice. Hydroxyproline content of wounds was examined at days 5, 7,10, and 14 after injury, n = 5. (C) No significant difference in wound capillary density was observed between the mice with sustained thrombocytopenia and control mice, n = 6. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Levels of growth factors in wounds of mice with sustained thrombocytopenia. Wound fluid was collected and examined by ELISA for (A) FGF-2 (n = 6 at 2 and 24 h; n = 7 at 6 h), (B) VEGF content (k = 6), and (C) TGF-Pl (« = 6). No significant difference was observed between control and thrombocytopenic mice for any of the three factors. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Relative mRNA levels of (A) EGF and (B) KGF in wounds of mice with sustained thrombocytopenia, n = 3. RT-PCR was performed on total RNA isolated from wound tissue of control mice and mice with sustained thrombocytopenia at indicated times postinjury. To determine relative changes in mRNA levels densitometry values for each gel were corrected to P-actin expression at each time and normalized by setting the highest value to 100. (Q A representative PCR for each gene is shown. Lanes 1, 3, 5, control mice; lanes 2, 4, 6, thrombocytopenic mice. Journal of Investigative Dermatology 2003 120, 1130-1137DOI: (10.1016/S0022-202X(18)32320-0) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions