Karen S. Uray

Intravenous Multipotent Adult Progenitor Cell Therapy Attenuates Activated Microglial/Macrophage Response and Improves Spatial Learning After Traumatic Brain Injury

We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood‐brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long‐term behavioral and anti‐inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI‐2) or 10 million cells per kilogram (CCI‐10). CCI‐10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI‐10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI‐10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI‐10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long‐term improvements in spatial learning as well as attenuation of neuroinflammation.

Hypertonic saline modulation of intestinal tissue stress and fluid balance.

Crystalloid-based resuscitation of severely injured trauma patients leads to intestinal edema. A potential mechanism of intestinal edema-induced ileus is a reduction of myosin light chain phosphorylation in intestinal smooth muscle. We sought to determine if the onset of edema initiated a measurable, early mechanotransductive signal and if hypertonic saline (HS) can modulate this early signal by changing intestinal fluid balance. An anesthetized rat model of acute interstitial intestinal edema was used. At laparotomy, the mesenteric lymphatic was cannulated to measure lymph flow and pressure, and a fluid-filled micropipette was placed in the intestinal submucosa to measure interstitial pressure. Rats were randomized into four groups (n = 6 per group): sham, mesenteric venous hypertension + 80 mL/kg 0.9% isotonic sodium chloride solution (ISCS 80), mesenteric venous hypertension + 80 mL/kg 0.9% ISCS + 4 mL/kg 7.5% saline (ISCS 80 + HS), or 4 mL/kg 7.5% saline (HS alone) to receive the aforementioned intravenous fluid administered over 5 min. Measurements were made 30 min after completion of the preparation. Tissue water, lymph flow, and interstitial pressure were measured. Resultant applied volume induced stress on the smooth muscle (σravi-muscularis) was calculated. Mesenteric venous hypertension and crystalloid resuscitation caused intestinal edema that was prevented by HS. Intestinal edema caused an early increase in intestinal interstitial pressure that was prevented by HS. Hypertonic saline did not augment lymphatic removal of intestinal edema. σravi-muscularis was increased with onset of edema and prevented by HS, paralleling the interstitial pressure data. Intestinal edema causes an early increase in interstitial pressure that is prevented by HS. Prevention of the edema-induced increase in interstitial pressure serves to blunt the mechanotransductive signal of σravi-muscularis.

Resuscitation-induced intestinal edema and related dysfunction: State of the science

High volume resuscitation and damage control surgical methods, while responsible for significantly decreasing morbidity and mortality from traumatic injuries, are associated with pathophysiologic derangements that lead to subsequent end organ edema and dysfunction. Alterations in hydrostatic and oncotic pressures frequently result in intestinal edema and subsequent dysfunction. The purpose of this review is to examine the principles involved in the development of intestinal edema, current and historical models for the study of edema, effects of edema on intestinal function (particularly ileus), molecular mediators governing edema-induced dysfunction, potential role of mechanotransduction , and therapeutic effects of hypertonic saline. We review the current state of the science as it relates to resuscitation induced intestinal edema and resultant dysfunction.

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.

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.

A novel physiologic model for the study of Abdominal Compartment Syndrome (ACS)

BACKGROUND : Current abdominal compartment syndrome (ACS) models rely on intraperitoneal instillation of fluid, air, and other space-occupying substances. Although this allows for the study of the effects of increased abdominal pressure, it poorly mimics its pathogenesis. We have developed the first reported large animal model of ACS incorporating hemorrhagic shock/resuscitation. METHODS : Hemorrhagic shock was induced and maintained (1 hour) in 12 Yorkshire swine by bleeding to a mean arterial pressure (MAP) of 50 mm Hg. The collected blood plus two additional volumes of crystalloid was then reinfused. Mesenteric venous hypertension was induced by tightening a previously placed portal vein snare in a nonocclusive manner to mimic the effects of abdominal packing. Crystalloids were infused to maintain MAP. Hemodynamic measurements, abdominal pressure, peak inspiratory pressures, urine output, and blood chemistries were measured sequentially. Animals were studied for 36 hours after decompression. RESULTS : ACS (intra-abdominal pressure of > or =20 mm Hg with new organ dysfunction) developed in all animals. There were significant increases in peak inspiratory pressure, central venous pressure, and pulmonary artery pressure and decreases in MAP upon development of ACS. Urine output was significantly decreased before decompression. Mean blood lactate decreased and base excess increased significantly after decompression. CONCLUSIONS : We have created the first reported physiologic animal ACS model incorporating hemorrhagic shock/resuscitation and the effects of damage control surgery.

Nuclear factor-κB activation by edema inhibits intestinal contractile activity

Objective:To investigate the molecular mechanisms leading to edema-induced decreases in intestinal smooth muscle myosin light-chain phosphorylation. Intestinal interstitial edema often develops during abdominal surgery and after fluid resuscitation in trauma patients. Intestinal edema causes decreased intestinal contractile activity via decreased intestinal smooth muscle myosin light-chain phosphorylation, leading to slower intestinal transit. Interstitial edema development is a complex phenomenon, resulting in many changes to the interstitial environment surrounding intestinal smooth muscle cells. Thus, the mechanism(s) by which intestinal edema development causes intestinal dysfunction are likely to be multifactorial. Design:Randomized animal study. Setting:University laboratory. Subjects:Male Sprague-Dawley rats, weighing 250-350 g. Intervention:Studies were performed in a rat model in which a combination of mesenteric venous hypertension and administration of resuscitative fluids induces intestinal edema, mimicking the clinical setting of damage control resuscitation. Measurements and Main Results:Microarray analysis of edematous intestinal smooth muscle combined with an in silico search for overrepresented transcription factor binding sites revealed the involvement of nuclear factor-&kgr;B in edema-induced intestinal dysfunction. Nuclear factor-&kgr;B deoxyribonucleic acid binding activity was significantly increased in edematous intestinal smooth muscle compared with controls. Inhibition of nuclear factor-&kgr;B activation blocked edema-induced decreases in basal intestinal contractile activity. Inhibition of nuclear factor-&kgr;B activation also attenuated edema-induced decreases in myosin light-chain phosphorylation. Conclusions:We conclude that intestinal edema activates nuclear factor-&kgr;B, which, in turn, triggers a gene regulation program that eventually leads to decreased myosin light-chain phosphorylation and, thus, decreased intestinal contractile activity.

SIRT1 inhibits the mouse intestinal motility and epithelial proliferation

Sirtuin 1 (SIRT1), a NAD(+)-dependent histone deacetylase, is involved in a wide array of cellular processes including glucose homeostasis, energy metabolism, proliferation and apoptosis, and immune response. However, it is unknown whether SIRT1 plays any physiological role in the regulation of intestinal homeostasis and motility. Thus the aim was to define SIRT1 expression and function in the gastrointestinal (GI) tract under physiological conditions. Forty 12-14-wk-old SIRT1 knockout (KO) and wild-type (WT) mice were fasted 21 h and/or refed 3 h. Fasted mice were injected intraperitoneally with bromodeoxyuridine (120 mg/kg body wt) 2 h before euthanasia. SIRT1 protein was localized to gastric and intestinal epithelial nuclei and was responsive to the nutritional status. SIRT1 was required for intestinal epithelial homeostasis. The SIRT1 KO mice showed enhanced crypt proliferation and suppressed villous apoptosis, resulting in increased intestinal villous height. In the SIRT1 KO intestine, the abundance of Forkhead box protein O1 and p53 protein decreased, whereas the subcellular localization of β-catenin protein accumulated mainly in the crypts. The SIRT1 KO mice showed accelerated gastric emptying rate with increased abundance of ghrelin mRNA and protein in the stomach. Moreover, the SIRT1 KO mouse intestine showed enhanced ex vivo spontaneous contraction. We concluded that, SIRT1 plays a critical role in the control of intestinal homeostasis (by promoting apoptosis and inhibiting proliferation) and gastrointestinal motility (by reducing gastric emptying and intestinal contractile activity), implicating a novel role for SIRT1.

Hypertonic saline alters hydraulic conductivity and up-regulates mucosal/submucosal aquaporin 4 in resuscitation-induced intestinal edema

Objective:To characterize membrane conductivity by applying mathematical modeling techniques and immunohistochemistry and to localize and predict areas of the bowel where aquaporins may be associated with edema resolution/prevention associated with hypertonic saline. Intestinal edema induced by resuscitation and mesenteric venous hypertension impairs intestinal transit/contractility. Hypertonic saline decreases intestinal edema and improves transit. Aquaporins are water transport membrane proteins that may be up-regulated with edema and/or hypertonic saline. Design:Laboratory study. Setting:University research laboratory. Subjects:Male Sprague Dawley rats, weighing 270 to 330 g. Interventions:Rats were randomized to control (with and without hypertonic saline) and mesenteric venous hypertension with either 80 mL/kg normal saline (RESUS + VH + VEH) or 80 mL/kg normal saline with hypertonic saline (RESUS + VH + HTS). After 6 hrs, intestinal wet/dry ratios, urine output, peritoneal fluid, and intraluminal fluid were measured. Hydraulic conductivity was calculated from our previously known and published pressure-flow data. The cDNA microarray, Western blot, polymerase chain reaction, and immunohistochemistry studies were conducted for candidate aquaporins and distribution in intestinal edema resolution. Measurements and Main Results:Hypertonic saline decreased edema and increased urine, intraluminal, and peritoneal fluid volume. RESUS + VH favors fluid flux into the interstitium. Hypertonic saline causes increased hydraulic conductivity at the seromuscular and mucosal surfaces at the same time limiting flow into the interstitium. This is associated with increased aquaporin 4 expression in the intestinal mucosa and submucosa. Conclusions:Hypertonic saline mitigates intestinal edema development and promotes fluid redistribution secondary to increased membrane conductivity at the mucosal and seromuscular surfaces. This is associated with up-regulation of aquaporin 4 gene expression and protein. Aquaporin 4 may be a useful therapeutic target for strategies to enhance edema resolution.

Effects of traumatic brain injury on intestinal contractility.

Patients with traumatic brain injury (TBI) often suffer from gastrointestinal dysfunction including intolerance to enteral feedings. However, it is unclear how TBI affects small intestinal contractile activity. The purpose of this study was to determine if TBI affects intestinal smooth muscle function.