Severe trauma and chronic stress activates extramedullary erythropoiesis

Abstract

Background: Severe traumatic injury is associated with bone marrow dysfunction that manifests as impaired erythropoiesis and prolonged hematopoietic progenitor cell (HPC) mobilization from the bone marrow. Extramedullary erythropoiesis, the development of red blood cells outside the bone marrow, has not been studied after severe injury and critical illness. This study examined the influence of lung contusion/hemorrhagic shock (LCHS) followed by chronic stress (CS) on the rodent spleen and to investigate the involvement of the splenic erythropoietin (EPO)/EPO receptor and BMP4 signaling.

Methods: Male Sprague-Dawley rats were subjected to LCHS and LCHS/CS. Animals underwent 2 hours of daily restraint stress until the day of sacrifice. On day 7, the spleen was assessed for weight, growth of splenic colony-forming units (CFU)-granulocyte-, erythrocyte-, monocyte- megakaryocyte (GEMM), burst-forming unit-erythroid (BFU-E), and CFU-E colonies, the presence of HPCs, and splenic mRNA expression of bone morphogenetic protein 4 (BMP4), EPO and its receptor. Data were presented as mean ± SD; *p < 0.05 vs. naïve and **p < 0.05 vs. LCHS by t test.

Results: On day 7, the addition of CS to LCHS increased spleen weight by 22%. LCHS/CS increased splenic growth of CFU-GEMM, BFU-E, and CFU-E colonies by 28% to 39% versus LCHS alone. Seven days after LCHS/CS, splenic HPCs increased from 0.60% to 1.12 % compared with naïve animals. After LCHS/CS, both BMP4 and EPO expression increased significantly in the spleen. Splenic EPO receptor (EPOr) expression decreased after LCHS/CS in the presence of a persistent moderate anemia.

Conclusion: Extramedullary erythropoiesis, manifest by increased splenic weight, splenic erythroid colony growth, splenic HPCs, BMP4, and EPO expression, is present in the spleen after LCHS/CS. Splenic EPOr expression was significantly decreased after LCHS/CS. Extramedullary erythropoiesis may play a key role in identifying new therapies to aid the recovery from acute anemia after severe trauma and chronic stress.

Figure 1

Spleen size and erythroid progenitor colony growth following LCHS and LCHS/CS. 1A. LCHS/CS significantly increased spleen weight in relation to total body weight compared to LCHS alone.1B–D. Daily CS exposure following LCHS resulted in increased growth in all three erythroid progenitor colonies in the spleen. LCHS= lung contusion and hemorrhagic shock; LCHS/CS= lung contusion, hemorrhagic shock and chronic restraint stress; *p <0.05 vs. naïve; **p <0.05 vs. LCHS.

Figure 2

Mobilization of HPCs to peripheral blood and spleen following LCHS and LCHS/CS. 2A. The addition of CS to LCHS results in significantly increased early HPC mobilization to the peripheral blood. 2B. Following LCHS/CS, there was an increase in early HPCs in the spleen compared to naive. 2C. The addition of CS following LCHS resulted in a high number of proliferating cells. LCHS= lung contusion and hemorrhagic shock; LCHS/CS= lung contusion, hemorrhagic shock and chronic restraint stress; ; S-G2M= Synthesis-Gap2 and Mitosis phase of the cell cycle; *p <0.05 vs. naïve; **p <0.05 vs. LCHS.

Figure 3

The addition of CS following LCHS significantly increased spleen BMP4 expression. LCHS= lung contusion and hemorrhagic shock; LCHS/CS= lung contusion, hemorrhagic shock and chronic restraint stress; *p <0.05 vs. naïve

Figure 4

Splenic EPO and EPOr expression and hemoglobin levels following LCHS and LCHS/CS. 4A. LCHS/CS increased EPO expression in the spleen. 4B. LCHS/CS resulted in significantly decreased EPOr expression compared to LCHS alone. 4C. LCHS/CS resulted in a persistent-injury associated anemia on day seven. LCHS= lung contusion and hemorrhagic shock; LCHS/CS= lung contusion, hemorrhagic shock and chronic restraint stress; *p <0.05 vs. naïve; **p <0.05 vs. LCHS.