Role of thromboxane in producing portal hypertension following trauma-hemorrhage

Thromboxane A2 (TXA2) and endothelin-1 (ET-1) have been proposed as the important vasoconstrictors that increase portal venous resistance in paracrine or autocrine fashion. We hypothesized that the hepatic damage following trauma-hemorrhage (T-H) is induced by the impaired hepatic circulation due to the increased production of vasoconstrictors such as ET-1 and TXA2 by the liver. To test this, male Sprague-Dawley rats (n = 6/group) were subjected to trauma (i.e., midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. At 2 or 5 h after the end of resuscitation, the liver was isolated and perfused and portal inflow pressure, bile flow, and release of ET-1 and thromboxane B2 (TXB2; a stable metabolite of TXA2) into the perfusate were measured. The level of portal pressure was higher at 5 h following T-H compared with 2 h after T-H and sham. The portal pressure was inversely correlated to the amount of bile production. Furthermore, the bile flow was significantly correlated to the hepatic damage as evidenced by release of lactate dehydrogenase into the perfusate. The level of ET-1 at 5 h following T-H in the perfusate after 30 min of recirculation did not show any difference from sham. However, the levels of TXB2 in the T-H group were significantly higher than those in sham at that interval. These results indicate that the increased release of TXA2 but not ET-1 following T-H might be responsible for producing the increased portal resistance, decreased bile production, and hepatic damage.     

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging

Previous studies have demonstrated that hemorrhagic shockproduces immunodepression in young male mice, whereas theimmunoresponsivness in young proestrus female mice is enhanced undersuch conditions. This sexually dimorphic immune response to hemorrhageappears to be related to high estrogen and testosterone levels infemales and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected tolaparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-) release] were depressed in youngmales and enhanced in young females after trauma-hemorrhage. Incontrast, in the aged male and female groups these parameters ofsplenocyte function were reversed after trauma-hemorrhage (i.e.,increased proliferation and IL-2 release in aged males compared withsuppressed proliferation and IFN- release in aged females).Furthermore, the release of the immunosuppressive cytokine IL-10inversely correlated with the age- and gender-related changes insplenocyte responses after trauma-hemorrhage. Thus the sexuallydimorphic immune response in young males and females totrauma-hemorrhage appears to reverse as sex hormone levels decline with age.

     

Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and alpha(B)-crystallin, in response to stress. Activation of HSP27 or alpha(B)-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17beta-estradiol (E(2)) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E(2) under those conditions are mediated via p38 MAPK remains unknown. Male rats (275-325 g body wt) were subjected to soft tissue trauma and hemorrhage (35-40 mmHg mean blood pressure for approximately 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E(2) (1 mg/kg body wt), E(2) + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E(2) administration, and phosphorylation of cardiac p38 MAPK, HSP27, and alpha(B)-crystallin was increased. The E(2)-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and alpha(B)-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E(2) on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and alpha(B)-crystallin.     

The in vitro behaviour and patterns of colony formation of murine epithelial stem cells

OBJECTIVE: The mechanisms of renewal of skin and mucosal epithelia in vivo are associated with hierarchies of stem and amplifying cells organized in distinct spatial patterns. Stem and amplifying characteristics persist after isolation and growth of human keratinocytes in vitro but the pattern for murine keratinocytes has been less clear. MATERIALS AND METHODS: Murine keratinocytes were grown in low calcium media and examined for their patterns of colony morphologies. RESULTS: We consistently identified three types of colonies, one of which contains concentric zones of amplifying and differentiated cells surrounding a central zone of cells that have patterns of expression and behavioural characteristic of stem cells. This zonal organization facilitated analysis of stem cell formation and loss. Cells in the central stem cell zone undergo rapid symmetric divisions but expansion of this population is partially limited by their peripheral transition into amplifying cells. A striking feature of central zone cells is their enhanced apoptotic susceptibility and stem cell expansion limited by consistently high background rates of apoptosis. This occasionally reaches catastrophic levels with elimination of the entire central zone. CONCLUSION: In vitro amplification of stem cells for the generation of engineered tissue has tended to focus on control of asymmetric division but these findings suggest that development of mechanisms protecting stem cells from apoptotic changes are also likely to be of particular value.     

Alcohol ingestion before burn injury decreases splanchnic blood flow and oxygen delivery

Recent studies from our laboratory have shown that alcohol and burn injury impair intestinal barrier and immune functions. Although multiple factors can contribute to impaired intestinal barrier function, such an alteration could result from a decrease in intestinal blood flow (BF) and oxygen delivery (DO2). Therefore, in this study, we tested the hypothesis that alcohol ingestion before burn injury reduces splanchnic blood flow and oxygen delivery. Rats (250 g) were gavaged with alcohol to achieve a blood ethanol level in the range of 100 mg/dl before burn or sham injury (25% total body surface area). Day 1 after injury, animals were anesthetized with methoxyflurane. Blood pressure, cardiac output (CO), +/-dP/dt, organ BF (in ml.min(-1).100 g(-1)), and DO2 (in mg.ml(-1).100 g(-1)) were determined. CO and organ BF were determined using a radioactive microsphere technique. Our results indicate that blood pressure, CO, and +dP/dt were decreased in rats receiving a combined insult of alcohol and burn injury compared with rats receiving either burn injury or alcohol alone. This is accompanied by a decrease in BF and DO2 to the liver and intestine. No significant change in BF to the coronary arteries (heart), brain, lung, skin, and muscles was observed after alcohol and burn injury. In conclusion, the results presented here suggest that alcohol ingestion before burn injury reduces splanchnic BF and DO2. Such decreases in BF and DO2 may cause hypoxic insult to the intestine and liver. Although a hypoxic insult to the liver would result in a release of proinflammatory mediators, a similar insult to the intestine will likely perturb both intestinal immune cell and barrier functions, as observed in our previous study.     

Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL^+/+ and transgenic (PRL^–/–) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL^–/– mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL^+/+ mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL^+/+ mice. Splenocyte proliferation in male PRL^–/– mice was significantly lower than in PRL^+/+ mice after T-H. Marginal differences between PRL^+/+ and PRL^–/– mice were observed in the release of IL-2 and IFN- by splenocytes, while the release of IL-10 was significantly higher in PRL^–/– than in PRL^+/+ mice. A significant observation of our study is the release of a 25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL^+/+ and PRL^–/– mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL^–/– transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice. Nb2 cells; cytokines; immune functions     

MAP kinases differentially regulate the expression of macrophage hyperactivity after thermal injury

Thermal injury increases the capacity of macrophages (Mphi) to produce various inflammatory mediators, (i.e., Mphi hyperactivity), which is believed to be involved in the development of subsequent immunosuppression, sepsis, and multiple organ failure. The signal transduction pathways involved in the expression of Mphi hyperactivity post-burn, however, remain to be clearly elucidated. To study this C57BL/6 female mice were subjected to a 25% TBSA burn and splenic Mphis were isolated 7 days later. LPS-stimulated inflammatory mediator production and MAPK expression (P38 ERK 1/2 and JNK) were determined. Burn injury increased LPS-induced P38 MAPK, suppressed JNK activation and ERK 1/2 activation was unaltered. These changes in MAPK activation were paralleled by the increased production of PGE(2), TNF-alpha, IL-1beta, IL-6, and IL-10. Differential sensitivity to the inhibition of the MAPK pathways was observed with regard to the mediator evaluated and the presence or absence of burn injury. In general cytokine production in the burn group was in part resistant to the inhibition of a single MAPK pathway as compared with shams. Thus, burn injury increases cross-talk between the MAPKs pathways, suggesting that alterations MAPK activation and signal transduction contribute to the development Mphi hyperactivity post-injury.