Abstract
Purpose::
The purpose of this study was to examine the pattern of apoptosis in the cornea of P. aeruginosa infected C57BL/6 (B6, susceptible) vs. BALB/c (resistant) mice. Ancillary to this purpose was to determine the role of the anti-apoptotic neuropeptide, Substance P (SP), expressed at higher levels in cornea in B6 over BALB/c mice.
Methods::
Mice were infected with P. aeruginosa and disease severity was graded using clinical score. TUNEL staining was used to selectively identify apoptotic cells in cornea. Microarray was used to determine fold changes in apoptotic genes at 1 day p.i. These data were confirmed and extended using real time PCR to measure mRNA levels for caspase 3 (effector), caspase 9 (initiator) and Bcl-2 (anti-apoptotic). In another series of experiments B6 mice were injected with PBS (control) or Spantide I to block SP interaction with its major receptor, NK-1R before infection. Corneas were similarly analyzed as above.
Results::
: In BALB/c mouse cornea, intense TUNEL positive staining was seen at 1 day p.i., but was decreased substantially by 3 days p.i. In contrast, in B6 mice, intense Tunel staining was not observed until 3 days p.i. Real time PCR confirmed the gene array data and showed that at 1 day p.i., corneal mRNA levels for caspases 3 and 9 were significantly upregulated in BALB/c compared to B6 mice. B6 over BALB/c mice exhibited increased corneal Bcl-2 expression at 18 h p.i., agreeing with the caspase data. mRNA for caspase 3 was significantly greater in B6 vs. BALB/c mice at 3 days p.i., consistent with the apoptotic pattern revealed by TUNEL staining. In the next series of experiments, treatment of B6 mice with Spantide I vs. PBS resulted in fewer perforated corneas at 5 days p.i., earlier (1 day p.i.) TUNEL positive staining and increased mRNA levels in cornea for caspases 3 and 9.
Conclusions::
Apoptosis is delayed in the cornea of susceptible B6 vs. resistant BALB/c mice after bacterial infection. Blocking the effects of the anti-apoptotic neuropeptide SP in B6 mice with Spantide I enhanced earlier apoptosis in the infected cornea and resulted in improved disease outcome.
Keywords: apoptosis/cell death • neuropeptides • Pseudomonas