Amy V. Gore

Early propranolol administration to severely injured patients can improve bone marrow dysfunction.

BACKGROUND Bone marrow (BM) dysfunction is common in severely injured trauma patients, resulting from elevated catecholamines and plasma granulocyte colony-stimulating factor (G-CSF) as well as prolonged mobilization of hematopoietic progenitor cells (HPCs). We have previously shown that propranolol (&bgr;-blocker [BB]) reduces HPC mobilization in a rodent model of injury and hemorrhagic shock. We hypothesize that BB would prevent BM dysfunction in humans following severe injury. METHODS Forty-five severely injured trauma patients were studied in a prospective, randomized pilot trial. Twenty-five patients received BB, and 20 served as untreated controls. The dose of propranolol was adjusted to decrease the heart rate by 10% to 20% from baseline. Blood was analyzed for the presence of HPC (blast-forming unit erythroid cells [BFU-E] and colony-forming unit erythroid cells) and G-CSF. Demographic data, Injury Severity Score (ISS), hemoglobin, reticulocyte number, and outcome data were obtained. RESULTS The mean age of the study population was 33 years; 87% were male, with a mean ISS of 29. There is a significant increase in BFU-E in peripheral blood immediately following traumatic injury, and this mobilization persists for 30 days. The use of BB significantly decreases BFU-E and colony-forming unit erythroid cells at all time points. G-CSF is significantly elevated in both groups on admission; the use of BB decreases G-CSF levels by 51% as compared with 37% for controls. The average hemoglobin is nearly 1 g higher on the day of discharge with propranolol treatment (BB, 9.9 ± 0.4 g/dL vs. no BB, 9.1 ± 0.6 g/dL). CONCLUSION Following severe trauma, early treatment with propranolol following resuscitation is safe. The use of propranolol blunts early tachycardia, reduces HPC mobilization, and results in a faster return to baseline of the G-CSF peak seen after injury. There is also a trend toward faster recovery and resolution of anemia. Propranolol may be the first therapeutic agent to show improved BM function after severe injury. LEVEL OF EVIDENCE Therapeutic study, level III.

Can mesenchymal stem cells reverse chronic stress-induced impairment of lung healing following traumatic injury?

BACKGROUND One week following unilateral lung contusion (LC), rat lungs demonstrate full histologic recovery. When animals undergo LC plus the addition of chronic restraint stress (CS), wound healing is significantly delayed. Mesenchymal stem cells (MSCs) are pluripotent cells capable of immunomodulation, which have been the focus of much research in wound healing and tissue regeneration. We hypothesize that the addition of MSCs will improve wound healing in the setting of CS. METHODS Male Sprague-Dawley rats (n = 6–7 per group) were subjected to LC/CS with or without the injection of MSCs. MSCs were given as a single intravenous dose of 5 × 106 cells in 1 mL Iscove’s Modified Dulbecco’s Medium at the time of LC. Rats were subjected to 2 hours of restraint stress on Days 1 to 6 following LC. Seven days following injury, rats were sacrificed, and the lungs were examined for histologic evidence of wound healing using a well-established histologic lung injury score (LIS) to grade injury. LIS examines inflammatory cells/high-power field (HPF) averaged over 30 fields, interstitial edema, pulmonary edema, and alveolar integrity, with scores ranging from 0 (normal) to 11 (highly damaged). Peripheral blood was analyzed by flow cytometry for the presence of T-regulatory (C4+CD25+FoxP3+) cells. Data were analyzed by analysis of variance followed by Tukey’s multiple comparison test, expressed as mean (SD). RESULTS As previously shown, 7 days following isolated LC, LIS has returned to 0.83 (0.41), with a subscore of zero for inflammatory cells/HPF. The addition of CS results in an LIS of 4.4 (2.2), with a subscore of 1.9 (0.7) for inflammatory cells/HPF. Addition of MSC to LC/CS decreased LIS to 1.7 (0.8), with a subscore of zero for inflammatory cells/HPF. Furthermore, treatment of animals undergoing LC/CS with MSCs increased the %T-regulatory cells by 70% in animals undergoing LC/CS alone (12.9% [2.4]% vs. 6.2% [1.3%]). CONCLUSION Stress-induced impairment of wound healing is reversed by the addition of MSCs given at the time of injury in this rat LC model. This improvement in lung healing is associated with a decrease in the number of inflammatory cells and an increase in the number of T-regulatory cells. Further study into the mechanisms by which MSCs hasten wound healing is warranted.

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.

Mesenchymal stem cells increase T-regulatory cells and improve healing following trauma and hemorrhagic shock.

BACKGROUND Rodent lungs undergo full histologic recovery within 1 week following unilateral lung contusion (LC). However, when LC is followed by hemorrhagic shock (HS), healing is impaired. We hypothesize that the intravenous administration of mesenchymal stem cells (MSCs) in animals undergoing combined LC followed by HS (LCHS) will improve wound healing. METHODS Male Sprague-Dawley rats (n = 5–6 per group) were subjected to LCHS with or without the injection of a single intravenous dose of 5 × 106 MSCs following return of shed blood after HS. Rats were sacrificed 7 days following injury. Flow cytometry was used to determine the T-regulatory cell (Treg) population in peripheral blood. Lung histology was graded using a well-established lung injury score (LIS). Components of the LIS include average inflammatory cells per high-power field over 30 fields, interstitial edema, pulmonary edema, and alveolar integrity, with total scores ranging from 0 to 11. Data were analyzed by analysis of variance followed by Tukey’s multiple comparison test, expressed as mean (SD). p < 0.05 was considered significant. RESULTS Seven days following isolated LC, animals demonstrated lung healing with an LIS unchanged from naive. The addition of HS resulted in a persistently elevated LIS score, whereas the addition of MSCs to LCHS decreased the LIS score back to naive levels. The change in LIS was driven by a significant decrease in edema scores. In rats undergoing LC alone, 10.5% (3.3%) of CD4+ cells were Tregs. The addition of HS caused no significant change in Treg population (9.3% [0.7%]), whereas LCHS + MSC significantly increased the population to 18.2% (6.8%) in peripheral blood (p < 0.05 vs. LCHS). CONCLUSION Impaired wound healing following trauma and HS is improved by a single dose of MSCs given immediately after injury. This enhanced healing is associated with an increase in the Treg population and a significant decrease in lung edema score as compared with animals undergoing LCHS. Further study into the role of Tregs in MSC-mediated wound healing is warranted.

Mesenchymal stem cells enhance lung recovery after injury, shock, and chronic stress

BACKGROUND Normal lung healing is impaired when lung contusion (LC) is followed by hemorrhagic shock (HS), and chronic stress (CS). Mesenchymal stem cells (MSCs) are immunomodulatory, pluripotent cells that are under investigation for use in wound healing and tissue regeneration. We hypothesized that treatment with MSCs can reverse the impaired healing seen after LC combined with HS and CS (LCHS/CS). METHODS Male Sprague-Dawley rats (n = 6/group) underwent LCHS with or without a single intravenous dose of 5 × 10(6) Sprague-Dawley rat MSCs after resuscitation. Thereafter, rats were subjected to 2 hours of CS daily on days 1-6 and were humanely killed on day 7. Lung histology was scored according to a well-established lung injury score (LIS) that included interstitial and pulmonary edema, alveolar integrity, and inflammatory cells. Scoring ranges from 0 (normal lung) to 11 (most severely injured). Whole blood was analyzed for the presence of CD4(+)CD25(+)FoxP3(+) T-regulatory cells (Treg) by flow cytometry. RESULTS Seven days after isolated LC, LIS had returned to 0.8 ± 0.4; however, after LCHS/CS healing is significantly delayed (7.2 ± 2.2; P < .05). Addition of MSC to LCHS/CS decreased LIS to 2.0 ± 1.3 (P < .05) and decreased all subgroup scores (inflammatory cells, interstitial and pulmonary edema, and alveolar integrity) significantly compared with LCHS/CS (P < .05). The percentage of Tregs found in the peripheral blood of animals undergoing LCHS/CS did not change from LC alone (10.5 ± 3.3% vs 6.7 ± 1.7%; P > .05). Treatment with MSCs significantly increased the Treg population compared with LCHS/CS alone (11.7 ± 2.7% vs 6.7 ± 1.7%; P < .05) CONCLUSION: In this model, severe impairment of wound healing observed 1 week after LCHS/CS is reversed by a single treatment with MSCs immediately after resuscitation. This improvement in lung healing is associated with a decrease in the number of inflammatory cells and lung edema and a significant increase in peripheral Tregs. Further study into the timing of administration and mechanisms by which cell-based therapy using MSCs modulate the immune system and improve wound healing is warranted.

Mesenchymal stem cells reverse bone marrow dysfunction following injury and stress.

BACKGROUND Bone marrow (BM) dysfunction following experimental lung contusion (LC) resolves in 7 days; however, if followed by chronic stress (CS) following, BM dysfunction is persistent. Mesenchymal stem cells (MSCs) have protective immunomodulatory effects. We hypothesize that MSC can protect the BM against the deleterious effect of CS following LC. METHODS Male Sprague-Dawley rats (n = 6–7 per group) underwent LC or LC/CS ± MSC injection. CS consisted of a daily 2-hour period of restraint with repositioning and alarming every 30 minutes to prevent habituation. A single intravenous dose of 5 × 106 MSCs was given within 10 minutes following LC. Animals were sacrificed at Day 7, and peripheral blood (PB) and BM were collected. Flow cytometry was used to assess hematopoietic progenitor cells (HPCs) mobilized to PB. Plasma granulocyte colony-stimulating factor (G-CSF) levels were measured by enzyme-linked immunosorbent assay. BM cellularity and growth of BM HPC colonies (colony-forming unit–erythroid [CFU-E], burst-forming unit–erythroid [BFU-E], colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte [CFU-GEMM]) were also evaluated. RESULTS As previously reported, the addition of CS to LC resulted in a 32% decrease in BM cellularity; significant decreases in CFU-GEMM, BFU-E, and CFU-E; and marked increase in HPC in the PB as compared with the naive animals. The addition of MSC to LC/CS resulted in a 22% increase in BM cellularity and significant increases in CFU-GEMM, BFU-E, and CFU-E cultured from the BM. MSCs additionally reduced plasma G-CSF, prevented prolonged mobilization of HPC to PB, and restored colony growth to naive levels. CONCLUSION CS following LC results in persistent BM dysfunction manifested by a significant decrease in cellularity, HPC colony growth, and increased G-CSF levels and HPC mobilization to the PB at 7 days following injury. The addition of a single dose of MSCs following acute traumatic injury reverses the deleterious effects of CS on BM function. Further study is warranted to better understand the mechanisms behind MSC-mediated protection of BM function in the setting of CS.

Mesenchymal stem cells reverse trauma and hemorrhagic shock-induced bone marrow dysfunction

BACKGROUND Lung contusion (LC) followed by hemorrhagic shock (HS) causes persistent bone marrow (BM) dysfunction lasting up to 7 d after injury. Mesenchymal stem cells (MSCs) are multipotent cells that can hasten healing and exert protective immunomodulatory effects. We hypothesize that MSCs can attenuate BM dysfunction after combined LCHS. MATERIALS AND METHODS Male Sprague-Dawley rats (n = 5-6 per group) underwent LC plus 45 min of HS (mean arterial pressure of 30-35). Allogeneic MSCs (5 × 10(6) cells) were injected intravenously after resuscitation. At 7 d, BM was analyzed for cellularity and growth of hematopoietic progenitor cell (HPC) colonies (colony-forming unit-erythroid; burst-forming unit-erythroid; and colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte). Flow cytometry measured %HPCs in peripheral blood; plasma granulocyte colony-stimulating factor (G-CSF) levels were measured via enzyme-linked immunosorbent assay. Data were analyzed by one-way analysis of variance followed by the Tukey multiple comparison test. RESULTS As previously shown, at 7 d, LCHS resulted in 22%, 30%, and 24% decreases in colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte, burst-forming unit-erythroid, and colony-forming unit-erythroid colony growth, respectively, versus naive. Treatment with MSCs returned all BM parameters to naive levels. There was no difference in %HPCs in peripheral blood between groups; however, G-CSF remained increased up to 7 d after LCHS. MSCs returned G-CSF to naive levels. Plasma from animals receiving MSCs was not suppressive to the BM. CONCLUSIONS One week after injury, the persistent BM dysfunction observed in animals undergoing LCHS is reversed by treatment with MSCs with an associated return of plasma G-CSF levels to normal. Plasma from animals undergoing LCHS plus MSCs was not suppressive to BM cells in vitro. Treatment with MSCs after injury and shock reverses BM suppression and returns plasma G-CSF levels to normal.