Whole body periodic acceleration improves survival and microvascular leak in a murine endotoxin model

Abstract

Sepsis is a life threatening condition which produces multi-organ dysfunction with profound circulatory and cellular derangements. Administration of E.Coli endotoxin (LPS) produces systemic inflammatory effects of sepsis including disruption of endothelial barrier, and if severe enough death. Whole body periodic acceleration (pGz) is the headward-footward motion of the body. pGz has been shown to induce pulsatile shear stress to the endothelium, thereby releasing vascular and cardio protective mediators. The purpose of this study was to determine whether or not pGz performed as a pre-treatment or post-treatment strategy improves survival in a lethal murine endotoxin model.This study was designed as a prospective randomized controlled study in mice. pGz was performed in mice as pre-treatment (pGz-LPS, 3 days prior to LPS), post-treatment (LPS- pGz, 30 min after LPS) strategies or Control (LPS-CONT), in a lethal murine model of endotoxemia. Endotoxemia was induced with intraperitoneal injection of E.Coli LPS (40mg/kg). In a separate group of mice, a nonspecific nitric oxide synthase inhibitor (L-NAME) was provided in their drinking water and pGz-LPS and LPS-pGz performed to determine the effect of nitric oxide (NO) inhibition on survival. In another subset of mice, micro vascular leakage was determined. Behavioral scoring around the clock was performed in all mice at 30 min intervals after LPS administration, until 48 hrs. survival or death. LPS induced 100% mortality in LPS-CONT animals by 30 hrs. In contrast, survival to 48 hrs. occurred in 60% of pGz-LPS and 80% of LPS-pGz. L-NAME abolished the survival effects of pGz. Microvascular leakage was markedly reduced in both pre and post pGz treated animals and was associated with increased tyrosine kinase endothelial-enriched tunica interna endothelial cell kinase 2 (TIE2) receptor and its phosphorylation (p-TIE2). In a murine model of lethal endotoxemia, pGz performed as a pre or post treatment strategy significantly improved survival, and markedly reduced microvascular leakage. The effect was modulated, in part, by NO since a non-selective inhibitor of NO abolished the pGz survival effect.

Fig 1. Behavioral scoring and survival Kaplan-Meyers curve in mice after lethal dose of LPS.

(A) Mean behavioral scores in mice every 30 mins, using scoring criteria of Schrum et al [24] in LPS-CONT, pGz pre-treatment (pGz-LPS) and pGz post-treatment (LPS-pGz). Scoring was begun 30 min after LPS injection and continued until death or survival for up to 48 hrs. (B) Survival in mice exposed to a lethal dose of LPS over the initial 48 hrs. Fifty percent survival in LPS treated mice was reached at 20 hrs. after LPS in control animals (LPS-CONT), in contrast all LNAME pretreated animals reached 50% survival at 12 hrs. 80% of LPS-pGz and 60% of pGz-LPS animals survived beyond 48 hrs. Data are mean ± SEM (†p<0.01 pGz-LPS or LPs-pGz vs. LPS-CONT ǂ p< 0.01 LPS-pGz vs. pGz-LPS, * p< 0.01 L-NAME-LPS-pGz or L-NAME-pGz-LPS or L-NAME-LPS-CONT vs LPS-CONT).

Fig 2. Microvascular leakage after LPS in mesenteric, lung and liver vasculature.

Mesenteric, lung and liver microvascular leakage determined by spectrophotometric optical density (OD) of Evans Blue, in Sham, LPS-CONT and pGz pretreated (pGz-LPS) (A) and (B) post-treated mice (LPS-pGz) mice. LPS-CONT (n = 8) pGz-LPS (n = 8), LPS-pGz (n = 8), or Sham (received Evans Blue but did not received LPS or pGz, n = 4). Data are mean ± SEM. (* p<0.01 vs. Sham, Ɨ p<0.01 vs. LPS-CONT).

Fig 3. Expression and phosphorylation of TIE2 in mice after LPS.

Expression of the tyrosine kinase receptor tunica interna endothelial cell kinase 2 (TIE2) and its phosphorylation in mice, 6 hrs. after LPS. LPS did not significantly change expression of TIE2, but significantly decreased TIE2 phosphorylation. (A) pGz pre-treatment (pGz-LPS) and (B) pGz post-treatment (LPS-pGz) significantly increased both TIE2 expression and phosphorylation. Inserts are representative western blots of protein expression of TIE2, p-TIE and GADPH in Sham, LPS-Control, and pGz-LPS and LPS-pGz. LPS-CONT (n = 8) pGz-LPS (n = 8), LPS-pGz (n = 8), or Sham (received equal volume of phosphate buffer but did not received LPS or pGz, n = 4). Data are mean ± SEM (* p<0.01 vs Sham, Ɨ p<0.01 vs. LPS-CONT).