Povidoneiodine Application Induces Epithelium and Stromal Keratocytes Apoptosis in Pig Corneas

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Povidone-iodine Application Induces Epithelium
and Stromal Keratocytes Apoptosis in Pig Corneas

• Shu-Wen Chang, M.D.
• Department of Ophthalmology
• Far-Eastern Memorial Hospital

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Introduction

• Topical Povidone-iodine (PI) application before
  intraocular surgery and before removal of the
  corneo-scleral rim from the donor eye ball is
  widely used to reduce the incidence of
  endophthalmitis.
• However, PI solution causes cellular necrosis and
  apoptosis in vitro, and topical application of
  10 PI in vivo induces moderate corneal
  epithelial edema within 5 min of application.
• Although the cytotoxicity of PI on the corneal
  endothelium has been documented, its effect on
  the corneal epithelium and the stromal keratocyte
  is less well studied.

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Materials and Methods

• One hundred and sixty porcine eyes
• Group Ia 5 PI x 10 sec, with epithelium
• Group Ib 5 PI x 10 sec, without epithelium
• Group IIa 5 PI x 10 min, with epithelium
• Group IIb 5 PI x 10 min, without epithelium
• Corneo-scleral rims preserved in Optisol-GS, 4C
• One half of each cornea stained for HE, TUNEL,
  and caspase 3 to characterize apoptosis.
• The other half of each cornea ligation-mediated
  polymerase chain reaction (LM-PCR) to confirm the
  DNA laddering pattern indicative of apoptosis.

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• Results
• TUNEL staining of corneal epithelium is less
  noticeable in group Ia (Figure 1A1-A4) than that
  in group IIa (A5-A9). In group IIa, cytoplasmic
  vacuolization in the corneal epithelium is
  observed on day 3 (A7). The superficial corneal
  epithelium separates from the remaining
  epithelium on day 5 (A8, arrowheads). Wing cell
  necrosis, evident on day 7 (data not shown), is
  aggravated on day 14 in IIa (A9, arrowheads).
  Changes in keratocyte apoptosis (arrows) are more
  significant when the corneal epithelium was
  removed before preservation (B1-B8) than when
  preserved with intact epithelium (A1-A9).

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• Increased keratocyte apoptosis with longer
  preservation time is noticed in superficial
  stroma (Figure 2A1), mid-stroma (A2), and deep
  stroma (A3) of group Ia and IIa. The change is
  more obvious in group IIa than in group Ia.

• The percentage of apoptotic keratocytes is
  greater in the absence of the corneal epithelium
  (B1-B3). However, the difference between group Ib
  and IIb is less significant than that between
  group Ia and IIa.

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• Immediately following 5 povidone treatment,
  caspase 3 stained micrographs reveal negligible
  corneal epithelial apoptosis in group Ia (Figure
  3A1), while corneal epithelial apoptosis was
  evident in group IIa (B1, arrowheads). Corneal
  epithelial staining increases with longer
  preservation time in both group Ia (A2-4,
  arrowheads) and IIa (B1-4, arrowheads). The
  epithelial staining becomes less distinguishable
  after day 5 in both groups. Separation of the
  superficial epithelial sheet from the cornea is
  noticed after day 2 in group IIa (B3, thick
  arrows). Wing cell necrosis became significant
  after day 7 (B5-6, thick arrows). More stromal
  keratocyte apoptosis is observed in groups IIa
  and IIb (B1-6, C5-8, thin arrows) than in groups
  Ia and Ib (A1-7, C1-4, thin arrows). (
  represents 100 µm)

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• Figure 4. (A) Ligation mediated polymerase chain
  reaction detects the DNA laddering pattern,
  indicating corneal epithelial and keratocyte
  apoptosis in both groups Ia and IIa. DNA
  laddering in the corneal epithelium is less
  obvious on day 14 in group IIa (A), when the
  keratocyte laddering is still prominent in the
  stroma (B). DNA laddering pattern is noticed in
  keratocytes in all four groups (B).

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Discussion

• Povidone is hydrophilic and acts as a carrier of
  the iodine moiety to cell membranes. Once the
  povidone-iodine complex reaches the cell wall,
  the free iodine released is rapidly cytotoxic,
  killing the prokaryotic cell within 10 seconds.
  The groups undergoing PI treatment for 10 seconds
  could be regarded as treatment duration control
  group for the prolonged-10 minutes treatment
  group.
• In this study, we showed that application of 5
  PI for 10 minutes induced more corneal epithelium
  and keratocyte alterations than PI application
  for 10 seconds.
• Although it has been demonstrated that corneal
  epithelium and keratocyte death occur in the
  preservation medium, we showed for the first time
  that prolonged PI application before preservation
  aggravated epithelial and keratocyte apoptosis.
• Our results further suggest that the length of PI
  treatment before preservation also affects the
  integrity of corneal epithelium during
  preservation.
• We suggest that prolonged application of PI on
  the cornea should be avoided during preoperative
  preparation to minimize potential cytotoxicity.