Kolenkode B. Kannan

Chronic restraint stress after injury and shock is associated with persistent anemia despite prolonged elevation in erythropoietin levels.

BACKGROUND Following severe traumatic injury, critically ill patients have a prolonged hypercatacholamine state that is associated with bone marrow (BM) dysfunction and persistent anemia. However, current animal models of injury and shock result in a transient anemia. Daily restraint stress (chronic stress [CS]) has been shown to increase catecholamines. We hypothesize that adding CS following injury or injury and shock in rats will prolong the hypercatecholaminemia and prolong the initial anemia, despite elevated erythropoietin (EPO) levels. METHODS Male Sprague-Dawley rats (n = 6–8 per group) underwent lung contusion (LC) or combined LC/hemorrhagic shock (LCHS) followed by 6 days of CS. CS consisted of a 2-hour restraint period interrupted with repositioning and alarms every 30 minutes. At 7 days, urine was assessed for norepinephrine (NE) levels, blood for EPO and hemoglobin (Hgb), and BM for erythroid progenitor growth. RESULTS Animals undergoing LC or combined LCHS predictably recovered by Day 7; urine NE, EPO, and Hgb levels were normal. The addition of CS to LC and LCHS models was associated with a significant elevation in NE on Day 6. The addition of CS to LC led to a persistent 20% to 25% decrease in the growth of BM hematopoietic progenitor cells. These findings were further exaggerated when CS was added following LCHS, resulting in a 20%q to 40% reduction in BM erythroid progenitor colony growth and a 20% decrease in Hgb when compared with LCHS alone. CONCLUSION Exposing injured animals to CS results in prolonged elevation of NE and EPO, which is associated with worsening BM erythroid function and persistent anemia. Chronic restraint stress following injury and shock provides a clinically relevant model to further evaluate persistent injury-associated anemia seen in critically ill trauma patients. Furthermore, alleviating CS after severe injury is a potential therapeutic target to improve BM dysfunction and anemia.

Characterization of erythropoietin and hepcidin in the regulation of persistent injury-associated anemia.

BACKGROUND The cause of persistent injury-associated anemia is multifactorial and includes acute blood loss, an altered erythropoietin (EPO) response, dysregulation of iron homeostasis, and impaired erythropoiesis in the setting of chronic inflammation/stress. Hepcidin plays a key role in iron homeostasis and is regulated by anemia and inflammation. Erythropoietin is a main regulator of erythropoiesis induced by hypoxia. A unique rodent model of combined lung injury (LC)/hemorrhagic shock (HS) (LCHS)/chronic restraint stress (CS) was used to produce persistent injury-associated anemia to further investigate the roles of EPO, hepcidin, iron, ferritin, and the expression of EPO receptors (EPOr). METHODS Male Sprague-Dawley rats were randomly assigned into one of the four groups of rodent models: naive, CS alone, combined LCHS, or LCHS/CS. Plasma was used to evaluate levels of EPO, hepcidin, iron, and ferritin. RNA was isolated from bone marrow and lung tissue to evaluate expression of EPOr. Comparisons between models were performed by t tests followed by one-way analysis of variance. RESULTS After 7 days, only LCHS/CS was associated with persistent anemia despite significant elevation of plasma EPO. Combined LCHS and LCHS/CS led to a persistent decrease in EPOr expression in bone marrow on Day 7. The LCHS/CS significantly decreased plasma hepcidin levels by 75% on Day 1 and 84% on Day 7 compared to LCHS alone. Hepcidin plasma levels are inversely proportional to EPO plasma levels (Pearson R = −0.362, p < 0.05). CONCLUSION Tissue injury, hemorrhagic shock, and stress stimulate and maintain high levels of plasma EPO while hepcidin levels are decreased. In addition, bone marrow EPOr and plasma iron availability are significantly reduced following LCHS/CS. The combined deficit of reduced iron availability and reduced bone marrow EPOr expression may play a key role in the ineffective EPO response associated with persistent injury-associated anemia.

Daily propranolol administration reduces persistent injury-associated anemia following severe trauma and chronic stress.

BACKGROUND After severe trauma, patients develop a norepinephrine-mediated persistent, injury-associated anemia. This anemia is associated with suppression of bone marrow (BM) erythroid colony growth, along with decreased iron levels, and elevated erythropoietin (EPO) levels, which are insufficient to promote effective erythropoiesis. The impact of norepinephrine on iron regulators, such as ferroportin, transferrin, and transferrin receptor-1 (TFR-1), is unknown. Using a clinically relevant rodent model of lung contusion (LC), hemorrhagic shock (HS), and chronic stress (CS), we hypothesize that daily propranolol (BB), a nonselective &bgr; blocker, restores BM function and improves iron homeostasis. METHODS Male Sprague-Dawley rats were subjected to LCHS ± BB and LCHS/CS ± BB. BB was achieved with propranolol (10 mg/kg) daily until the day of sacrifice. Hemoglobin, plasma EPO, plasma hepcidin, BM cellularity and BM erythroid colony growth were assessed. RNA was isolated to measure transferrin, TFR-1 and ferroportin expression. Data are presented as mean ± SD; *p < 0.05 versus untreated counterpart by t test. RESULTS The addition of CS to LCHS leads to persistent anemia on posttrauma day 7, while the addition of BB improved hemoglobin levels (LCHS/CS: 10.6 ± 0.8 vs. LCHS/CS + BB: 13.9 ± 0.4* g/dL). Daily BB use after LCHS/CS improved BM cellularity, colony-forming units granulocyte, erythrocyte, monocyte megakaryocyte, burst-forming unit erythroid and colony-forming unit erythroid cell colony growth. LCHS/CS + BB significantly reduced plasma EPO levels and increased plasma hepcidin levels on day 7. The addition of CS to LCHS resulted in decreased liver ferroportin expression as well as decreased BM transferrin and TFR-1 expression, thus, blocking iron supply to erythroid cells. However, daily BB after LCHS/CS improved expression of all iron regulators. CONCLUSION Daily propranolol administration after LCHS/CS restored BM function and improved anemia after severe trauma. In addition, iron regulators are significantly reduced after LCHS/CS, which may contribute to iron restriction after injury. However, daily propranolol administration after LCHS/CS improved iron homeostasis.

The Postinjury Inflammatory State and the Bone Marrow Response to Anemia

Rationale: The pathophysiology of persistent injury‐associated anemia is incompletely understood, and human data are sparse. Objectives: To characterize persistent injury‐associated anemia among critically ill trauma patients with the hypothesis that severe trauma would be associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, and decreased erythropoiesis. Methods: A translational prospective observational cohort study comparing severely injured, blunt trauma patients who had operative fixation of a hip or femur fracture (n = 17) with elective hip repair patients (n = 22). Bone marrow and plasma obtained at the index operation were assessed for circulating catecholamines, systemic inflammation, erythropoietin, iron trafficking pathways, and erythroid progenitor growth. Bone marrow was also obtained from healthy donors from a commercial source (n = 8). Measurements and Main Results: During admission, trauma patients had a median of 625 ml operative blood loss and 5 units of red blood cell transfusions, and Hb decreased from 10.5 to 9.3 g/dl. Compared with hip repair, trauma patients had higher median plasma norepinephrine (21.9 vs. 8.9 ng/ml) and hepcidin (56.3 vs. 12.2 ng/ml) concentrations (both P < 0.05). Bone marrow erythropoietin and erythropoietin receptor expression were significantly increased among patients undergoing hip repair (23% and 14% increases, respectively; both P < 0.05), but not in trauma patients (3% and 5% increases, respectively), compared with healthy control subjects. Trauma patients had lower bone marrow transferrin receptor expression than did hip repair patients (57% decrease; P < 0.05). Erythroid progenitor growth was decreased in trauma patients (39.0 colonies per plate; P < 0.05) compared with those with hip repair (57.0 colonies per plate; P < 0.05 compared with healthy control subjects) and healthy control subjects (66.5 colonies per plate). Conclusions: Severe blunt trauma was associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, erythroid progenitor growth suppression, and persistent injury‐associated anemia. Clinical trial registered with www.clinicaltrials.gov (NCT 02577731).

Clonidine reduces norepinephrine and improves bone marrow function in a rodent model of lung contusion, hemorrhagic shock, and chronic stress.

Background. Propranolol has been shown previously to restore bone marrow function and improve anemia after lung contusion/hemorrhagic shock. We hypothesized that daily clonidine administration would inhibit central sympathetic outflow and restore bone marrow function in our rodent model of lung contusion/hemorrhagic shock with chronic stress. Methods. Male Sprague‐Dawley rats underwent 6 days of restraint stress after lung contusion/hemorrhagic shock during which the animals received clonidine (75 &mgr;g/kg) after the restraint stress. On postinjury day 7, we assessed urine norepinephrine, blood hemoglobin, plasma granulocyte colony stimulating factor, and peripheral blood mobilization of hematopoietic progenitor cells, as well as bone marrow cellularity and erythroid progenitor cell growth. Results. The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased urine norepinephrine levels, improved bone marrow cellularity, restored erythroid progenitor colony growth, and improved hemoglobin (14.1 ± 0.6 vs 10.8 ± 0.6 g/dL). The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased hematopoietic progenitor cells mobilization and restored granulocyte colony stimulating factor levels. Conclusion. After lung contusion/hemorrhagic shock with chronic restraint stress, daily administration of clonidine restored bone marrow function and improved anemia. Alleviating chronic stress and decreasing norepinephrine is a key therapeutic target to improve bone marrow function after severe injury.

Persistent injury-associated anemia in aged rats

Background: Hypercatecholaminemia and bone marrow dysfunction have been implicated in the pathophysiology of persistent‐injury associated anemia. The elderly may be vulnerable to this phenomenon due to high basal and peak catecholamine levels, impaired erythroid progenitor growth, and baseline anemia. We hypothesized that aged F344‐BN rats subjected to severe trauma and chronic stress would have persistent injury‐associated anemia. Methods: Male F344‐BN rats age 25 months were randomly allocated to: naïve (n = 8), lung contusion (LC, n = 9), LC followed by daily chronic restraint stress (LC/CS, n = 9), LC followed immediately by hemorrhagic shock (LCHS, n = 8), and LCHS followed by daily CS (LCHS/CS, n = 8). Urine norepinephrine was measured on days one and seven. Locomotor testing was performed on day five. Bone marrow cellularity, hematopoietic progenitor growth, and peripheral blood hemoglobin levels were assessed at sacrifice on day seven. Data are presented as mean ± standard deviation, *p < 0.05 vs. naïve. Results: Norepinephrine levels (ng/mL) were significantly elevated one day after LCHS (420 ± 239* vs. naïve: 97 ± 71) and LCHS/CS (375 ± 185*), and remained significantly elevated on day seven for LCHS/CS (359 ± 99*), but not LCHS (212 ± 130). On locomotor testing, groups subjected to CS traveled shorter distances at lower velocities and spent less time in the center of the cage. Colony forming units‐erythroid (colonies/plate), representing late erythroid progenitors, were significantly decreased after LC/CS (40 ± 1* vs. naïve: 47 ± 4), LCHS (40 ± 1*), and LCHS/CS (38 ± 3*). LCHS/CS animals had significantly lower hemoglobin (g/dL) than naïve animals (13.3 ± 1.3* vs. naïve: 15.2 ± 0.9). Conclusions: Persistent injury‐associated anemia occurs in aged rats. Further research is needed to determine whether the pathophysiology of this phenomenon differs from that of younger rats, and to translate these findings to elderly trauma patients. HighlightsHigh catecholamine levels and bone marrow dysfunction cause anemia after trauma.The elderly have high catecholamine levels and bone marrow dysfunction at baseline.Aged rats were subjected to severe traumatic injury and daily stress for seven days.Animals had high catecholamines, impaired erythroid progenitor growth, and anemia.