Flow Cytometric Analysis of Hematopoietic Populations in Rat Bone Marrow. Impact of Trauma and Hemorrhagic Shock

Abstract

Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data are largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 h following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, and IgM) or myeloid (CD11b, CD45, and RP‐1) lineages. Subsequently, cell populations were fluorescence‐activated cell sorted for morphological assessment. Stage‐specific cell populations were identified using a limited number of antibodies, and leucopoietic changes were determined 6 h following trauma and HS. Myeloid subpopulations could be identified by varying levels CD11b expression, CD45, and RP‐1. Trauma and HS resulted in a significant reduction in total CD11b + myeloid cells including both immature (RP‐1(−)) and mature (RP‐1+) granulocytes. Multiple B‐cell lymphoid subsets were identified. The total percentage of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(−) cells suggesting movement into the periphery. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Therapeutic Potential of Mesenchymal Stromal Cell-Derived Extracellular Vesicles in the Prevention of Organ Injuries Induced by Traumatic Hemorrhagic Shock

“…Bone marrow stroma failed to grow to confluence by day 14 in >90% of trauma patients. These data indicate that trauma induces a bone marrow dysfunction that releases immature white blood cells into circulation and may also contribute to a failure to clear infection and an increased propensity to organ failure (123,124). Moreover, in pathophysiological conditions such as trauma, a partial blockade in the differentiation of immature myeloid cells into mature myeloid cells results in an expansion of this population called myeloid-derived suppressor cells (MDSCs), which have remarkable ability to suppress T-cell responses and to modulate macrophage cytokines (125).…”

Murine Myeloid Progenitors Attenuate Immune Dysfunction Induced by Hemorrhagic Shock

“…In rodent models of hemorrhagic shock, it has been demonstrated that, in addition to suppressive neutrophil mobilization, there is an increase in hematopoietic progenitor cell (HPC) mobilization from the bone marrow, which may serve to modulate the inflammatory response to hemorrhage ( Francis et al., 2019 ; Xiang et al., 2012 ). In fact, injection of bone marrow-derived hematopoietic stem cells (HSCs) has been shown to decrease mortality and organ dysfunction in response to cecal ligation and puncture ( Mei et al., 2010 ; Nemeth et al., 2009 ).…”

Pharmacological inhibition of the lipid phosphatase PTEN ameliorates heart damage and adipose tissue inflammation in stressed rats with metabolic syndrome

“…Second, we did not examine the bone marrow. Since both cells of the monocyte‐macrophage lineage and B cells are generated in the bone marrow after birth, flow cytometric analysis of hematopoietic (myeloid or lymphoid) populations in the bone marrow may also be useful in rats (Francis et al, 2019 ) Future studies may be considered to determine the effects of bpV(pic) on immune cells including macrophages and B cells in the bone marrow. Finally, although our data support the notion that bpV(pic) induces activation of Akt through inhibition of PTEN, we have no data for Akt activity with or without bpV(pic) in LV and fat tissue of stressed and nonstressed animals.…”

“…In conclusion, inhibition of PTEN by bpV(pic) ameliorated LV and AT pathology in DS/obese rats subjected to restraint stress. Such amelioration was likely mediated by two key mechanisms: (Baumgarth, 2011 ) reduced blood pressure and promotion of coronary capillary formation via activation of a PI3K–Akt–HIF‐1α–VEGF–eNOS/NO pathway, resulting in suppression of LV inflammation, fibrosis, and diastolic dysfunction and (Francis et al, 2019 ) alteration of the distribution of immune cells in AT, without a change in fat mass, induced by activation of PI3K–Akt signaling, and resulting in attenuation of AT inflammation. Further studies are warranted to uncover the precise molecular mechanisms underpinning these actions of PTEN inhibition in MetS associated with stress.…”