Isolated Perfused Porcine Skin Flap

Ronald E. Baynes1

1 North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 5.4
DOI:  10.1002/0471140856.tx0504s08
Online Posting Date:  August, 2001
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Abstract

The isolated porcine skin flap is used to determine the rate and extent of absorption of chemicals through skin. This ex vivo model system allows cutaneous toxicology and pharmacology studies to be conducted in viable skin that has a normal structure and intact microvasculature. Porcine skin is also anatomically and physiologically similar to human skin, therefore data from studies using this ex vivo system can in some cases to predict dermatopharmacokinetics and dermotoxicity to test compounds in humans.

     
 
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Table of Contents

  • Basic Protocol 1: Operation of the Isolated Perfused Porcine Skin Flap
  • Support Protocol 1: Surgical Preparation of Porcine Skin Flap
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Operation of the Isolated Perfused Porcine Skin Flap

  Materials
  • Porcine skin flap (see protocol 2)
  • Oxygenated Krebs‐Ringer bicarbonate buffer
  • 4.5% bovine serum albumin medium (see recipe)
  • Air supply: 95% oxygen/5% carbon dioxide
  • Skin Bond (e.g., Skin‐Bond, Smith & Nephew)
  • Test chemical solution, with or without radiolabel
  • 1% (v/v) soap solution (e.g., ivory soap)
  • Ethyl acetate
  • OCT Compound (Tissue‐Tek, Miles)
  • Liquid nitrogen
  • 10% (v/v) neutral buffered formalin
  • Soluene 350 (Packard Instrument)
  • Sodium hypochlorite
  • Isopropyl alcohol
  • Custom‐designed flap chamber made of 0.5‐in thick Plexiglass and containing:
    • Custom‐designed humidifier
    • Custom‐designed media reservoirs
    • Hygrometer
    • Thermometer
    • Peristaltic pump
    • Silastic and tygon tubing
    • Pressure transducer
    • pH meter
    • Magnetic stirers
  • 3 × 7–cm flexible template (e.g., Stomahesive, ConvaTec‐Squibb)
  • Glucose analyzer (e.g., Glucose Analyzer 2, Beckman)
  • 5‐ml and 20‐ml capped scintillation vials (e.g., Sarstedt)
  • 2 × 2–in. gauze
  • Cellophane tape
  • Razor blade or scalpel
  • Forceps
  • Aluminum foil boat

Support Protocol 1: Surgical Preparation of Porcine Skin Flap

  Materials
  • 8‐week‐old weanling female Yorkshire pigs (Looper Farms, Hickory, NC)
  • Preanesthetic: 0.04 mg/kg atropine sulfate; 1.5 mg/kg xylazine hydrochloride; 11 mg/kg ketamine hydrochloride
  • Anesthesia: halothane
  • Betadine
  • Alcohol
  • Heparinized saline: 0.5 ml heparin in 50 ml physiological saline
  • Gowns and towels, disposable
  • Endotracheal tube (size 5.5 mm)
  • Hair clippers
  • Scalpels
  • Suture material (no. 0 and no. 2 chromic catgut; Prolene no. 3.0; Ethicon no. 3.0)
  • Surgical wrap and bandage (e.g., Teflon pad and elasticon)
  • Tygon tubing (0.023‐in i.d., 0.038‐in o.d.)
  • Hemostats
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Figures

Videos

Literature Cited

Literature Cited
   Allen, D.G., Riviere, J.E., and Monteiro‐Riviere, N.A. 2000. Induction of early biomarkers of inflammation produced by keratinocytes exposed to jet fuels Jet‐A, JP‐8, and JP‐8(100). J. Biochem. Molecular Toxicol. 14:231‐237.
   Baynes, R.E., Brownie, C., Freeman, H., and Riviere, J.E. 1996. In vitro percutaneous absorption of benzidine in complex mechanistically defined chemical mixtures. Toxicol. Appl. Pharmacol. 141:497‐506.
   Baynes, R.E., Monteiro‐Riviere, N.A., Qiao, G.L., and Riviere, J.E. 1997. Cutaneous toxicity of the benzidine dye direct red 28 applied as mechanistically‐defined chemical mixtures (MDCM) in perfused porcine skin. Toxicol. Lett. 93:159‐169.
   Behrendt, H. and Kampffmeyer, H.G. 1989. Absorption and ester cleavage of methyl salicylate by skin of single‐pass perfused rabbit ears. Xenobiotica 19:131‐141.
   Chang, S.K., Williams, P.L., Dauterman, W.C., and Riviere, J.E. 1994. Percutaneous absorption, dermatopharmacokinetics and related bio‐transformation studies of carbaryl, lindane, malathion, and parathion in isolated perfused porcine skin. Toxicology 91:269‐280.
   Dick, I.P. and Scott, R.C. 1992. Pig ear skin as an in vitro model for human skin permeability. J. Pharm. Pharmacol. 44:640‐645.
   Heit, M., Williams, P.L., Jayes, F.L., Chang, S.K., and Riviere, J.E. 1993. Transdermal iontophoretic peptide delivery. In vitro and in vivo studies with luteinizing hormone releasing hormone (LHRH). J. Pharm. Sci. 82:240‐243.
   King, J.R. and Monteiro‐Riviere, N.A. 1991. Effects of organic solvents on the viability and morphology of isolated perfused porcine skin. Toxicology 69:11‐26.
   Knecht, C.H., Allen, A.R., Williams, D.J., and Johnson, J.H., eds. 1987. Fundamental Techniques in Veterinary Surgery, 3rd ed. W.B. Saunders Co., Philadelphia, PA.
   Monteiro‐Riviere, N.A. 1991. Comparative anatomy, physiology, and biochemistry of mammalian skin. In Dermal and Ocular Toxicology: Fundamentals and Methods (D.W. Hobson, ed.) pp. 3‐71. CRC Press, Boca Raton, Fla.
   Monteiro‐Riviere, N.A. 1993. The use of isolated perfused skin in dermatotoxicology. In Vitro Toxicol. 5:219‐233.
   Riviere, J.E., Bowman, K.F., Monteiro‐Riviere, N.A., Dix, L.P., and Carver, M.P. 1986. The isolated perfused porcine skin flap (IPPSF). I. A novel in vitro model for percutaneous absorption and cutaneous toxicology studies. Fundam. Appl. Toxicol. 7:444‐453.
   Riviere, J.E., Sage, B.S., and Williams, P.L. 1991. The effects of vasoactive drugs on transdermal lidocaine iontophoresis. J. Pharm. Sci. 80:615‐620.
   Riviere, J.E., Williams, P.L., Hillman, R., and Mishky, L. 1992. Quantitative prediction of transdermal iontophoretic delivery of arbutamine in humans using the in vitro isolated perfused porcine skin flap IPPSF . J. Pharm. Sci. 81:504‐507.
   Riviere, J.E., Monteiro‐Riviere, N.A., and Williams, P.L. 1995. Isolated perfused porcine skin flap as an in vitro model for predicting transdermal pharmacokinetics. Eur. J. Pharm. Biopharm. 41:152‐162.
   Spoo, J.W., Rogers, R.A., and Monteiro‐Riviere, N.A. 1992. Effects of formaldehyde, DMSO, benzoyl peroxide, and sodium lauryl sulfate on isolated perfused porcine skin. In Vitro Toxicol. 5:251‐260.
   Vaden, S.L., Page, R.L., Williams, P.L., and Riviere, J.E. 1994. Effects of hyperthermia on cisplatin and carboplatin in the isolated, perfused tumor and skin flap. Int. J. Hyperthermia 10:563‐572.
   Wester, R.C., Melendres, J., Sedik, L., Maibach, H., and Riviere, J.E. 1998. Percutaneous absorption of salicylic acid, theophylline, 2, 4‐dimethylamine, diethyl hexyl phthalic acid, and p‐aminobenzoic acid in the isolated perfused porcine skin flap compared to man in vivo. Toxicol. Appl. Pharmacol. 151:159‐165.
   Zhang, A., Riviere, J.E., and Monteiro‐Riviere, N.A. 1995. Topical sulfur mustard induces changes in prostaglandins and interleukin 1alpha in isolated perfused porcine skin . In Vitro Toxicol. 8:149‐157.
Key Reference
   Riviere, et al., 1986. See above.
   The first paper to describe development of the skin flap and its application in dermal absorption and cutaneous toxicology studies.
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