Changes in the Expression of AQP4 and AQP9 in the Hippocampus Following Eclampsia-Like Seizure.

Eclampsia is a hypertensive disorder of pregnancy that is defined by the new onset of grand mal seizures on the basis of pre-eclampsia. Until now, the mechanisms underlying eclampsia were poorly understood. Brain edema is considered a leading cause of eclamptic seizures; aquaporins (AQP4 and AQP9), the glial water channel proteins mainly expressed in the nervous system, play an important role in brain edema. We studied AQP4 and AQP9 expression in the hippocampus of pre-eclamptic and eclamptic rats in order to explore the molecular mechanisms involved in brain edema. Using our previous animal models, we found several neuronal deaths in the hippocampal CA1 and CA3 regions after pre-eclampsia and that eclampsia induced more neuronal deaths in both areas by Nissl staining. In the current study, RT-PCR and Western blotting data showed significant upregulation of AQP4 and AQP9 mRNA and protein levels after eclamptic seizures in comparison to pre-eclampsia and at the same time AQP4 and AQP9 immunoreactivity also increased after eclampsia. These findings showed that eclamptic seizures induced cell death and that upregulation of AQP4 and AQP9 may play an important role in this pathophysiological process.

Cyclosporin A significantly improves preeclampsia signs and suppresses inflammation in a rat model.

Preeclampsia is associated with an increased inflammatory response. Immune suppression might be an effective treatment. The aim of this study was to examine whether Cyclosporin A (CsA), an immunosuppressant, improves clinical characteristics of preeclampsia and suppresses inflammation in a lipopolysaccharide (LPS) induced preeclampsia rat model. Pregnant rats were randomly divided into 4 groups: group 1 (PE) rats each received LPS via tail vein on gestational day (GD) 14; group 2 (PE+CsA5) rats were pretreated with LPS (1.0 µg/kg) on GD 14 and were then treated with CsA (5mg/kg, ip) on GDs 16, 17 and 18; group 3 (PE+CsA10) rats were pretreated with LPS (1.0 µg/kg) on GD 14 and were then treated with CsA (10mg/kg, ip) on GDs 16, 17 and 18; group 4 (pregnant control, PC) rats were treated with the vehicle (saline) used for groups 1, 2 and 3. Systolic blood pressure, urinary albumin, biometric parameters and the levels of serum cytokines were measured on day 20. CsA treatment significantly reduced LPS-induced systolic blood pressure and the mean 24-h urinary albumin excretion. Pro-inflammatory cytokines IL-6, IL-17, IFN-γ and TNF-α were increased in the LPS treatment group but were reduced in (LPS+CsA) group (P<0.05). Anti-inflammatory cytokine IL-4 was decreased in the LPS group but was increased in (LPS+CsA) group (P<0.05). Cyclosporine A improved preeclampsia signs and attenuated inflammatory responses in the LPS induced preeclampsia rat model which suggests that immunosuppressant might be an alternative management option for preeclampsia.

[Influence of iron on siderophore and photosynthetic pigments biosynthesis by siderophore-producing Rhodopesudomonnas palustris].

OBJECTIVE: To explore the regulation of iron on siderophore production, cell growth and photosynthetic pigments biosynthesis by siderophore-producing anoxygenic phototrophic bacteria. METHODS: Siderophore production was determined using Chrome Azurol S (CAS) assay. The siderophore types were determined by Arnow method, Csaky test and Shenker test. The compositions and contents of photosynthetic pigments were determined by spectrophotometry and HPLC analysis. RESULTS: Rhodopseudomonas palustris (Rps. palustris) CQV97 was capable of producing hydroxamate-type of siderophore. Siderophore production reached the highest yield in the absence of ferric chloride. With increasing ferric chloride concentrations, the lag phase of cell growth was shortened, and the cell growth rate, final biomass and the total amounts of carotenoid and bacteriochlorophyll a were increased significantly. The characteristic absorption maxima of carotenoids from pigment extracts were blueshifted. Iron concentration had little effect on the compositions and relative contents of bacteriochlorophylls a, whereas predominately affected carotenoid compositions, rhodopin was present as major carotenoid component instead of spirillxanthin. Culture tends to accumulate the Cars having shorter conjugated double bonds at the expense of longer conjugated double bonds as the ferric chloride concentration increased. The changes in carotenoid composition were consistent with those of the blue shift of absorption spectra of pigment extracts. CONCLUSION: Rps. palustris CQV97 can produce siderophore and the changes in microbial growth, siderophore production and photosynthetic pigments accumulation of anoxygenic phototrophic bacteria are related to the iron concentration in the medium.

Cyclosporin A ameliorates eclampsia seizure through reducing systemic inflammation in an eclampsia-like rat model.

Our previous studies have shown that the maternal hyperinflammatory response in pre-eclampsia lowered the eclampsia-like seizure threshold. Cyclosporin A (CsA), which is an effective immunosuppressant, could attenuate the inflammatory responses in LPS-induced pre-eclampsia rats. Here, we hypothesized that CsA may ameliorate seizure severity through reducing systemic inflammation in pre-eclampsia/eclampsia. In the current study, the effects of CsA on pre-eclampsia manifestation, eclampsia-like seizure activities and systemic inflammation were examined in a pre-eclampsia model. Pregnant rats were given an intraperitoneal injection of the epileptogenic drug pentylenetetrazol (PTZ) following a tail vein injection of lipopolysaccharide to establish the eclampsia-like seizure model. CsA (5 mg/kg) was administered intravenously through the tail after LPS infusion. Mean systolic blood pressure and proteinuria in pre-eclampsia were detected. After PTZ injection, seizure activity was assessed, inflammatory responses were determined and pregnancy outcomes were analyzed. The results showed that CsA treatment significantly decreased blood pressure and proteinuria and increased the fetal and placental weight (P < 0.01). Meanwhile, CsA treatment significantly reduced serum IL-1ß, TNF-α, and IL-17 levels (P < 0.01), decreased the seizure scores and prolonged the latency to seizure (P < 0.01). CsA effectively attenuated pre-eclampsia manifestation and eclampsia-like seizure severity. In addition, CsA treatment significantly reduced the inflammatory cytokine levels and improved pregnancy outcomes following eclampsia-like seizures. The decreased inflammatory cytokines in pre-eclampsia are coincident with attenuated pre-eclampsia manifestation after CsA treatment, suggesting that CsA treatment might decrease the eclampsia-like seizure severity through decreasing systemic inflammation in pre-eclasmpsia/eclampsia.

Decreased seizure threshold in an eclampsia-like model induced in pregnant rats with lipopolysaccharide and pentylenetetrazol treatments.

OBJECTIVE: Eclampsia is a poorly understood but potentially fatal complication of pregnancy. Research to date on this disorder has been hampered by the lack of a suitable animal model. To correct this deficiency, this report describes the generation of a rat eclampsia-like model using pentylenetetrazol (PTZ) in a previously established rat preeclampsia model. METHOD: Rats were administered lipopolysaccharide (1.0 µg/kg) by tail vein injection on gestational day 14 to establish preeclampsia (PE). PE and control rats (non-pregnant, NP; normal-pregnant, P) were injected intraperitoneally (i.p.) with PTZ (40 mg/kg) to induce seizures. In separate experiments, MgSO4 (270 mg/kg IP) was injected in advance of PTZ into PE rats to observe its effect on PTZ-induced seizures. RESULTS: PE conditions were verified in rats after LPS administration by significantly higher blood pressure (P<0.01) and urinary albumin excretion (P<0.05), elevated sFlt-1 (P<0.05) and decreased PlGF serum levels (P<0.05), and evidence of hepatic dysfunction compared to control groups. PTZ successfully induced seizure activity in all groups studied. Latency to seizure was significantly (P<0.01) less in the PE-PTZ group (73.2 ± 6.6 sec.) than in PTZ-treated controls (107.0 ± 7.4 sec.). Pretreatment with MgSO4 prolonged (P<0.05) latency to seizure, shortened seizure duration and decreased seizure rates. Significant increased (P<0.05) in the serum levels of the inflammatory cytokines TNF-α and IL-1ß in PE and PE-PTZ groups, and decreased (P<0.05) in their levels following MgSO4 administration. CONCLUSION: This PTZ-induced eclampsia-like rat model is comparable to the human condition of eclampsia and may serve as a useful research tool for future studies of this disease. The increased inflammatory cytokines in preeclampsia are coincident with a decreased threshold for PTZ-induced seizures, suggesting that an inflammatory mechanism may contribute to the susceptibility to seizure activity and inflammation might have an important role in eclampsia.

PP014. A pioneer explore of PTZ-induced eclampsia in preeclampsia rats.

INTRODUCTION: Eclampsia is the most serious state of hypertensive disorder in pregnancy, but the pathogenesis of eclampsia seizures is poorly understood. OBJECTIVES: An eclampsia animal model was developed by PTZ injection on preeclampsia rats. METHOD: Rats received endotoxin (1.0µg/kg body weight) form tail vein in an hour on GD14 to build the preeclampsia (PE) model. PTZ i.p injection were given to PE rats on GD16, 17, 18 to induce seizures and build an eclampsia model. All experiment rats were divided into three groups, PE, normal, pregnant (P), non-pregnant (NP) group (n=18 in each group). Each group gave two doses of PTZ (40 or 35mg/kg) as subgroups. Blood pressure, urinary albumin, biochemical indexes (AST, ALT, SCr, Urea) were measured. Seizure severity was defined by Racine' standard and seizure time were recorded. RESULTS: PTZ-induced eclampsia shows a increasing blood pressure, urinary albumin, liver function damage and different stages of seizures. The latency of seizure in PE rats with 40mg/kg PTZ is obviously shortened when compared with the P and NP group (p<0.05) and the duration is prolonged (p<0.05). The same trend is still existence at the low dose of PTZ injection. Rate of tonic-clonic seizures is elevated in PE and P group when compared with NP group, but nonsignificantly. CONCLUSION: We use 40mg/kg PTZ to endotoxin induced preeclampsia model to build an eclampsia model, which mimicked the multiple organ function disorder in human eclampsia. Meanwhile, we found the PE status may decreases threshold for PTZ induced-seizures.

PP022. An animal model for eclampsia.

BACKGROUND/AIMS: Eclampsia, a leading cause of maternal mortality. Little is known about the causes of eclampsia and there is no effective treatment. Development of an animal model may help to expand our understanding and may hold great potential for the design of effective treatment. METHODS: Experimental preeclampsia model was build first, rats were received a infusion of endotoxin (LPS) (1.0µg/kg body weight) or saline solution through the tail vein during 1h on gestational day 14, blood pressure and albuminuria were measured. On gestational day 18 all experimental rats were received electric stimulation, the seizure behaviors were observed and EEG were recorded in these rats. Terminations of pregnancy were performed on day 21 of gestation, resorptions and pups birth weights were recorded. RESULTS: PE model rats develop many features of human PE that correlates with bad pregnancy outcomes. Both clinical behavior and EEG records documented seizure activity developed in 100% of pregnancy rats and 58.3% of non-pregnancy rats (P=0.01). The PE model rats had a 31.25% decrease of the latency (3.3±1.4min) to evoke a full motor seizure compared with normal pregnancy rats (4.8±2.2min), and had a significant decrease contrast to non-pregnancy rats (10.6±7.1min), £¨P<0.05). EEG recordings showed seizure activities when rats had clinically generalized tonic-clonic convulsions. CONCLUSION: We described a rat model of eclampsia, were the relevant predominant features of human eclampsia.

PP023. Soluble Fms-like tyrosine kinase-1 and placental growth factor expression in a rat model of pre-eclampsia.

BACKGROUND/AIMS: Soluble Fms-like tyrosine kinase-1(sFlt-1) and placental growth factor (PIGF) have been used clinically to predict preeclampsia (PE). This study investigated these factors in a rat model of preeclampsia induced by ultra-low-dose endotoxin. METHODS: The experimental PE rat model was generated on gestational day 14. Rats were anesthetized and divided into a normal pregnancy group (NP, n=7) (which received a normal saline infusion) and a PE model group (n=9) (which received an infusion of lipopolysaccharide (LPS) endotoxin (1.0µg/kg body weight). Infusions were given through the tail vein for 1 hour. Blood pressure was monitored and albuminuria, serum ALT, AST, creatinine and BUN were measured. As well, concentrations of sFlt-1 and PIGF in serum and amniotic fluid were measured by enzyme-linked immuno sorbent assay (ELISA). RESULTS: Arterial pressure was increased (135±7 versus 116±3mmHg; P<0.03) in the PE model rats compared with the NP rats. The concentration of sFlt-1 in the PE model group (162.7±73.9pg/ml) was significantly higher than NP group (123±64pg/ml) (P<0.05). The serum level of PIGF in PE model group (9.7±6.2pg/ml) was significantly lower compared to the NP group (23.4±12.4) (P<0.05). The plasma sFlt-1/PIGF ratio in the PE model group (18.3±5.6) was greater than that in the NP group (6.7±2.1) (P<0.05). Similar changes were also present in the amniotic fluid. CONCLUSION: sFlt-1 and PIGF levels in this rat PE model induced by ultra-low-dose endontoxin showed changes consistent with findings in preeclamptic patients, indicating that this animal model mimics human preeclampsia well in these aspects of the disorder.

PP024. Effects of intravenous magnesium sulfate on the characteristics of eclamptic seizures induced by electrical stimuli in a rat preeclampsia/eclampsia model.

BACKGROUND/AIMS: Eclampsia is a serious complication of pregnancy and remains a leading cause of maternal mortality worldwide. Magnesium sulfate is commonly used in the prophylaxis and treatment of eclampsia. However, uncertainty remain regarding its anticonvulsant mechanism(s) of action. This study examined the effects of intravenous magnesium sulfate on the characteristics of eclamptic seizures in a rat preeclampsia/eclampsia model. METHODS: All rats were implanted with stainless nickel-cadmium alloy bipolar electrodes one week before fertilization. Next, an experimental rat preeclampsia (PE) model was induced on gestational day 14 by anaesthetising rats and infusing over 1 hour into their tail veins lipopolysaccharide (LPS) (1.0µg/kg body weight) (with control rats receiving normal saline). The rats were then divided into three groups: a normal pregnancy (NP) group (n=6) which received a continuous infusion of saline; a control PE model group (n=7) (which had previously received the LPS treatment) which also received a continuous infusion of saline; and a treated PE model group (n=8) (which had previously received the LPS treatment) which received a continuous infusion of magnesium sulfate (60mg/kg/day). The continuous infusions in all three groups were delivered by implanted osmotic minipumps . Measurements were made of blood pressure, albuminuria, serum ALT, AST, and creatinine, BUN and serum magnesium concentrations. On gestational day 18, all experimental rats received a standardized electrical stimulus. Seizure activity was assessed using electroencephalogram (EEG) recordings. Terminations of pregnancy were performed on gestational day 21. Resorptions and pup birth weights were recorded. RESULTS: The pregnant LPS treated rats developed many features of human PE (e.g. hypertension, proteinuria, liver and kidney dysfunctions). The mean concentration of Mg(2+) in the magnesium sulfate therapy group (0.86±0.24mmol/L) was significantly higher (p<0.05) than in both the control PE model group (0.61±0.12mmol/L) and the NP group (0.62±0.09mmol/L). The magnesium sulfate therapy group had a significantly (p<0.05) increased latency period (21.7±8.9min) to evoke a full motor seizure compared to both the NP group (4.8±2.2min)and the control PE model group (3.3±1.4min), there being no significant difference (p>0.05) between the latency periods of the NP group and the control PE model group. Overall, the magnesium sulfate therapy regimen completely prevented seizure activity in 3/8 (37.5%) of the treated PE model rats compared to 6/6 (100%) of the NP rats and 7/7 (100%) of the control PE rats. The treated PE model group also had significantly (p<0.05) reduced seizure duration (26±4s) compared to both the NP (40±7s) and the control PE model (45±9s) groups. As well, there was a significantly (p<0.05) shorter EEG seizure amplitude change in the treated PE model group (58±6µv). CONCLUSION: In this rat preeclamsia/eclampsia model, the anticonvulsant characteristics of magnesium sulfate have been shown to include significantly increasing seizure latency period, reducing seizure duration and decreasing seizure EEG amplitude.

[Expression of aquaporin-4 protein in the brain of preeclampsia model rats].

OBJECTIVE: To study the expression of aquaporin-4 (AQP4) protein in preeclampsia(PE) model rats. METHODS: Adult SD rats were divided into PE model group (n=7) induced by endotoxin, normal pregnancy group (n=6) and non-pregnancy group (n=12) treated with an equal volume of saline. AQP4 protein expression in the brain of the rats was detected with immunohistochemical staining and Western blotting. RESULTS: The blood pressure in PE model rats (135∓9 mmHg) was significantly higher than that in normal pregnancy rats (116∓8 mmHg) and non-pregnancy rats (112∓6 mmHg) (P<0.02). The rats in PE model group showed obvious proteinuria compared with the other two groups (3.8∓0.5 vs 2.6∓0.6 and 2.3∓0.4, P<0.05). Immunohistochemistry showed that AQP4 protein was localized primarily around the brain parenchymal blood vessels. Western analysis revealed a significantly elevated AQP4 protein expression in PE model group (39.6∓4.9) compared with that in normal pregnancy group (26.5∓4.3) and non-pregnancy group (9.7∓2.1) (P<0.01). CONCLUSION: The up-regulation of AQP4 protein around the intraparenchymal blood vessels of the brain may play a role in the development of eclampsia.