Association analysis of the RET proto-oncogene with Hirschsprung disease in the Han Chinese population of southeastern China.

Hirschsprung disease (HSCR) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. This study investigated a possible role of the RET proto-oncogene in sporadic HSCR patients in the Han Chinese population. Our results indicated that rs1800858, rs1800860, rs1800863, and rs2075912, located in exons 2, 7, 15, and intron 19 of RET, are strongly associated with the disease (P < 0.01), with rs1800860 and rs1800863 playing a protective role in the pathogenesis of HSCR in the Chinese population. We also showed that the haplotype consisting of four SNPs is significantly associated with HSCR. We did not find a significant difference in the CA-repeat in intron 5 of RET between cases and controls. Our study provided further evidence that the RET gene is involved in the susceptibility to HSCR in the Han Chinese population.

Association analysis of the PHOX2B gene with Hirschsprung disease in the Han Chinese population of Southeastern China.

BACKGROUND: Hirschsprung disease (HSCR, OMIM 142623) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. The PHOX2B gene is involved in neurogenesis and disruption of Phox2b in mice results in a HSCR-like phenotype. The first association study of the PHOX2B gene with HSCR derived from Chinese population in Hong Kong; here, we address the question of whether PHOX2B acts as a predisposing factor in HSCR pathogenesis in Chinese population in mainland. METHODS: To investigate the contribution of PHOX2B to the HSCR phenotype, polymerase chain reaction amplification and direct sequencing were used to screen PHOX2B coding regions and intron/exon boundaries for mutations and polymorphisms in 102 patients with HSCR and 96 ethnically matched controls, in Han Chinese populations of Southeastern China. RESULTS: In this study, we genotyped 4 single nucleotide polymorphisms (SNPs) (including 1 novel SNP) located within the PHOX2B gene. Statistically significant differences were found for c.701 A > G and IVS2 + 100 A > G, and the log-additive model was accepted as the best inheritance model (odds ratio [OR], 1.79; 95% confidence interval [CI], 1.11-2.87) for IVS2 + 100 A > G. We also showed that the haplotype-A G A N composed of 4 SNPs exhibited significant association with the disease (P = .03); this haplotype was more frequently observed in cases than in controls (OR, 2.31; 95% CI, 1.11-4.82). CONCLUSIONS: Our study provided further evidence that the PHOX2B gene is involved in the susceptibility to HSCR in the Han Chinese population. Our findings are in accordance with the involvement of PHOX2B in the signaling pathways governing the development of enteric neurons.

[Effects of tannic acid pretreatment on cardiovascular function during hemorrhagic shock in rats].

OBJECTIVE: To investigate the effects of tannic acid pretreatment on cardiovascular function during hemorrhagic shock in rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into two groups of shock and tannic acid pretreatment+shock. (1) In vivo experiment: the model of hemorrhagic shock in rats was reproduced by bleeding to 40 mm Hg (1 mm Hg = 0.133 kPa) being maintained for 120 minutes. Tannic acid in the dosage of 5 mg/kg was injected intravenously 10 minutes before hemorrhagic shock in tannic acid pretreatment+shock group. The mean arterial pressure (MAP), myocardial contractility and vascular reactivity were measured before hemorrhagic shock and at 180 minutes after hemorrhagic shock. In another experiment, the rats subjected to hemorrhagic shock and blood reinfusion were injected with norepinephrine (NE) intravenously at 60,120 and 180 minutes, and the vascular reactivity was observed. (2) In vitro experiment: the heart was harvested after shock and fixed on a Langendorff system. The perfusion pressure was maintained at 100 mm Hg. The effects of tannic acid pretreatment on myocardial contractility was observed. RESULTS: (1)In vivo experiment showed that tannic acid pretreatment significantly increased MAP at 60 minutes and 150 minutes, and left ventricular systolic pressure (LVSP) at 60 minutes, and the heart rate was obviously slowed at 120 minutes, and left ventricular end diastolic pressure (LVEDP) was lowered (all P < 0.05). The vascular reactivity was significantly improved at 120 minutes in tannic acid pretreatment+shock group compared with shock group (P < 0.05). (2) In vitro experiment proved that tannic acid pretreatment significantly slowed heart rate at 90 minutes as well as increased +dp/dtmax at 10 minutes and 20 minutes and -dp/dtmax at 10 minutes (all P < 0.05). CONCLUSION: Pretreatment with tannic acid improves cardiovascular function following hemorrhagic shock to some extent in rats.

HSD is a better resuscitation fluid for hemorrhagic shock with pulmonary edema at high altitude.

To investigate the fluid tolerance of hemorrhagic shock with pulmonary edema (HSPE) at high altitude in unacclimated rats and the beneficial effect of 7.5% hypertonic saline/6% dextran (HSD). One hundred seventy-six Sprague-Dawley rats, transported to LaSa, Tibet, 3,760 m above the sea level, were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) within 1 week. Hemorrhagic shock with pulmonary edema was induced by bloodletting (50 mmHg for 1 h) plus intravenous injection of oleic acid (50 microL/kg). Seventy-seven rats were equally divided into 11 groups (n = 7/group) including sham-operated control group; hemorrhagic shock control group; HSPE control group; HSPE plus 0.5-, 1.0-, 1.5-, 2.0-, or 3.0-fold volumes of lactated Ringer's solution (LR) groups; and HSPE plus 4, 6, and 8 mL/kg of HSD groups. Hemodynamic parameters including mean arterial blood pressure, left intraventricular systolic pressure, and the maximal change rate of intraventricular pressure rise or decline (+/-dp/dtmax) were observed at baseline and at 15, 30, 60, and 120 min after infusion; blood gases were measured at 30 and 120 min after infusion, and the water content of lung and brain was determined at 120 min after infusion. Additional 99 rats were used to observe the effect of these treatments on the survival time of HSPE rats; 0.5 volume of LR infusion slightly increased the mean arterial blood pressure, left intraventricular systolic pressure, and +/-dp/dtmax and prolonged the survival time of HSPE animals as compared with the HSPE group (P < 0.05 - 0.01); it did not increase the water content of lung and brain and had no marked influences on blood gases. One volume of LR infusion had somewhat improved the hemodynamic parameters for HSPE animals, but had no apparent effect on the survival time and the water content of lung and brain. Lactate Ringer's solution infusion, 1.5, 2, and 3 volumes, significantly deteriorated the hemodynamic parameters, increased the water content of lung, and decreased the survival time of HSPE animals. Hypertonic saline/6% dextran (4 - 8 mL/kg) significantly increased the hemodynamic parameters, improved the blood gases, decreased the water content of lung and brain, and prolonged the survival time of HSPE rats. Among the three dosages of HSD, 6 mL/kg of HSD had the best effect. The tolerance of fluid infusion for hemorrhagic shock with pulmonary edema at high altitude is significantly decreased. More than one volume of LR infusion would aggravate the pulmonary edema and exacerbate the resuscitation effect, but only one volume of LR cannot reach the effective volume resuscitation. Small volume of HSD could better resuscitate hemorrhagic shock with pulmonary edema at high altitude.

[Effect of poly-adenosine diphosphate ribosyl-polymerase on vascular hyporeactivity in rats with hemorrhagic shock].

OBJECTIVE: To study the effects of poly-adenosine diphosphate ribosyl-polymerase (PARP) on vascular hyporeactivity during hemorrhagic shock in rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: shock, 3-aminobenzamide (3-AB) pretreatment+shock, and sham operation. Bleeding from the femoral artery to induce hemorrhagic shock model. The blood pressure changes following 3 microg/kg norepinephrine (NE) injection were observed in vivo. The response of vascular rings of superior mesenteric artery (SMA) to NE was determined ex vivo. The nitrogen monoxide (NO) contents of plasma and tissue homogenate of SMA were measured using the assay kit based on the nitrate reductase reaction. RESULTS: The maximum increase of mean arterial pressure in response to NE immediately following shock in the shock group was significantly lower than in the sham operation group (P<0.01) and the value at 1 hour after blood reinfusion in the shock group was obviously lower than in the 3-AB pretreatment+shock group (P<0.05) and in the sham operation group (P<0.01). The maximum concentration force in the sham operation group [(0.367 1+/-0.221 3)g/mm] was significantly increased than in the 3-AB pretreatment+shock group [(0.286 4+/-0.153 2) g/mm, P<0.05] and in the shock group [(0.185 6+/-0.111 3)g/mm, P<0.01]. The cumulative dose-response curves of SMA to NE shifted to the left, and the contraction force was markedly increased as NE concentration reaching 10(-6), 10(2+) and 10(-5) mol/L in the 3-AB pretreatment+shock group compared to the shock group (all P<0.05). There were no significant difference on plasma NO content among the three groups. However, the NO contents of plasma and tissue homogenate of SMA in the 3-AB pretreatment+ shock group were slightly lower than in the shock group (P>0.05). CONCLUSION: PARP is involved in the vascular hyporeactivity in hemorrhagic-shocked rats.

[Experimental study on changes in myocardial systolic function and arterial blood gases after impact injury to liver in rabbits].

OBJECTIVE: To explore the effects of impact injury of liver on myocardial systolic function and arterial blood gases in rabbits. METHODS: A model of hepatic impact injury was reproduced in rabbits by hitting the xiphoid area with two falling steel balls. Myocardial systolic function and arterial blood gases were measured respectively before injury, immediately and 0.5, 1, 2 hours after injury, and before the death. RESULTS: After injury, heart rate (HR), left ventricular systolic pressure (LVSP), the maximum change rate of left ventricular pressure rise and fall (+/-dp/dt max) descended significantly, while left ventricular end-diastolic pressure (LVEDP) showed no statistically significant change. At the same time, blood pH and HCO(3)(-) fell gradually, and negative base excess (BEecf) showed a significant increase, indicating metabolic acidosis gradually developed. There was no obvious significant difference in partial pressure of carbon dioxide in artery (PaCO(2)) between the values before and after injury. Partial pressure of oxygen in artery (PaO(2)) decreased gradually, but no significant differences were found between the values of 0.5 hour and 1 hour after injury and that before injury. CONCLUSION: After hepatic impact injury, myocardial systolic function deteriorates, metabolic acidosis appears.

[Activity of nuclear factor kappa B in rat pulmonary tissue after entrance of amniotic fluid into blood and inhibition with dexamethasone].

OBJECTIVE: To investigate the relationship between nuclear factor kappa B (NF-kappaB) and pulmonary injury in amniotic fluid embolism model of rat. METHODS: Seventy female Wistar rats were divided into five groups randomly: control group (6), amniotic fluid group (16), amniotic fluid + dexamethasone group (14), meconium group (20) and meconium + dexamethasone group (14). Different amniotic fluid was injected into jugular vein (dexamethasone was injected at 0.1 mg/100 g after entrance of amniotic fluid into blood) and blood pressure was examined. Pulmonary tissue was taken at 60 minutes. NF-kappaB activity was measured by Western-blot and percentage of NF-kappaB p65 positive cells in pulmonary tissue was determined by immunohistochemistry (HE). RESULTS: Dropsy, bleeding and neutrophil (PMN), macrophage, leukomonocyte infiltration were seen in four experimental groups. But none was found in control group. NF-kappaB activity in meconium group was 438,698 +/- 13,092, higher than those in amniotic fluid group, 377,982+/- 7,445, and in control group, 267,691 +/- 12 382 (F = 11.3, P < 0.01). With dexamethasone treatment, NF-kappaB activity was decreased, which was 308,826 +/- 13,771 in amniotic group and 339,516 +/- 17,358 in meconium group, respectively (t = 20.4 and t = 13.84, P < 0.01). Percentage of NF-kappaB p65 positive cells was higher in meconium group, 49.1 +/- 7.0, than in amniotic fluid group, 33.3 +/- 2.7, and control group, 13.3 +/- 2.1 (F = 1.17, P < 0.01). With dexamethasone treatment, the percentage decreased significantly to 22.9 +/- 3.0 and 21.4 +/- 3.6, respectively (t = 6.75 and t = 10.1, P < 0.05). CONCLUSIONS: NF-kappaB activity and percentage of NF-kappaB p65 positive cells are increased significantly, which is associated with pulmonary injury after entrance of amniotic fluid into blood and dexamethasone could inhibit NF-kappaB translocation to the nucleus to degrade NF-kappaB activity and alleviate pulmonary injury. NF-kappaB may be relevant to the occurrence and development of multiple organ dysfunction syndrome.

[Changes in systemic and local vascular reactivity in hemorrhagic shock and the therapeutic effect of delta opioid receptor antagonist ICI174,864].

OBJECTIVE: To investigate the changes in systemic and local vascular reactivity following hemorrhagic shock of different severity and the therapeutic effect of alpha opioid receptor antagonist ICI174,864. METHODS: Fifty-six Wistar rats were used in two experiments. In experiment I, 32 rats were equally divided into sham operation group, 1 hour, 2 hours and 3 hours hypotension groups. In the latter groups, rats were bled to a mean arterial blood pressure (MAP) of 40 mm Hg (1 mm Hg=0.133 kPa) and maintained at this level for 1, 2, 3 hours, respectively. The pressor response of blood pressure and the contractile response of superior mesenteric artery (SMA) to norepinephrine(NE, 3 ug/kg) were observed after shed blood was reinfused. In experiment II, 24 rats were divided into shock control, ICI174,864 0.5 mg/kg and 1.0 mg/kg groups. The response of blood pressure and SMA contractility to NE (3 microg/kg) were observed at 1, 2, and 4 hours after ICI174,864 administration. RESULTS: Following hemorrhagic shock, the systemic and local (SMA) vascular responsiveness was significantly decreased significantly and it was time dependent. Shed blood reinfusion alone did not restore the decreased vascular reactivity. ICI174,864 improved the decreased vascular reactivity in dose-dependent manner. CONCLUSION: Hemorrhagic shock can induce systemic and local vascular hyporeactivity. The decreased vascular reactivity is closely associated with the severity and duration of shock. Loss of systemic vascular reactivity parallels to that of the regional vessel. delta opioid receptor antagonist ICI174,864 has some beneficial effect in improving vascular hyporeactivity.

[Activity and role of tryptase after entrance of amniotic fluid into blood in rats].

OBJECTIVE: To investigate the activity and role of tryptase after entrance of amniotic fluid into blood in rats. METHODS: Thirty female Wistar rats (20 day pregnancy) were divided into the control group (10, injected with normal saline), amniotic group (10, injected with amniotic fluid), meconium group (10, injected with 1% meconium). After injection, pulmonary tissue was taken out. Tryptase activity was measured by special substrate. The histology of pulmonary tissue was determined by immunohistochemistry (HE). RESULTS: (1) Dropsy, hemorrhage, and infiltration of neutrophil (PMN), macrophage, leukomonocyte were observed in two experimental groups, but no such changes were found in control group. (2) After injection, tryptase activity in meconium group 176.4 +/- 8.6 and amniotic fluid group 165.4 +/- 7.4 was significantly higher than preexperimental groups 146.8 +/- 8.9 and 147.8 +/- 9.5, respectively (t = 7.58 and t = 4.64, P < 0.01); tryptase activity in control group was 145.3 +/- 10.6 before injection and 146.9 +/- 9.4 after injection, respectively, there was no difference (t = 0.37, P > 0.05). After injection, tryptase activity in meconium and amniotic fluid groups was significantly increased than that in control group (F = 30.66, P < 0.05). CONCLUSION: The activity of tryptase was significantly increased after entrance of amniotic fluid into blood in rats. Degranulation of mast cells to release tryptase may be the important cause of the pathophysiologic change after entrance of amniotic fluid into blood. These results suggest a role for mast cell activation in the mechanism of amniotic fluid embolism. This method is sensitive and effective for diagnosis of amniotic fluid embolism in clinic.

[Beneficial effect of thyrotropin-releasing hormone in combination with HSD on hemorrhagic shock with pulmonary edema at high altitude in the rat].

OBJECTIVE: To study the effects of thyrotropin-releasing hormone (TRH) in combination with hypertonic saline/dextran (7.5% NaCl + 6% Dextran 40, HSD ) on hemorrhagic shock with pulmonary edema in the rats which were recently brought to high altitude. METHODS: Forty-nine SD rats, transported to Lasa, Tibet, which was 3,760 meters above the sea level, were anesthetized one week later with sodium pentobarbital (30 mg/kg, intraperitoneal). Hemorrhagic shock with pulmonary edema was induced by hemorrhage (50 mm Hg maintained for 1 hour,1 mm Hg=0.133 kPa) plus intravenous injection of oleic acid (50 microl/kg). They were equally divided into seven groups (n=7): normal control, hemorrhagic shock, hemorrhagic shock with pulmonary edema (HSPE), HSPE plus TRH (5 mg/kg), HSPE plus HSD (4 ml/kg), and HEPE plus TRH and HSD in combination. Hemodynamic parameters including mean arterial blood pressure(MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (+/- dp/dt max) were observed at 15, 30, 60 and 120 minutes, blood gases were analyzed at 30 and 120 minutes, and the water content of lung and brain was determined at 120 minutes after drug administration. RESULTS: TRH or HSD used alone or in combination significantly increased MAP, LVSP and +/- dp/dt max (P<0.05 or P<0.01 ), ameliorated acid-base imbalance, and decreased the water content of lung and brain. The effect of the two in combination was superior to either drug used alone. CONCLUSION: TRH in combination with HSD can be used in the treatment of hemorrhagic shock with pulmonary edema at high altitude.

[Effect of different volumes of fluid resuscitation on hemorrhagic shock with pulmonary edema at high altitude in the unacclimated rat].

OBJECTIVE: To study the effects of different volumes of fluid resuscitation on hemorrhagic shock with pulmonary edema at high altitude in the unacclimated rat. METHODS: One hundred and twenty-six SD rats transported to Lasa, Tibet, 3 760 meters above the sea level, were anesthetized one week later with sodium pentobarbital (30 mg/kg, intraperitoneal). Hemorrhagic shock with pulmonary edema model was induced by hemorrhage (50 mm Hg for 1 hour, 1 mmHg=0.133 kPa) plus intravenous injection of oleic acid (50 microl/kg). Experiments were then conducted in two parts. Sixty-three rats in part I were equally divided into nine groups (n=7): normal control, hemorrhagic shock control, hemorrhagic shock with pulmonary edema (HSPE) without fluid infusion, HSPE plus infusing lactated Ringer's solution (LR) with 0.5-, 1-, 1.5-, 2- or 3- fold volume shed blood, and 1 volume of LR plus mannitol (10 ml/kg). Hemodynamic parameters including mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (+/- dp/dt max) were observed at 15, 30, 60 and 120 minutes after infusion, blood gases were measured at 30 and 120 minutes after infusion and the water content of lung and brain was determined at 120 minutes after infusion. In part II, additional 63 rats were used to observe the effect of different volumes of fluid resuscitation on survival time of HSPE rats. RESULTS: 0.5 volume of LR infusion significantly improved MAP, LVSP and +/- dp/dt max, prolonged the survival time of HSPE animals (all P<0.01), while it did not increase the water content of lung and brain and had no marked influence on blood gases. One volume of LR infusion slightly improved hemodynamic parameters, prolonged the survival time and increased the water content of lung. More than 1 volume of LR infusion including 1.5-, 2- and 3- fold volume LR deteriorated the hemodynamic parameters and decreased the survival time of shocked animal, meanwhile they apparently increased the water content of lung. One volume of LR plus mannitol (10 ml/kg) infusion did not improve the hemodynamic parameters and blood gases; also it did not decrease the water content of lung. CONCLUSION: The tolerance to fluid infusion for the unacclimated animal subjected to hemorrhagic shock with pulmonary edema at high altitude is significantly decreased. 0.5-1 volume of LR infusion appears to be beneficial effect on resuscitation at high altitude, while over 1 volume of LR infusion would aggravate pulmonary edema and exacerbate fluid resuscitation effect.

Protective effects of apocynin on "two-hit" injury induced by hemorrhagic shock and lipopolysaccharide.

AIM: To evaluate the protective effects of apocynin on "two-hit" injury in rats. METHODS: "Two-hit" injury model of rat was induced by hemorrhagic shock (40 mmHg for 45 min) followed by iv administration of lipopolysaccharide (LPS, 150 microg/kg). Rats were randomized into seven groups: Sham, LPS, hemorrhage, hemorrhage/LPS, and hemorrhage/LPS+apocynin (2.5, 5.0, and 10.0 mg/kg). Apocynin was dissolved in the resuscitation fluid (normal saline, NS) and administered iv for 2 h. After LPS or NS administration, the survival rates at 8 h, 16 h, 24 h, and 48 h were monitored. The content of malondialdehyde (MDA) was measured in lung at 3 h and 6 h after iv LPS and in serum before hemorrhage, after hemorrhage, and at 0, 0.5, 1, 2, 4, and 6 h after iv LPS. Myeloperoxidase (MPO) activity in lung and liver was examined at 3 h and 6 h after iv LPS/NS. RESULTS: After "two-hit" injury, the survival rates of rats at 8 h, 16 h, 24 h, and 48 h were 64.3 %, 35.7 %, 28.6 %, and 14.3 % respectively, there were significant differences as compared to sham group (P<0.05 or P<0.01, respectively), the MDA level in lung and serum were significantly enhanced (P<0.01) as compared to sham group, and MPO activity in lung and liver after "two-hit" injury was also significantly increased (P<0.01). Apocynin treatment enhanced the mean arterial pressure (MAP) of hemorrhagic shock rats dose-dependently (P<0.05), increased the survival rate of "two-hit" injury rats, decreased the serum and lung MDA content, and downregulated MPO activity in lung and liver. CONCLUSION: Apocynin could preventively ameliorate "two-hit" injury in rats induced by hemorrhagic shock and LPS insult.