Mesenchymal stem cells and their conditioned medium as potential therapeutic strategies in managing comorbid anxiety in rat sepsis induced by cecal ligation and puncture

Abstract

Objectives: Sepsis-associated encephalopathy (SAE) is a common brain dysfunction following sepsis. Due to the beneficial effects of mesenchymal stem cells (MSCs) therapy on anxiety, an extreme and early manifestation of SAE, we hypothesized that MSCs-derived conditioned medium (CM) may be able to attenuate anxiety in cecal ligation and puncture (CLP)-induced sepsis.

Materials and methods: Rats were assigned into 4 groups: sham, CLP, MSC, and CM. All animals, except in the sham group, underwent the CLP procedure to induce sepsis. Two hours after sepsis induction, the rats in MSC and CM groups, received 1×10⁶ MSCs and CM derived from the same number of cells, respectively. 48 hr after the treatments, anxiety-related behaviors were assessed, and brain and right hippocampal tissues were collected.

Results: MSCs and CM enhanced the percentages of open arm entries and time spent in the open arms of the elevated plus-maze and the time spent in the light side of the light-dark box. MSCs and CM decreased the Evans blue content and decreased the IL-6 and TNF-α levels in the brain tissue samples. Reductions in the expression of 5-HT2A receptors and phosphorylation of ERK1/2 and an increase in the expression of 5-HT1A receptors in the hippocampal tissue samples were observed in the MSC and CM groups.

Conclusion: MSCs and MSCs-derived CM attenuated anxiety-related behaviors to an equal extent by reducing inflammation, modifying 5-HT receptor expression changes, and inhibiting the ERK pathway. Therefore, MSCs-derived CM may be considered a promising therapy for comorbid anxiety in septic patients.

Keywords: Extracellular signal-regulated kinases; Inflammation; Mesenchymal stem cells; Sepsis; Sepsis-associated-encephalopathy; Serotonin.

Figure 1

Experimental protocol of the study

Figure 2

Flow cytometry analysis of cell surface markers of mesenchymal stem cells (MSCs) derived from Wistar rat adipose tissues. Non-stained MSCs (a). Passage 2 MSCs were positive for cluster of differentiation (CD) 44 and CD90 (mesenchymal markers) (b and c) while being negative for CD34 and CD45 (hematopoietic markers) (d and e)

Figure 3

Characterization of Wistar rat adipose tissue-derived mesenchymal stem cells (MSCs). The adherent cells were mostly spindle (fibroblastic morphology) (a). Differentiation into osteocytes was evaluated by Alizarin Red staining for calcified deposits (b). Adipocyte differentiation was detected by Oil Red O staining for lipid droplets and morphological changes from spindle into round (c). Magnification of all images: × 100

Figure 4

Changes of percentages of the open arm entries (a) and time spent in the open arms (b) as well as total entries (c) in the different groups. The data are expressed as mean±SEM (n= 12 in each group). * P<0.05 versus the sham group. ^##P<0.01 and ^###P<0.001 versus the CLP group

Figure 5

Changes in the time spent in the light side of the light-dark box in different groups. Data are expressed as mean±SEM (n=12 in each group). *P<0.05 versus the sham group. ^###P<0.001 versus the CLP group

Figure 6

Changes of Evans blue content in the brain tissue samples in the different groups. The data are expressed as mean±SEM (n=4 in each group). *P<0.05 versus the sham group. ^#P<0.05 versus the CLP group

Figure 7

Changes in the levels of interleukin 6 (IL-6) (a) and tumor necrosis factor-alpha (TNF-α) (b) in the brain tissue samples in the different groups. The data are expressed as mean±SEM (n=6 in each group). **P<0.01 and ***P<0.001 versus the sham group. ^##P<0.01 and ^###P<0.001 versus the CLP group

Figure 8

Changes in the protein expression levels of 5-HT1A receptors (a) and 5-HT2A receptors (b) as well as phosphorylation levels of extracellular signal-regulated kinases (ERK) 1/2 (c) in the hippocampal tissue samples in different groups. The data are expressed as mean±SEM (n=4 in each group). * P<0.05 versus the sham group. ^#P<0.05 versus the CLP group

Figure 9

Schematic design of the beneficial effects of MSCs and MSCs-derived CM in attenuating anxiety-related behaviors following sepsis. It is possible that MSCs and MSCs-derived CM are able to decrease inflammation, modify 5-HT receptor expression changes and inhibit the ERK pathway