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Updated: Jul 4, 2026
Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis
Published on: May 12, 2020
Substance P delays apoptosis, enhancing keratitis after Pseudomonas aeruginosa infection
Zimei Zhou1, Ronald P Barrett, Sharon A McClellan
1Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Purpose:
Apoptosis was examined after Pseudomonas aeruginosa corneal infection in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice.
Methods:
TUNEL staining, real-time RT-PCR, polymorphonuclear neutrophils (PMNs) and macrophage (Mphi) depletion, and immunostaining were used.
Results:
Intense TUNEL staining was seen in BALB/c versus B6 cornea at 1 versus 3 days after infection (PI) and correlated with mRNA levels for caspase-3. TUNEL staining (with or without PMN depletion) and PMN immunostaining revealed the PMN as the major apoptotic cell for both groups. Next, B6 mice with high corneal levels of the antiapoptosis neuropeptide, substance P (SP), were treated with the SP antagonist, Spantide I (with/without Mphi depletion), resulting in earlier apoptosis and diminished disease only when M(phi)s were present. SP interactions with M(phi)s were explored further by eliciting cells from both groups and stimulating them with lipopolysaccharide (LPS), with or without SP. LPS with SP treatment decreased the number of apoptotic M(phi)s in B6 but not BALB/c mice and correlated with reduced mRNA expression of NK-1R (major SP receptor) on BALB/c cells. In addition, mRNA expression for IL-12 was upregulated in LPS-stimulated B6 M(phi)s, although cells from BALB/c mice expressed more IL-10.
Conclusions:
These studies provide evidence that PMN apoptosis is delayed in the cornea of B6 versus BALB/c mice after bacterial infection; that in B6 mice, blocking SP interaction with the NK-1R promotes earlier apoptosis and improves disease outcome; that M(phi)s regulate PMN apoptosis; and that M(phi)s from B6 versus BALB/c mice differ in expression of the NK-1R and cytokines produced after LPS challenge.
Insights
Polymorphonuclear neutrophil (PMN) apoptosis is delayed in susceptible mice after Pseudomonas aeruginosa infection. Blocking substance P (SP) interactions with macrophages improves outcomes in these mice.
Area of Science:
- Ophthalmology
- Immunology
- Microbiology
Background:
- Pseudomonas aeruginosa corneal infection causes significant ocular morbidity.
- Understanding the host immune response, particularly apoptosis, is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the role of apoptosis in Pseudomonas aeruginosa corneal infection in susceptible (C57BL/6) and resistant (BALB/c) mice.
- To elucidate the mechanisms regulating polymorphonuclear neutrophil (PMN) and macrophage (Mphi) apoptosis during infection.
Main Methods:
- Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptosis detection.
- Real-time reverse transcription polymerase chain reaction (RT-PCR) for gene expression analysis.
- Depletion of PMNs and Mphis, and immunostaining to identify cell types and their roles.
Main Results:
- PMN apoptosis was significantly delayed in susceptible B6 mice compared to resistant BALB/c mice.
- Substance P (SP) and its antagonist Spantide I influenced PMN and Mphi apoptosis in B6 mice, with Mphis playing a regulatory role.
- Differential expression of the SP receptor (NK-1R) and cytokines (IL-12, IL-10) by Mphis from B6 and BALB/c mice was observed.
Conclusions:
- PMN apoptosis is a key factor in the differential susceptibility to Pseudomonas aeruginosa keratitis.
- Targeting the SP/NK-1R pathway in macrophages presents a potential therapeutic strategy to enhance apoptosis and improve disease outcomes in susceptible individuals.
- Macrophages are critical regulators of PMN apoptosis and immune responses during bacterial keratitis.
