The effects of propranolol and clonidine on bone marrow expression of hematopoietic cytokines following trauma and chronic stress.

BACKGROUND: Attenuating post-injury neuroendocrine stress abrogates persistent injury-associated anemia. Our objective was to examine the mechanisms by which propranolol and clonidine modulate this process. We hypothesized that propranolol and clonidine would decrease bone marrow expression of high-mobility group box-1 (HMGB1) and increase expression of stem cell factor (SCF) and B-cell lymphoma-extra large (Bcl-xL). METHODS: Male Sprague-Dawley rats were allocated to naïve control, lung contusion followed by hemorrhagic shock (LCHS), or LCHS plus daily chronic restraint stress (LCHS/CS) ±propranolol, ±clonidine. Day seven bone marrow expression of HMGB1, SCF, and Bcl-xL was assessed by polymerase chain reaction. RESULTS: Following LCHS, HMGB1 was decreased by propranolol (49% decrease, p = 0.012) and clonidine (54% decrease, p < 0.010). SCF was decreased following LCHS/CS, and was increased by propranolol (629% increase, p < 0.001) and clonidine (468% increase, p < 0.001). Bcl-xL was decreased following LCHS/CS, and was increased by propranolol (59% increase, p = 0.006) and clonidine (77% increase, p < 0.001). CONCLUSIONS: Following severe trauma, propranolol and clonidine abrogate persistent injury-associated anemia by modulating bone marrow cytokines, favoring effective erythropoiesis.

Clonidine restores vascular endothelial growth factor expression and improves tissue repair following severe trauma.

BACKGROUND: We hypothesized that clonidine and propranolol would increase VEGF and VEGF-receptor expression and promote lung healing following severe trauma and chronic stress. METHODS: Sprague-Dawley rats were subjected to lung contusion (LC), lung contusion/hemorrhagic shock (LCHS), or lung contusion/hemorrhagic shock/daily restraint stress (LCHS/CS). Clonidine and propranolol were administered daily. On day seven, lung VEGF, VEGFR-1, VEGFR-2, and HMGB1 were assessed by PCR. Lung injury was assessed by light microscopy (*p < 0.05). RESULTS: Clonidine increased VEGF expression following LCHS (43%*) and LCHS/CS (46%*). Clonidine increased VEGFR-1 and R-2 expression following LCHS/CS (203%* and 47%*, respectively). Clonidine decreased HMGB1 and TNF-alpha expression following LCHS/CS (22%* and 58%*, respectively.) Clonidine decreased inflammatory cell infiltration and total Lung Injury Score following LCHS/CS. Propranolol minimally affected VEGF and did not improve lung healing. CONCLUSIONS: Clonidine increased VEGF and VEGF-receptor expression, decreased HMGB1 expression, decreased lung inflammation, and improved lung tissue repair.

Persistent injury-associated anemia: the role of the bone marrow microenvironment.

BACKGROUND: The regulation of erythropoiesis involves hematopoietic progenitor cells, bone marrow stroma, and the microenvironment. Following severe injury, a hypercatecholamine state develops that is associated with increased mobilization of hematopoietic progenitor cells to peripheral blood and decreased growth of bone marrow erythroid progenitor cells that manifests clinically as a persistent injury-associated anemia. Changes within the bone marrow microenvironment influence the development of erythroid progenitor cells. Therefore, we sought to determine the effects of lung contusion, hemorrhagic shock, and chronic stress on the hematopoietic cytokine response. MATERIALS AND METHODS: Bone marrow was obtained from male Sprague-Dawley rats (n = 6/group) killed 7 d after lung contusion followed by hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1ß, interleukin-10, stem cell factor, transforming growth factor-ß, high-mobility group box-1 (HMGB-1), and B-cell lymphoma-extra large. RESULTS: Seven days following LCHS and LCHS/CS, bone marrow expression of prohematopoietic cytokines (interleukin-1ß, interleukin-10, stem cell factor, and transforming growth factor-ß) was significantly decreased, and bone marrow expression of HMGB-1 was significantly increased. B-cell lymphoma-extra large bone marrow expression was not affected by LCHS or LCHS/CS (naïve: 44 ± 12, LCHS: 44 ± 12, LCHS/CS: 37 ± 1, all P > 0.05). CONCLUSIONS: The bone marrow microenvironment was significantly altered following severe trauma in a rodent model. Prohematopoietic cytokines were downregulated, and the proinflammatory cytokine HMGB-1 had increased bone marrow expression. Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia.

Effects of trauma, hemorrhagic shock, and chronic stress on lung vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) regulate vascular permeability and endothelial cell survival. We hypothesized that hemorrhagic shock (HS) and chronic stress (CS) would increase expression of lung VEGF and its receptors, potentiating pulmonary edema in lung tissue. MATERIALS AND METHODS: Male Sprague-Dawley rats aged 8-9 wk were randomized: naïve control, lung contusion (LC), LC followed by HS (LCHS), and LCHS with CS in a restraint cylinder for 2 h/d (LCHS/CS). Animals were sacrificed on days 1 and 7. Expressions of lung VEGF, VEGFR-1, and VEGFR-2 were determined by polymerase chain reaction. Lung Injury Score (LIS) was graded on light microscopy by inflammatory cell counts, interstitial edema, pulmonary edema, and alveolar integrity (range: 0 = normal; 8 = severe injury). RESULTS: Seven days after LC, lung VEGF and VEGFR-1 were increased, and lung tissue healed (LIS: 0.8 ± 0.8). However, 7 d after LCHS and LCHS/CS, lung VEGF and VEGFR-1 expressions were decreased. VEGFR-2 was also decreased after LCHS/CS. LIS was elevated 7 d after LCHS and LCHS/CS (6.5 ± 1.0 and 8.2 ± 0.8). Increased LIS after LCHS and LCHS/CS was because of higher inflammatory cell counts, increased interstitial edema, and loss of alveolar integrity, whereas pulmonary edema was unchanged. CONCLUSIONS: Elevation of lung VEGF and VEGFR-1 expressions after LC alone was associated with healing of injured lung tissue. Expressions of VEGF, VEGFR-1, and VEGFR-2 were reduced after LCHS and LCHS/CS, and injured lung tissue did not heal. Persistent lung injury after severe trauma was because of inflammation rather than pulmonary edema.

Severe trauma and chronic stress activates extramedullary erythropoiesis.

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.

Daily propranolol administration reduces persistent injury-associated anemia after 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 ß 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.

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 µg/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.

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.

Accuracy of intra-articular glenohumeral injections: the anterosuperior technique with arthroscopic documentation.

PURPOSE: Our objective was to assess the accuracy rate of needle placement with the anterosuperior technique of glenohumeral joint injection that uses familiar palpable superficial landmarks as a guide instead of diagnostic imaging. METHODS: Between April 2007 and October 2007 at our institution, 42 patients met the study inclusion criteria of being aged 18 years or older and undergoing shoulder arthroscopy. For the injection (performed by 1 surgeon), anesthetized patients were placed in the beach-chair position with the arm in adduction and internal rotation. The surgeon was allowed to redirect the needle only once without withdrawing the needle from the entry site. After injection, arthroscopic confirmation of needle position in the joint and the presence of backflow from the posterior portal cannula were used to determine accuracy and the relation of the needle to adjacent anatomy. RESULTS: Of the 42 injections, 38 needles were inserted accurately into the glenohumeral joint (91% accuracy rate), most through the rotator interval (21) or the long head of the biceps tendon (9). Four needles were placed inaccurately into the anterior synovium and subacromial space. Adhesive capsulitis was the diagnosis in 3 of those 4 shoulders but in only 5 of the 38 shoulders in the group with accurate placement (P < .05). Body mass index was not statistically different between the accurate and inaccurate injection groups (P > .05). CONCLUSIONS: Anterosuperior glenohumeral joint injection without image guidance provides an accuracy rate of 91%. The anterosuperior technique for glenohumeral injections yields an accuracy rate higher than that of the standard anterior techniques and comparable to that of posterior injection. LEVEL OF EVIDENCE: Level IV, diagnostic study.