Wound Healing Kinetics of the Genetically Diabetic Mouse

  • 1/1/2008
  • 0 Comments
  • 15843 reads
Author(s): 
Sandra Saja Scherer, MD;1 Giorgio Pietramaggiori, MD;1,2 Jasmine C. Mathews;1 Rodney Chan, MD;1 Paolo Fiorina, MD, PhD;3 Dennis P. Orgill MD, PhD1

Histological Analysis

Wound tissues for histological assessment were collected in the period when 50% closure was reached (8–13 days post operation).26

The level of contraction of a wound can be measured by the dermal gap. The dermal gap of 1.0 cm2 reduced in size of 8.3 mm ± 1 mm on day 9. Microscopic quantification of the epithelial gap (the open portion of the wound) on the same day resulted in 5.8 mm ± 2 mm (Figure 2A, 2B).30,31

Microscopic analysis of wounds mid-healing demonstrated less inflammatory infiltrates in diabetic wounds than wild-type wounds.26 The granulation tissue formed by the db/db mouse in the middle of the lesion of a 1.0 cm2 full-thickness wound was 78% less when compared to wild type animals. Similar results were seen when granulation tissue thickness was measured—79% reduction (Figure 2A, 2C).26,30,31 The number of blood vessels on the same day was 5.7 ± 5.8 per high power field at

40-x magnification.

Blood vessel density in the granulation tissue reached 4 ± 3% and cell proliferation was found up to 20 ± 7% on day 9, as assessed by immunohistochemistry.

Diabetic mice and hyperglycemia. Db/db mice showed a diabetic-hyperglycemic phenotype with significantly greater blood sugar levels (590 mg/dL ± 6 mg/dL) compared to their corresponding wild-type counterparts (285 mg/dL ± 7 mg/dL).26

Genotype analysis. Genotype analysis revealed a band at 135 bp in wild type mice (Figure 6A). Mice heterozygous (db/+) for the point mutation, showed a 135 bp and 108 bp bands (Figure 6B). Mice homozygous for leptin receptor mutation (db/db) showed only the 108 bp band (Figure 6C).


Discussion

Considering the increased prevalence of diabetes type 2 in the general population, the development of a reliable animal model, and the standardization of measurement techniques and study designs, are crucial aspects to facilitate improvements in therapies that seek to provide an efficient clinical translation. Among all animal models used, the db/db mouse model is the one that is most commonly used in wound care.

Different mechanisms contribute to wound closure kinetics, such as wound contraction and re-epithelialization. In the db/db mouse, the possibility to capture the different wound closure mechanisms makes it possible to quantify early effects of treatments, whereas vigorous wound contraction in wild type mice easily plaster effects. In the authors’ experience with the db/db mouse it is crucial to study the relative contribution to healing from re-epithelialization and contraction in order to better understand the effects of a therapy. For example, while for some wounds, such as a chronic foot ulcer, the most important goal is to achieve faster wound closure regardless from its mechanism, others would benefit mainly from inhibition of contraction (eg, joint wounds, wounds in cosmetic relevant positions, or burn wounds covering a larger surface).

References: 

1. International Diabetes Federation. Available at: http://www.idf.org/home/index.cfm. Accessed 2006.
2. Galiano RD, Tepper OM, Pelo CR, et al. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am J Pathol. 2004;164(6):1935–1947.
3. Goulimari P, Kitzing TM, Knieling H, Brandt DT, Offermann S, Grosse R. Galpha12/13 is essential for directed cell migration and localized Rho-Dia1 function. J Biol Chem. 2005;280(51):42242–42251.
4. Gottrup F, Agren MS, Karlsmark T. Models for use in wound healing research: a survey focusing on in vitro and in vivo adult soft tissue. Wound Repair Regen. 2000;8(2):83–96.
5. Harrop AR, Ghahary A, Scott PG, Forsyth N, Uji-Friedland A, Tredget EE. Regulation of collagen synthesis and mRNA expression in normal and hypertrophic scar fibroblasts in vitro by interferon-gamma. J Surg Res. 1995;58(5):471–477.
6. MacDonald IM, Pannu R, Kovithavongs K, Peters C, Tredget EE, Ghahary A. Effect of retinoic acid on expression of transforming growth factor-beta by retinal pigment epithelial cells in culture. Can J Ophthalmol. 1995;30(6):301–305.
7. Woodley DT, Wynn KC, O'Keefe EJ. Type IV collagen and fibronectin enhance human keratinocyte thymidine incorporation and spreading in the absence of soluble growth factors. J Invest Dermatol. 1990;94(1):139–143.
8. Frank S, Stallmeyer B, Kämpfer H, Kolb N, Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair. Faseb J. 1999;13(14):2002–2014.
9. Hager B, Bickenbach JR, Fleckman P. Long-term culture of murine epidermal keratinocytes. J Invest Dermatol. 1999;112(6):971–976.
10. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000;6(4):389–395.
11. Abdollahi A, Hahnfeldt P, Maercker C, et al. Endostatin’s antiangiogenic signaling network. Mol Cell. 2004;13(5):649–663.
12. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA. Definition of two angiogenic pathways by distinct alpha v integrins. Science. 1995;270(5241):1500–1502.
13. Bettinger D, Gore D, Humphries Y. Evaluation of calcium alginate for skin graft donor sites. J Burn Care Rehabil. 1995;16(1):59–61.
14. Calderon M, Lawrence WT, Banes AJ. Increased proliferation in keloid fibroblasts wounded in vitro. J Surg Res. 1996;61(2):343–347.
15. Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci U S A. 1979;76(3):1274–1278.
16. Grinnell F. Fibroblasts, myofibroblasts, and wound contraction. J Cell Biol. 1994;124(4):401–404.
17. Morykwas MJ. In vitro properties of crosslinked, reconstituted collagen sheets. J Biomed Mater Res. 1990;24(8):1105–1110.
18. Morykwas MJ, David LR, Schneider AM, et al. Use of subatmospheric pressure to prevent progression of partial-thickness burns in a swine model. J Burn Care Rehabil. 1999;20(1 Pt 1):15–21.
19. Morykwas MJ, Kennedy A, Argenta JP, Argenta LC. Use of subatmospheric pressure to prevent doxorubicin extravasation ulcers in a swine model. J Surg Oncol. 1999;72(1):14–17.
20. Buján J, Pascual G, Corrales C, Gomez-Gil V, Garcia-Honduvilla N, Bellon JM. Muscle-derived stem cells used to treat skin defects prevent wound contraction and expedite reepithelialization. Wound Repair Regen. 2006;14(2):216–223.
21. Reid RR, Mogford JE, Butt R, deGiorgio-Miller A, Mustoe TA. Inhibition of procollagen C-proteinase reduces scar hypertrophy in a rabbit model of cutaneous scarring. Wound Repair Regen. 2006;14(2):138–141.
22. Lee JP, Jalili RB, Tredget EE, Demare JR, Ghahary A. Antifibrogenic effects of liposome-encapsulated IFN-alpha2b cream on skin wounds in a fibrotic rabbit ear model. J Interferon Cytokine Res. 2005;25(10):627–631.
23. Said HK, Hijjawi J, Roy N, Mogford J, Mustoe T. Transdermal sustained-delivery oxygen improves epithelial healing in a rabbit ear wound model. Arch Surg. 2005;140(10):998–1004.
24. Lin MP, Marti GP, Dieb R, et al. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model. Wound Repair Regen. 2006;14(5):618–624.
25. Hata K, Fujitani M, Yasuda Y, et al. RGMa inhibition promotes axonal growth and recovery after spinal cord injury. J Cell Biol. 2006;173(1):47–58.
26. Chan RK, Liu PH, Pietramaggiori G, Ibrahim SI, Hechtman HB, Orgill DP. Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice. J Burn Care Res. 2006;27(2):202–205.
27. Falanga V. Wound healing and its impairment in the diabetic foot. Lancet. 2005;366(9498):1736–1743.
28. Ferrara N. VEGF as a therapeutic target in cancer. Oncology. 2005;69(Suppl 3):11–16.
29. Greenhalgh DG. Models of wound healing. J Burn Care Rehabil. 2005;26(4):293–305.
30. Pietramaggiori A, Kaipainen A, Ho D, et al. Trehalose lyophilized platelets for wound healing. Wound Repair Regen. 2007;15(2):213–220.
31. Pietramaggiori G, Kaipainen A, Czeczuga JM, Wagner CT, Orgill DP. Freeze-dried platelet-rich plasma shows beneficial healing properties in chronic wounds. Wound Repair Regen. 2006;14(5):573–580.
32. Sullivan SR, Underwood RA, Gibran NS, et al. Validation of a model for the study of multiple wounds in the diabetic mouse (db/db). Plast Reconstr Surg. 2004;113(3):953–960.
33. Falanga V, Schrayer D, Cha J, et al. Full-thickness wounding of the mouse tail as a model for delayed wound healing: accelerated wound closure in Smad3 knock-out mice. Wound Repair Regen. 2004;12(3):320–326.
34. Rerup CC. Drugs producing diabetes through damage of the insulin secreting cells. Pharmacol Rev. 1970;22(4):485–518.
35. Goodson WH 3rd, Hung TK. Studies of wound healing in experimental diabetes mellitus. J Surg Res. 1977;22(3):221–227.
36. Seifter E, Rettura G, Padawer J, Stratford F, Kambosos D, Levenson SM. Impaired wound healing in streptozotocin diabetes. Prevention by supplemental vitamin A. Ann Surg. 1981;194(1):42–50.
37. Cianfarani F, Zambruno G, Brogelli L, et al. Placenta growth factor in diabetic wound healing: altered expression and therapeutic potential. Am J Pathol. 2006;169(4):1167–1182.
38. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994;372(6505):425–432.
39. Goodson WH 3rd, Hunt TK. Wound collagen accumulation in obese hyperglycemic mice. Diabetes. 1986;35(4):491–495.
40. Chen H, Charlat O, Tartaglia LA, et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996;84(3):491–495.
41. Coleman DL. Diabetes-obesity syndromes in mice. Diabetes. 1982;31(Suppl 1 Pt 2):1–6.
42. Kaplan MM, Young JB. Abnormal thyroid hormone deiodination in tissues of ob/ob and db/db obese mice. Endocrinology. 1987;120(3):886–893.
43. Stallmeyer B, Kampfer H, Podda M, Kaufmann R, Pfeilschifter J, Frank S. A novel keratinocyte mitogen: regulation of leptin and its functional receptor in skin repair. J Invest Dermatol. 2001;117(1):98–105.
44. Stallmeyer B, Pfeilschifter J, Frank S. Systemically and topically supplemented leptin fails to reconstitute a normal angiogenic response during skin repair in diabetic ob/ob mice. Diabetologia. 2001;44(4):471–479.
45. Man LX, Park JC, Terry MJ, et al. Lentiviral gene therapy with platelet-derived growth factor B sustains accelerated healing of diabetic wounds over time. Ann Plast Surg. 2005;55(1):81–86.
46. Obara K, Ishihara M, Fujita M, et al. Acceleration of wound healing in healing-impaired db/db mice with a photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2. Wound Repair Regen. 2005;13(4):390–397.
47. Mulder G. Electroporatic delivery of TGF-beta1 gene works synergistically with electric therapy to enhance diabetic wound healing in db/db mice. J Invest Dermatol. 2004;123(4):xi.
48. Lee PY, Chesnoy S, Huang L. Electroporatic delivery of TGF-beta1 gene works synergistically with electric therapy to enhance diabetic wound healing in db/db mice. J Invest Dermatol. 2004;123(4):791–798.
49. Muangman P, Muffley LA, Anthony JP, et al. Nerve growth factor accelerates wound healing in diabetic mice. Wound Repair Regen. 2004;12(1):44–52.
50. Thenen SW, Mayer J. Adipose tissue glycerokinase activity in genetic and acquired obesity in rats and mice. Proc Soc Exp Biol Med. 1975;148(4):953–957.
51. Hamann A, Matthaei S. Regulation of energy balance by leptin. Exp Clin Endocrinol Diabetes.1996;104(4):293–300.
52. Rahmouni K, Haynes WG, Morgan DA, Mark AL. Intracellular mechanisms involved in leptin regulation of sympathetic outflow. Hypertension. 2003;41(3 Pt 2):763–767.
53. Kennedy WR, Wendelschafer-Crabb G, Johnson T. Quantitation of epidermal nerves in diabetic neuropathy. Neurology. 1996;47(4):1042–1048.
54. Pereira L, Matthes J, Schuster I, et al. Mechanisms of [Ca2+]i transient decrease in cardiomyopathy of db/db type 2 diabetic mice. Diabetes. 2006;55(3):608–615.
55. Greer JJ, Ware DP, Lefer DJ. Myocardial infarction and heart failure in the db/db diabetic mouse. Am J Physiol Heart Circ Physiol. 2006;290(1):H146–H153.
56. Carley AN, Semeniuk LM, Shimoni Y, et al. Treatment of type 2 diabetic db/db mice with a novel PPARgamma agonist improves cardiac metabolism but not contractile function. Am J Physiol Endocrinol Metab. 2004;286(3):E449–E455.
57. Garris DR. Variable onset determinants and consequences of diabetes (db/db) obesity mutation expression: adrenergic promotion of utero-ovarian dysfunction. Horm Metab Res. 2004;36(5):312–318.
58. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science. 1995;269(5223):546–549.
59. Halaas JL, Gajiwala KS, Maffei M, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 1995;269(5223):543–546.
60. Stephens TW, Basinski M, Bristow PK, et al. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 1995;377(6549):530–532.
61. Kieffer TJ, Heller RS, Leech CA, et al. Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells. Diabetes. 1997;46(6):1087–1093.
62. Mikhail AA, Beck EX, Shafer A, et al. Leptin stimulates fetal and adult erythroid and myeloid development. Blood. 1997;89(5):1507–1512.
63. Carlson MA, Longaker MT, Thompson JS. Wound splinting regulates granulation tissue survival. J Surg Res. 2003;110(1):304–309.
64. Reid RR, Said HK, Mogford JE, Mustoe TA. The future of wound healing: pursuing surgical models in transgenic and knockout mice. J Am Coll Surg. 2004;199(4):578–585.
65. Greenhalgh DG, Sprugel KH, Murray MJ, Ross R. PDGF and FGF stimulate wound healing in the genetically diabetic mouse. Am J Pathol. 1990;136(6):1235–1246.
66. Yannas I. Tissue and Organ Regeneration in Adults. New York, NY: Springer; 2001.
67. Michaels JT, Dobryansky M, Galiano RD, et al. Topical vascular endothelial growth factor reverses delayed wound healing secondary to angiogenesis inhibitor demonstration. Wound Repair Regen. 2005;13(5):506–512.
68. Klingbeil CK, Cesar LB, Fiddes JC. Basic fibroblast growth factor accelerates tissue repair in models of impaired wound healing. Prog Clin Biol Res. 1991;365:443–458.
69. Pietramaggiori G, Yang HJ, Scherer S, et al. Effects of poly-N-acetyl glucosamine (pGlcNAc) patch on wound healing in db/db mouse. J Trauma. In press.
70. Senter LH, Legrand EK, Laemmerhirt KE, Kiorpes TC. Assessment of full-thickness wounds in the genetically diabetic mouse for suitability as a wound healing model. Wound Repair Regen. 1995;3(3):351–358.

image description image description 





  


  
Post new comment


  



 
 




 
 




 
 



  • Lines and paragraphs break automatically.
  • Web page addresses and e-mail addresses turn into links automatically.
  • Use  to create page breaks.
More information about formatting options





Image CAPTCHA

 
 
 
Enter the characters shown in the image.