Modulation of Cannabinoid System and Proinflammatory Cytokine mRNAs in Central and Peripheral Tissue in a Rodent E. coli Sepsis Model

Cytokine production is regulated by the endogenous cannabinoid system (ECS) in many pro‐inflammatory conditions. Sepsis is a highly morbid condition that results from a dysregulated host response to infection. Our previous work showed that ventilatory pattern variability (VPV) decreased concomitantly with increased pro‐inflammatory cytokine production. However, the ECS has not been integrated with transcriptional regulation of pro‐inflammatory cytokines nor with VPV during the progression to sepsis. We hypothesized that decreases in VPV correlate with increased cannabinoid receptor ( Cnr ) 1 & 2 and pro‐inflammatory cytokine mRNAs. We implanted a fibrin clot that contained either 0 or 25×10 6 E. coli in the peritoneal cavity of adult, male rats (n=24). Respiratory measures were obtained prior to clot implantation and again 1 hour (h) prior to euthanasia. At 3, 6, or 12 h following the clot implantation the rostral and caudal medulla, lung, heart and liver were harvested. VPV was computed with a group of linear and nonlinear dynamic measurements. Total RNA was extracted using Trizol reagent, and cDNA was synthesized using reverse transcription PCR. Quantitative real‐time PCR was performed in duplicate for each sample using TaqMan Gene® Expression Assays specific for 18S ribosomal RNA ( 18S ), Glyceraldehyde 3‐phosphate dehydrogenase ( Gapdh ), beta actin ( Actb ), Cnr1 & 2 , interleukin ( IL ) 1‐beta ( Il1b) & Il6 and tumor necrosis factor‐alpha ( Tnfa ). The most stable reference gene combination was determined using NormFinder software and each target gene was normalized to the geometric mean of these reference genes. Relative quantitation of gene expression was performed using the comparative CT method (2 − ΔΔ CT method) with the 0 E. coli fibrin clot groups (n=4/group) at each time point as the comparative control. At 12 h after inoculation, expiratory duration decreased [F(2,18) = 4.01, p = 0.036] and the ventilatory waveform became more predictable (increased mutual information [F(2,18) = 5.56, p = 0.013] and decreased sample entropy [F(2,18) = 4.25, p = 0.031]). The fold change in cytokine and ECS gene expression varied with tissue. At 12 h after inoculation gene expression increased in heart and rostral medulla, while gene expression decreased in lung, caudal medulla and liver. These preliminary data and analyses support our hypothesis that decreased VPV observed at 12 h is associated with increased expression of ECS and pro‐inflammatory cytokines in heart and rostral medulla. In lung and caudal medulla, however, expression of pro‐inflammatory cytokines decreased, while Cnr1 increased in caudal medulla and Cnr2 increased in lung tissue. Further research is needed to determine how modulation of the ECS can effectively regulate proinflammatory cytokine production and changes in VPV. Support or Funding Information NIH 5T32HL007913‐18, NIH U01 EB021960, VA Research Service I01BX004197