Paneth cell ontogeny in term and preterm ovine models

Abstract

Introduction: Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent.

Methods: Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry.

Results: Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon.

Discussion: Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

Keywords: Paneth cell; immature intestine; ovine; perinatal bradycardic stress; preterm.

Figure 1

Representative histology. (A) Graphical diagram of methodology. (B) Representative term intestine stained with H&E and Alcian Blue Periodic Acid Schiff stain at 20 x magnification.

Figure 2

Paneth cell density varies by region and gestation along the small intestine of a newborn sheep. (A) Representative histology of preterm ileal samples at 40x and 60x stained with AB-PAS and anti-lysozyme techniques. Paneth cells are designated by arrows at 60X. (B) The ileal portion of the small intestine contained the highest average density of Paneth cells (3.72 Paneth cells/crypt, n = 8), followed by the jejunal small intestine (2.3 Paneth cells/crypt, n = 18). The colonic samples had no visible Paneth cells (0 Paneth cells/crypt, n = 18) (p < 0.0001 for all comparisons). (C) Matched samples that contained all three intestinal segments (n = 7) showing a similar distribution to the entire study population. In the matched series, the ileal Paneth cell numbers significantly differed from the colonic samples (p = 0.0015). There was a nonsignificant trend for increased Paneth cells in the ileum compared to the jejunum (p = 0.5). (D) Full-term control samples (140–141 days of gestation, n = 8 from Figure 1) had an average density of 3.72 Paneth cells/Crypt as compared to preterm ileal samples (126–127 days of gestation, n = 15), which had an average density of 2.86 Paneth cells/crypt (p = 0.013). (E) A linear regression model was calculated to interrogate the correlation between gestational age and Paneth cell density. As shown, there is a direct correlation between increasing gestational age and Paneth cell density (p = 0.016, R² = 0.25, Sy.x = 0.7).

Figure 3

Prenatal bradycardic stress is associated with decreased Paneth cell density. Ileal Paneth cell density was quantified for preterm sheep who had experienced fetal bradycardia (127 days gestation, n = 6) and compared to the Paneth cell density of age-matched preterm controls (126–127 days gestation, n = 15, Figure 3A). (A) Representative histology from control and bradycardic stress groups using both H&E and AB/PAS staining. (B) Preterm sheep who had experienced perinatal bradycardia show statistically significant depletions of Paneth cell densities (1.62 Paneth cell/crypt compared to 3.13 Paneth cell/crypt, p = 0.002).