Traumatic injury induces a state of systemic inflammation associated with release of proinflammatory cytokines (Dinarello, 2000). Trauma patients exhibit elevations in circulating pro-inflammatory cytokines like C-reactive protein (CRP), interleukin-1α and 1β (IL-1α and IL-1β), interleukin-6 (IL-6), nitric oxide (NO), and tumor necrosis factor alpha (TNFα) immediately after injury (Volpin et al., 2014). This occurs in conjunction with decreased levels of anti-inflammatory cytokines such as interleukin-10 (IL-10), which stimulates erythroid colony growth (Wang et al., 1996). Proinflammatory signaling factors promote myeloid lineage expansion at the expense of erythroid progenitors (IL-1α, IL-1β), induce production of iron-sequestering peptide hepcidin (IL-6) that leads to inappropriate hypoferremia in the setting of acute blood loss anemia, and directly inhibit erythroid progenitor growth (TNFα) (Johnson et al., 1991; Rusten and Jacobsen, 1995; Nemeth et al., 2004; Raj, 2009; Pietras et al., 2016). Nitric oxide signaling results in increased IL-6 and HPC pro-mobilization factor, in addition to granulocyte-colony stimulating factor (G-CSF) (Hierholzer et al., 1998). Elevated CRP is an acute phase reactant and indicator of inflammation which is associated with anemia (Heidari et al., 2015). The alteration of these cytokine levels after trauma negatively impacts erythropoiesis in these patients.

Traumatic injury also causes damage to red blood cells. A study of trauma and hemorrhagic shock in rats demonstrated that red blood cells have decreased deformability, measured by elongation index, after shock which correlates with the degree of physiologic insult (Zaets et al., 2003). Another rat study of trauma and hemorrhagic shock showed that red blood cell damage also contributes to organ hypoperfusion leading to organ failure, possibly as a result of erythrophagocytosis in tissues, a phenomena observed in septic subjects (Machiedo et al., 2009). Finally, red blood cell metabolism is altered after hemorrhagic shock, with a dependence on glutamine for glutathione synthesis and alanine transamination (Reisz et al., 2017). These findings illustrate how trauma changes red blood cells and impacts organ perfusion independent of the hypovolemic state of hemorrhagic shock.

Trauma induces mobilization of HPCs from the bone marrow to the peripheral blood and sites of tissue injury (Livingston et al., 2003; Badami et al., 2007; Shah et al., 2009; Cook et al., 2013). In animal studies, HPC mobilization to injured tissues is essential for accelerated wound healing (Badami et al., 2007; Shah et al., 2009; Hannoush et al., 2011). Plasma from trauma patients demonstrate sustained peripheral blood HPC for up to 10 days after injury (Cook et al., 2013). In normal erythropoiesis, multipotent progenitor cells differentiate into multipotential colony-forming units (CFU-GEMM), erythroid burst-forming units (BFU-E), and erythroid colony-forming units (CFU-E), and then erythroblasts, ultimately developing into erythrocytes (Figure 1). Erythroid progenitors such as these are shown to be consistently suppressed in both trauma patients and animal studies (Livingston et al., 2003; Badami et al., 2007; Loftus et al., 2018b). In contrast, other proteins like stem cell factor (SCF) and B-cell lymphoma-extra-large (Bcl-xL) have been linked to the promotion of erythropoiesis (Alter et al., 1992; Afreen et al., 2020). The damage-associated high-mobility group box-1 (HMGB1) protein suppresses bone marrow cellularity and mediates an anemia that has been reversed with administration of anti-HMGB1 monoclonal antibodies in mice (Valdes-Ferrer et al., 2016). Suppression of CFU-E has also been observed when human bone marrow was cultured with HMGB1 in vitro (Dulmovits et al., 2022).

A variety of factors affect the balance between retention and mobilization of hematopoietic progenitor cells in the bone marrow. Granulocyte colony-stimulating factor (G-CSF) is well established to promote hematopoietic progenitor cell mobilization (Tesio et al., 2011). It induces stromal-cell derived factor-1 (SDF-1) secretion into the circulation to attract HPC (Saba et al., 2015). Additional factors which promote HPC mobilization include hepatocyte growth factor (HGF), tyrosine-protein kinase Met (c-Met), matrix-metalloproteinase-9 (MMP-9), and matrix-metalloproteinase-2 (MMP-2) (Heissig et al., 2002; Tesio et al., 2011; Klein et al., 2015; Tari et al., 2017). Tissue inhibitor of metalloproteinase-1 (TIMP-1) and tissue inhibitor of metalloproteinase-2 (TIMP-2) are metalloproteinase inhibitors; TIMP-1 binds MMP-9 and TIMP-2 binds MMP-2 and subsequently inhibit their actions such as HPC mobilization (Parks et al., 2004). In murine studies, plasma corticosterone, a glucocorticoid, promotes HPC migration through glucocorticoid receptor Nr3c1-dependent signaling (Pierce et al., 2017). Murine studies also demonstrate that high mobility group box-1 (HMGB1) mediates macrophage secretion of G-CSF, inducing HPC mobilization (Xiang et al., 2012). Stromal cell-derived factor-1 (SDF-1), also known as C-X-C motif chemokine ligand 12 (CXCL12), and its receptor C-X-C chemokine receptor type 4 (CXCR4) which is present on hematopoietic progenitor cells, mediate retention of HPC in bone marrow stroma (Greenbaum and Link, 2011; Bonig and Papayannopoulou, 2012). In addition, HPC express very late antigen-4 (VLA-4), an adhesion molecule which binds to vascular cell adhesion molecule-1 (VCAM-1) to retain HPC in bone marrow (Bonig and Papayannopoulou, 2012). Neutrophil elastase cleaves VCAM-1 to induce HPC mobilization (Levesque et al., 2001). These factors affecting HPC mobilization from the bone marrow play a pivotal role in understanding the underlying mechanisms of bone marrow dysfunction after trauma, manifesting as persistent anemia.

Norepinephrine and epinephrine are catecholamines secreted by the adrenal medulla and act on adrenergic receptors (sometimes referred to as adrenoreceptors), which are classified into α₁, α₂, β₁, β₂, and β₃ subtypes (Maestroni, 2020; Wu et al., 2021). Norepinephrine has a greater affinity for alpha receptors and epinephrine has a stronger affinity for beta receptors (Maestroni, 2020). Activation of α₁ receptors result in peripheral vasoconstriction, whereas centrally located α₂ receptor activation inhibits the release of additional norepinephrine (Wu et al., 2021). Beta-1 receptors are the predominant adrenergic receptor in the heart and activation results in increased rate of nodal firing and conduction as well as increased cardiac contractility (Wu et al., 2021). Beta-2 receptors are found at a lower density compared to beta-1 receptors in the heart; they are also located in smooth muscle, including smooth muscle of the airways where they play a clinical role in bronchoconstriction when activated (Wu et al., 2021). Beta-3 receptors are involved in lipolysis in adipose tissue; they are also present in the myocardium but have controversial effects on cardiac function (Arioglu-Inan et al., 2019; Schena and Caplan, 2019). Beta-3 receptor activation in human studies have either demonstrated minimal cardiac changes or negative inotropic effects (Arioglu-Inan et al., 2019). Hypercatecholamine states result in activation of these receptors and induction of downstream effects, including increased cardiac contractility and smooth muscle constriction.

In humans and animals, elevated catecholamine levels persist after injury (Fonseca et al., 2004; Loftus et al., 2018b). Rodent trauma models demonstrate significantly increased urine norepinephrine which persists 7 days after injury (Bible et al., 2015a; Alamo et al., 2017b; Loftus et al., 2018a). Trauma patients admitted to the surgical intensive care unit (SICU) demonstrate significantly increased urine norepinephrine for 10 days and epinephrine for 7 days compared to healthy controls (Fonseca et al., 2004). An observational study of blunt trauma patients also demonstrated significantly higher circulating norepinephrine compared to control patients who underwent total hip replacement (Loftus et al., 2018b). Similar to trauma, burn injury has been shown to lead to significant elevation of cortisol, catecholamines, cytokines and acute phase proteins for up to 3 years post-burn (Jeschke et al., 2011). This state of hypercatecholaminemia has varying physiologic implications, including increased expression of erythropoietic-inhibiting cytokines such as IL-1α, interleukin-2 (IL-2), IL-6, IL-10 and transforming growth factor beta (TGF-β) (Saba et al., 2017). Given this rise in catecholamine levels and its effects on cytokine release, hypercatecholaminemia has been a point of interest for further investigation as a contributing factor to persistent anemia after trauma.

Iron is an essential component in erythropoiesis (Weiss et al., 2019). Every second, almost two million erythrocytes are produced in the bone marrow, which requires up to 30 mg of iron daily for synthesis of hemoglobin (Gkouvatsos et al., 2012; Weiss et al., 2019). Erythropoiesis becomes iron dependent during hemoglobin synthesis, which begins at the proerythoblast stage (Weiss et al., 2019). As a result, erythropoiesis can be substantially impacted by states of functional iron deficiency.

Inflammatory states disrupt the iron homeostasis necessary for adequate erythropoiesis (Sangkhae and Nemeth, 2017). The liver plays a key role in these processes. It is the predominant producer of hepcidin, a peptide hormone that serves as a gatekeeper for release of free iron by causing internalization and degradation of iron-transport protein ferroportin (Figure 2) (Sangkhae and Nemeth, 2017; Lanser et al., 2021). It is also a major source of IL-6, which increases hepcidin expression and in turn promotes iron sequestration (Sangkhae and Nemeth, 2017). Additionally, IL-1β is another potent inducer of hepcidin expression (Gkouvatsos et al., 2012). The widespread inflammation and increase in systemic hepcidin seen in trauma patients lead to iron sequestration and reduced availability of the raw materials necessary for hemoglobin synthesis during erythropoiesis (Sangkhae and Nemeth, 2017; Loftus et al., 2018b).

In vitro studies demonstrate the direct effects of hypercatecholaminemia on erythropoiesis. One study which cultured iliac crest bone marrow from trauma patients demonstrated BFU-E and CFU-E growth decline at supraphysiologic catecholamine levels, starting at 10⁻⁷ M (Fonseca et al., 2004). This is similar to levels of circulating norepinephrine measured in severely injured trauma patients with a mean of 44.1 ng/ml (2.6 × 10⁻⁷ M) (Apple et al., 2020a). However, this decrease in erythroid progenitor cell growth was not observed in bone marrow depleted of stroma, suggesting that bone marrow stroma plays an essential role in the maturation of erythroid progenitor cells (Fonseca et al., 2004). A follow-up study of human bone marrow cells cultured in the presence of epinephrine or norepinephrine at increasing doses demonstrated a decline in BFU-E and CFU-E in a dose-dependent manner at supraphysiologic levels (Fonseca et al., 2005). It is known that the bone marrow is innervated with sympathetic fibers which affect erythropoiesis (Maestroni, 2020). When chemical sympathectomy was performed in rodents and norepinephrine pumps implanted for administration of increasing doses of norepinephrine for 5 days, rodents had decreased erythroid progenitor growth in a dose-dependent fashion when bone marrow was cultured in vitro (Penn et al., 2011). These in vitro studies suggest dose-dependent effects of norepinephrine and epinephrine on erythroid progenitor growth and erythropoiesis.

Effects of catecholamines on terminal erythropoiesis continue to be investigated. Terminal erythropoiesis is the process by which erythroblasts accumulate hemoglobin, mature, and finally expel their nuclei to become reticulocytes, as shown in Figure 1. This process occurs on the erythroblastic island (EBI), which is comprised of a central macrophage surrounded by developing CFU-E, erythroblasts, and recently enucleated reticulocytes (Chasis and Mohandas, 2008). These islands can be found within both bone marrow and spleen, where they serve as a niche for the proliferation, differentiation and enucleation of erythroid precursors ultimately leading to the production of erythrocytes (Chasis and Mohandas, 2008). Bone marrow-derived rabbit erythroid cells undergo an uncoupling of their beta adrenergic receptors when cultured in catecholamine-enriched media; this uncoupling occurs early in erythroblast development (Setchenska and Arnstein, 1983; Setchenska et al., 1983). Much remains unknown about what effects adrenergic modulation could exhibit on terminal erythropoiesis and EBIs.

Some mechanisms behind these effects of supraphysiologic levels of catecholamines on erythropoiesis have been identified. Sympathetic nervous system stimulation is associated with release of G-CSF which in turn promotes HPC mobilization (Katayama et al., 2006; Saba et al., 2015; Gao et al., 2021). Particularly, β-2 and β-3 adrenoreceptors are critical in HPC mobilization, likely associated with inhibition of SDF-1 (Mendez-Ferrer et al., 2010). In addition, it has been shown that HPC express α-1, α-2, and β-2 adrenergic receptors, of which there is higher expression of β-2 on HPC when mobilized via G-CSF, suggesting that mobilization may increase catecholamine sensitivity (Muthu et al., 2007; Spiegel et al., 2007).

The use of adrenergic modulation in the trauma population has established clinical relevance in the setting of hypercatecholaminemia. Elevated levels of epinephrine are associated with increased mortality after trauma (Johansson et al., 2012). The use of beta-adrenergic blockade in trauma patients improves mortality, possibly by decreasing hypermetabolism and cerebral oxygen requirement (Arbabi et al., 2007). Beta blockade in trauma patients has also decreases IL-6 levels (Friese et al., 2008). Burn patients treated with beta antagonists demonstrate improved wound healing and a decreased surface area required for skin grafting (Mohammadi et al., 2009; Ali et al., 2015). In addition, burn patients who are treated with beta blockade have attenuated hypermetabolism and reversal of muscle-protein catabolism (Herndon et al., 2001). With adrenergic modulation already in use in this patient population, and given the effects of hypercatecholaminemia on erythropoiesis, the application of alpha agonism and beta blockade as therapeutic interventions to prevent persistent anemia after injury were of particular interest. A thorough literature search of studies involving adrenergic modulation of erythropoiesis after trauma was performed to identify articles for this comprehensive review. While many of these studies originate from the authors, all studies that were relevant to this topic have been included.

Alpha agonist medications act by inhibiting sympathetic outflow from the central nervous system, which has been shown to decrease both cerebrospinal and peripheral norepinephrine and normetanephrine and reduce plasma epinephrine (Sullivan et al., 1986). Clonidine is a widely utilized medication that stimulates α-2 adrenoceptors in the central nervous system to inhibit sympathetic outflow, resulting in reductions in heart rate, blood pressure, and peripheral vascular resistance (Campese et al., 1980; Sullivan et al., 1986; Dorman et al., 1997). In general, patients who receive clonidine (not limited to trauma patients) exhibit reduced circulating plasma norepinephrine and epinephrine (Campese et al., 1980; Sullivan et al., 1986; Payen et al., 1990; Dorman et al., 1997). Clonidine is typically used to treat hypertension, but its efficacy in reducing the sympathetic response has made it a drug of interest among researchers seeking novel treatments for persistent anemia in injured patients.

Much of what is known about the effects of alpha agonism on erythropoiesis is based on preclinical studies, particularly on rat models of trauma. This involves either lung contusion (LC), lung contusion with chronic daily restraint stress (LC + CS), hemorrhagic shock (HS), lung contusion with hemorrhagic shock (LCHS), or lung contusion with hemorrhagic shock and chronic daily restraint stress (LCHS + CS). Chronic daily restraint stress alone (CS) is used to identify how stress, meant to simulate SICU stay, affects outcomes. Rodent trauma models represent different injuries, such as blunt trauma (lung contusion), hemorrhage and subsequent stay in a SICU (chronic daily restraint stress). Despite these parallels in the model to human blunt trauma and hospitalization, patients often have multiple injuries including fractures which are not represented by this model such as fractures and traumatic brain injury among others. Rat models also do not involve post-operative or post-injury infection, such as wound infection or pneumonia. In addition, rats who undergo hemorrhagic shock are resuscitated by re-transfusion of their own blood, different from human resuscitation with allogenic blood. Another consideration is that hemorrhagic shock in rats does not produce the same tachycardia observed in humans in hemorrhagic shock (Weber et al., 2019). A summary of animal studies which are reviewed can be found in Table 1. These models have been validated to effectively influencing a variety of factors involved in erythropoiesis as will be discussed in the following sections. The models of LC, LCHS and LCHS + CS affect expression of hematopoietic cytokines, HMGB1, SCF, Bcl-xL; urine norepinephrine; hemoglobin; plasma G-CSF; HPC mobilization; plasma corticosterone; bone marrow cellularity; and erythroid progenitor cell growth (Alamo et al., 2017b; Loftus et al., 2019; Loftus et al., 2020).

Clonidine demonstrates effects on factors attributed to bone marrow dysfunction and anemia in animal studies. As described previously, trauma results in elevated levels of urine norepinephrine in rodents (Bible et al., 2015a; Alamo et al., 2017b; Loftus et al., 2018a). Elevated levels of urine norepinephrine significantly decreased by nearly half among rats that received daily intraperitoneal clonidine injections after CS and LCHS + CS, with n = 6–8 per group (Alamo et al., 2017b). In a rodent model of LCHS with or without chronic restraint stress, rodents showed a significant increase in bone marrow expression of HMGB1 and decrease in bone marrow expression of SCF and IL-10 compared to control rats (Loftus et al., 2019). Bone marrow expression of Bcl-xL was not significantly altered after LCHS or LCHS + CS (Loftus et al., 2019). In groups which underwent LCHS or LCHS + CS followed by daily administration of clonidine, bone marrow expression of HMGB1 was significantly decreased by 47%–54% respectively compared to untreated rodents (Loftus et al., 2019). LCHS and LCHS + CS groups treated with clonidine demonstrated a significant increase of 468% and 1,062% in bone marrow SCF; bone marrow Bcl-xL also exhibited a significant increase of 77% in LCHS + CS after clonidine treatment compared to untreated counterparts (Loftus et al., 2019). This study demonstrated how clonidine decreased inhibitory cytokines and growth factors and increased pro-erythropoietic factors, supporting positive effects of this medication on erythropoiesis.

Given these results, another point of interest has been the effects of alpha agonism on HPC mobilization, erythroid progenitor growth, and hemoglobin. In a rodent model of trauma consisting of chronic stress, LCHS, or LCHS and chronic stress with or without daily clonidine administration, groups receiving daily stress who were treated with daily intraperitoneal clonidine demonstrated significantly decreased peripheral HPC by 66% and 84% compared to their untreated counterparts (Alamo et al., 2017b). This study also found significantly decreased growth of CFU-E, BFU-E, and CFU-GEMM in bone marrow-derived cells from all groups subjected to stress and/or trauma, but that such reductions could be prevented with clonidine administration, showing significantly increased levels of CFU-E, BFU-E, and CFU-GEMM when compared to untreated groups (Alamo et al., 2017b). Another study of LCHS with chronic stress also demonstrated an increase in peripheral HPC which was significantly decreased after clonidine administration (Loftus et al., 2020). Factors such as plasma corticosterone, HGF, MMP-9, c-Met, and G-CSF all play roles in HPC mobilization as previously described. Among rats subjected to LCHS with chronic restraint stress, plasma corticosterone levels were significantly elevated compared to controls; these levels were subsequently significantly decreased by 125 ng/ml when administered daily intraperitoneal clonidine compared to untreated counterparts (Loftus et al., 2020). In a rodent model of LC, LCHS, or LCHS and chronic stress with or without clonidine administration, rodents who underwent LC or LCHS + CS showed significantly elevated levels of bone marrow HGF and MMP-9 compared to controls (Loftus et al., 2020). Clonidine administration resulted in significant decline in bone marrow MMP-9 and c-Met compared to untreated rats in LCHS + CS and LC, respectively (Loftus et al., 2020). Levels of bone marrow HGF were not significantly different in rats treated with clonidine compared to untreated (Loftus et al., 2020). In LCHS + CS, plasma G-CSF was significantly elevated and was subsequently decreased by 44% when each group was treated with clonidine (Alamo et al., 2017b). Therefore, clonidine decreases HPC mobilization and inhibits pro-mobilizing factors such as plasma G-CSF, bone marrow c-Met and bone marrow MMP-9. Finally, rodent studies using lung contusion and hemorrhagic shock with or without daily restraint stress have identified significant reductions of 1–4 g/dl in hemoglobin compared to uninjured control rats, which was not observed with clonidine administration (Alamo et al., 2017b; Loftus et al., 2020). Rats treated with clonidine demonstrated mean hemoglobin levels which were 1–2 g/dl greater than untreated rats (Alamo et al., 2017b; Loftus et al., 2020). Alpha agonism not only contributes to suppression of the cytokines that affect erythropoiesis but also decreases mobilization of HPC and improves hemoglobin.

Few human studies have investigated the effects of clonidine on bone marrow dysfunction and anemia in trauma patients. In a retrospective cohort analysis of almost 300 human trauma patients requiring admission to a SICU, those who received clonidine and/or a non-selective beta blocker for at least 25% of their stay demonstrated a significant improvement in hemoglobin at time of discharge compared to trauma patients who had not received those medications, an absolute difference of almost 0.5 g/dl (Loftus et al., 2018c). However, patients in this study were pooled into one treatment group and not separated by medication, and only 7% of this group received clonidine alone (Loftus et al., 2018c). Therefore, the specific effect of clonidine on minimizing anemia cannot be determined from the findings of Loftus et al. (2018c). However, clonidine has the potential to play a role in the promotion of erythropoiesis in trauma patients.