Intravenous mesenchymal stem cell therapy for traumatic brain injury.

OBJECT: Cell therapy has shown preclinical promise in the treatment of many diseases, and its application is being translated to the clinical arena. Intravenous mesenchymal stem cell (MSC) therapy has been shown to improve functional recovery after traumatic brain injury (TBI). Herein, the authors report on their attempts to reproduce such observations, including detailed characterizations of the MSC population, non-bromodeoxyuridine-based cell labeling, macroscopic and microscopic cell tracking, quantification of cells traversing the pulmonary microvasculature, and well-validated measurement of motor and cognitive function recovery. METHODS: Rat MSCs were isolated, expanded in vitro, immunophenotyped, and labeled. Four million MSCs were intravenously infused into Sprague-Dawley rats 24 hours after receiving a moderate, unilateral controlled cortical impact TBI. Infrared macroscopic cell tracking was used to identify cell distribution. Immunohistochemical analysis of brain and lung tissues 48 hours and 2 weeks postinfusion revealed transplanted cells in these locations, and these cells were quantified. Intraarterial blood sampling and flow cytometry were used to quantify the number of transplanted cells reaching the arterial circulation. Motor and cognitive behavioral testing was performed to evaluate functional recovery. RESULTS: At 48 hours post-MSC infusion, the majority of cells were localized to the lungs. Between 1.5 and 3.7% of the infused cells were estimated to traverse the lungs and reach the arterial circulation, 0.295% reached the carotid artery, and a very small percentage reached the cerebral parenchyma (0.0005%) and remained there. Almost no cells were identified in the brain tissue at 2 weeks postinfusion. No motor or cognitive functional improvements in recovery were identified. CONCLUSIONS: The intravenous infusion of MSCs appeared neither to result in significant acute or prolonged cerebral engraftment of cells nor to modify the recovery of motor or cognitive function. Less than 4% of the infused cells were likely to traverse the pulmonary microvasculature and reach the arterial circulation, a phenomenon termed the "pulmonary first-pass effect," which may limit the efficacy of this therapeutic approach. The data in this study contradict the findings of previous reports and highlight the potential shortcomings of acute, single-dose, intravenous MSC therapy for TBI.

Edema-induced intestinal dysfunction is mediated by STAT3 activation.

Increased signal transducer and activator of transcription 3 (STAT3) activation has been shown to be associated with intestinal dysfunction. The purpose of this study was to investigate the role of STAT3 in edema-induced intestinal dysfunction. Intestinal edema was induced in male Sprague-Dawley rats by a combination of mesenteric venous hypertension and fluid resuscitation (RESUS + VH). Resuscitation fluid alone (RESUS), venous hypertension alone (VH), and sham-operated rats (CONTROL) were used as controls. Edema development, STAT3 DNA binding activity, nuclear translocation, and phosphorylation were measured in rat distal small intestinal muscularis. A significant amount of edema development was measured in the RESUS + VH rats compared with CONTROL and VH from 30 min to 6 h after surgery. Edema developed in the RESUS group at 30 min postsurgery but resolved before 2 h postsurgery. A significant increase in STAT3 DNA binding activity was observed from 30 min to 6 h after surgery in the edematous RESUS + VH group compared with nonedematous CONTROL. In addition, a significant increase in STAT3 nuclear translocation and phosphorylation was measured in the RESUS + VH group 2 and 6 h after surgery. No significant increases in STAT3 activation were observed in either the RESUS or VH groups compared with CONTROL. Rats in both the RESUS + VH and CONTROL groups were pretreated with AG490 (5 mg/kg, i.p.) to block STAT3 activation. Signal transducer and activator of transcription 3 inhibition attenuated edema-induced decrease in intestinal contractile activity and myosin light chain phosphorylation. We conclude from these data that edema-induced decreases in intestinal contractile activity are mediated, at least in part, by STAT3 activation.

Resuscitation-induced intestinal edema decreases the stiffness and residual stress of the intestine.

We have shown that acute edema impairs intestinal transit and we wanted to know whether this could be from changes in the physical characteristics of the intestine. Our hypothesis was that acute edema will change the physical characteristics of the intestine, which were measured by standardized engineering measures of elastic modulus, to determine stiffness and opening angle, and to determine residual stress. Rats were randomized to sham, mild edema (80 mL/ kg of normal saline resuscitation), and severe edema groups (80 mL/kg of normal saline resuscitation with intestinal venous hypertension). Segments of distal ileum were hung to a fixed point in a tissue bath and to a tensiometer and were stretched in increments of 1 mm, recording the new length and the corresponding force from the tensiometer to determine elastic modulus. Next, two transverse cuts were made yielding a 1- to 2-mm-thick ring-shaped segment of tissue and were then cut radially to open the ring. The opening angle was measured. Acute intestinal edema led to a decrease in transit, elastic modulus, and opening angle of the intestine in the absence of ischemic injury. Acute intestinal edema leads to a significant loss in stiffness and residual stress and is a plausible explanation for how acute edema impairs intestinal transit.

Effects of primary and secondary intra-abdominal hypertension on mesenteric lymph flow: implications for the abdominal compartment syndrome.

Intra-abdominal hypertension leading to abdominal compartment syndrome complicates trauma resuscitation. The purpose of this study was to determine the effect of primary (1 degrees) and secondary (2 degrees) intra-abdominal hypertension (IAH) on hemodynamics, intestinal fluid balance, and mesenteric lymph flow. Anesthetized dogs were instrumented with vascular catheters, intra-abdominal manometer, and mesenteric lymphatic fistulae. 1 degrees IAH was created by infusing 0.9% saline into the peritoneal cavity to increase abdominal pressure. 2 degrees IAH was created by elevating the inferior vena cava (IVC) pressure between 20 and 25 mmHg and crystalloid resuscitation to create intestinal edema to induce IAH. At baseline and at 30-min intervals, hemodynamics, lymph flow (QL), IVC, and intra-abdominal pressures were measured. Tissue water was determined using microgravimetry to assess gut edema. Results are reported as mean +/- SEM, with n = 7-8 dogs per group. 1 degrees IAH significantly increased CVP and decreased QL. 1 degrees IAH stopped mesenteric QL, thus transvascular fluid flux necessarily exceeded QL, contributing to gut edema formation. 2 degrees IAH significantly increased CVP and QL. 2 degrees IAH increased QL despite elevated IAP. Interstitial protein washdown maintained the plasma-to-interstitial oncotic gradient, thus increased transvascular fluid flux was due principally to increased capillary pressure. Transvascular fluid flux exceeded QL as manifested by increasing gut tissue water as QL plateaued. Modest elevations in IAP significantly affect mesenteric QL and the development of gut edema. The principle of early abdominal decompression to reduce mesenteric/IVC venous hypertension and capillary pressure is supported by these data.

Intestinal edema decreases intestinal contractile activity via decreased myosin light chain phosphorylation.

OBJECTIVE: The purpose of this study was to investigate the effects of interstitial edema on intestinal contractile activity. DESIGN: Randomized animal study. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTION: Intestinal edema was induced in rats by a combination of fluid infusion and mesenteric venous hypertension. Rats were divided into four groups: CONTROL, sham; RESUS, saline infusion only; RESUS+VH, saline infusion and venous hypertension; and VH, venous hypertension only. Edema development, basal contractile activity, maximum agonist-induced contractile response (measured as total force generation during the first 2 mins after carbachol treatment), and myosin light chain phosphorylation were measured in the distal small intestine. MEASUREMENTS AND MAIN RESULTS: The amount of interstitial fluid, indicated by the wet-to-dry ratio, increased significantly in both the RESUS and RESUS+VH groups as early as 30 mins after surgery compared with the CONTROL group. Whereas the tissue fluid remained significantly elevated in the RESUS+VH group up to 6 hrs after surgery, the RESUS group wet-to-dry ratios returned to CONTROL group levels by 2 hrs after surgery. Basal contractile activity was significantly less in the RESUS+VH group compared with either the RESUS group or the CONTROL group 6 hrs after surgery. Maximum contractile response decreased significantly in the RESUS+VH group compared with the RESUS group. Basal contractile activity and maximum contractile response did not change significantly in the VH group compared with the CONTROL group. The phosphorylated fraction of myosin light chain was significantly lower in the RESUS+VH group compared with the CONTROL group at 0.5, 2, and 6 hrs after surgery. CONCLUSION: We conclude that edema decreases myosin light chain phosphorylation, leading to decreased intestinal contractile activity.

Resuscitation-induced gut edema and intestinal dysfunction.

BACKGROUND: Mesenteric venous hypertension and subsequent gut edema play a pivotal role in the development of intra-abdominal hypertension. Although gut edema is one cause of intra-abdominal hypertension, its impact on gut function is unknown. The purpose of this study was to create a model of acute hydrostatic gut edema and to evaluate its effect on gut motility and barrier function. METHODS: The first study, group A, evaluated the effect of gut edema on transit over time using 20 mL/kg 0.9% saline. The second study, group B, focused on the 12-hour time period using 80 mL/kg 0.9% saline. Rats were randomized to superior mesenteric vein partial occlusion (venous hypertension) or sham surgery. At 6, 12, and 24 hours, group A underwent intestinal transit and tissue water weight measurements. At 12 hours, group B underwent tissue water, transit, ileal permeability and resistance, lactate and myeloperoxidase activity, and mucosal injury measurements. RESULTS: Venous hypertension with fluid resuscitation caused acute hydrostatic gut edema, delayed intestinal transit, increased mucosal permeability to macromolecules, and decreased tissue resistance over time. Mucosal injury was minimal in mesenteric venous hypertension. CONCLUSION: Acute mesenteric venous hypertension and resuscitation-induced gut edema, in the absence of ischemia/reperfusion injury, is associated with delayed intestinal transit and altered gut barrier function.