Effects of L-3-n-butylphthalide on caspase-3 and nuclear factor kappa-B expression in primary basal forebrain and hippocampal cultures after beta-amyloid peptide 1-42 treatment

BACKGROUND： L-3-n-butylphthalide （L-NBP） can inhibit phosphorylation of tau protein and reduce the neurotoxicity of beta-amyloid peptide 1-42 （Aβ1-42）. OBJECTIVE： To observe the neuroprotective effects of L-NBP on caspase-3 and nuclear factor kappa-B （NF- K B） expression in a rat model of Alzheimer＇s disease. DESIGN, TIME AND SETTING： A cell experiment was performed at the Central Laboratory of Provincial Hospital affiliated to Shandong University between January 2008 and August 2008. MATERIALS： L-NBP （purity 〉 98%） was provided by Shijiazhuang Pharma Group NBP Pharmaceutical Company Limited. Aβ1-42, 3-[4,5-dimethylthiazolo-2]-2,5 iphenyltetrazolium bromide （MTT）, and rabbit anti-Caspase-3 polyclonal antibody were provided by Cell Signaling, USA; goat anti-choactase and rabbit anti-NF- kB antibodies were provided by Santa Cruz, USA. METHODS： Primary cultures were generated from rat basal forebrain and hippocampal neurons at 17 or 19 days of gestation. The cells were assigned into five groups： the control group, the Aβ1-42 group （2 μmol/L）, the Aβ1-42 ＋ 0.1 μmol/L L-NBP group, the Aβ1-42 ＋ 1 μ mol/L L-NBP group, and the Aβ1-42 ＋ 10μmol/L L-NBP group. The neurons were treated with Aβ1-42 （2 μmol/L） alone or in combination with L-NBP （0.1, 1, 10 μmol/L） for 48 hours. Cells in the control group were incubated in PBS. MAIN OUTCOME MEASURES： Morphologic changes were evaluated using inverted microscopy, viability using the M-I-I- method, and the changes in caspase-3 and NF- k B expression using Western blot. RESULTS： Induction with Aβ1-42 for 48 hours caused cell death and soma atrophy, and increased caspase-3 and NF- K B expression （P 〈 0.05）. L-NBP blocked these changes in cell morphology, decreased caspase-3 and NF- k B expression （P 〈 0.05）, and improved cell viability, especially at the high dose （P 〈 0.05）. CONCLUSION： AI3^-42 is toxic to basal forebrain and hippocampal primary neurons; L-NBP protects against this toxicity and inhibits the induction of caspase-3 and NF- K B expression.

Mild hypothermia effects on matrix metalloproteinase-9 expression in the perihematomal region of rats following experimental intracerebral hemorrhage

BACKGROUND： Matrix metalloproteinase-9 （MMP-9） expression increases with intracerebral hemorrhage, and participates in the pathophysiological processes of secondary brain injury after intracerebral hemorrhage. OBJECTIVE： To investigate the effects of mild hypothermia on MMP-9 expression and brain edema in the perihematomal region of experimental intracerebral hemorrhage rats. DESIGN, TIME AND SETTING： The randomized, controlled experiment was performed at the Central Laboratory of Shandong Provincial Hospital between May and September 2007. MATERIALS： Seventy-two, Wistar, male rats, 12-weeks old, were used for this study. Rabbit anti-MMP-9 primary antibody was purchased from Boster, China. METHODS： Wistar rats were equally and randomly divided into normothermia and mild hypothermia groups. The two groups each comprised control, 6-hour intracerebral hemorrhage, 24-hour intracerebral hemorrhage, 48-hour intracerebral hemorrhage, 72-hour intracerebral hemorrhage, and l-week intracerebral hemorrhage subgroups, with six rats in each subgroup. Rat models of intracerebral hemorrhage were established by injecting 100 μL of autologous blood into the rat caudate nucleus. Rats in the mild hypothermia group received four hours of local mild hypothermia immediately following the injection. lntracerebral temperature was maintained at （33 ± 0.5） ℃. Subsequently, intracerebral temperature was spontaneously recovered at 25 ℃. Rats in the control subgroup were not injected with autologous blood and received only with intracerebral hemorrhage. MAIN OUTCOME MEASURES： Brain water content and MMP-9 expression surrounding the hematoma region. RESULTS： MMP-9 expression increased at 6 hours, and brain edema reached a peak at 48 hours after intracerebral hemorrhage. MMP-9 expression was significantly decreased in the mild hypothermia group compared with the normothermia group at each time point （P 〈 0.05）. CONCLUSION： Mild hypothermia can significantly inhibit MMP-9 overexpression and relieve brain edema following intracerebral hemorrhage.

Diazoxide preconditioning antagonizes cytotoxicity induced by epileptic seizures

Diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, can protect neurons and astrocytes against oxidative stress and apoptosis. In this study, we established a cellular mode of epilepsy by culturing hippocampal neurons in magnesium-free medium, and used this to investigate effects of diazoxide preconditioning on the expression of inwardly rectifying potassium channel （Kir） subunits of the ATP-sensitive potassium. We found that neuronal viability was significantly reduced in the epileptic cells, whereas it was enhanced by diazoxide preconditioning. Double immunofluorescence and western blot showed a significant increase in the expression of Kir6.1 and Kir6.2 in epileptic cells, especially at 72 hours after seizures. Diazoxide pretreatment completely reversed this effect at 24 hours after seizures. In addition, Kir6.1 expression was significantly upregulated compared with Kir6.2 in hippocampal neurons after seizures. These findings indicate that diazoxide pretreatment may counteract epileptiform discharge-induced cytotoxicity by suppressing the expression of Kir subunits.

Effects of hypoxic preconditioning on the changes of expression of neuroglobin mRNA and labeled positive cells following cerebral ischemia in gerbils

BACKGROUND： Neuroglobin （NGB）, as newly discovered the third member of the globin family binding oxygen, mainly exists in brain of human beings and vertebrates, and it is closely correlated with the oxygen supply in brain. OBJECTIVE： To observe the changes of the expression of neuroglobin and number of positive cells labeled immunohistochemically following cerebral ischemia in gerbils after hypoxic preconditioning. DESIGN ： A complete randomized grouping design and controlled experiment.SETTING ： Department of Neurology, Department of Anesthesia, Shandong Provincial Hospital, Shandong University. MATERIALS ： Sixty-six adult male Mongolian gerbils of clean degree, about 50-65 g, at an average of 57.5 g were provided by the Experimental Animal Center of Capital Medical University [certificate number of animal quality：SCXK（Beijing）2000-0012]. TRNzil （Tianwei Shidai Company, Beijing）, polymerase chain reaction （PCR） primers （synthetized by Invitrogen Company, Shanghai）; reverse transcription-PCR （RT-PCR） one-step kit （Toyobo Company）; PCR instrument （GeneAmp PCR System 240）; mice brain NGB monoclonal antibody （Academy of Military Medical Sciences）; DAB （Zhongshan Company, Beijing）. METHEDS ： The study was completed from December 2004 to June 2005 in Shandong Provincial Hospital. ① The 66 gerbils were randomly divided into sham-operated group （n =6）, cerebral ischemia group （n =30） and hypoxic preconditioning group （n =30）. The gerbils in the hypoxic preconditioning group were put in the environment which contained 02 （0.08 in volume fraction） and N2 （0.92 in volume fraction） at temperature of 25 ℃ for 2 hours. After 5 hours, the gerbils in the hypoxic preconditioning group and cerebral ischemia group were anesthetized, then bilateral common carotid arteries were ligated. In the sham-operated group, bilateral common carotid arteries were only isolated without ligation and hypoxic preconditioning. ② At 1, 5, 10, 30 and 60 minutes after cerebral ischemia, the expression of NGB mRNA was detected with RT-PCR, and the number of NGF positive cells was counted with immunohistochemical staining. The NGB mRNA expression and number of NGB positive cells at different time points after ischemia were compared between the gerbils treated with and without hypoxic preconditioning. MAIN OUTCOME MEASURES ： NGB mRNA expression and number of NGB positive cells at 1, 5, 10, 30 and 60 minutes after cerebral ischemia. RESULTS ： All the 66 Mongolian gerbils were involved in the analysis of results. ① Results of NGB mRNA： In the cerebral ischemia group, the NGB mRNA expression began to increase at 1 minute after cerebral ischemia, but had no obvious difference as compared with that in the sham-operated group （P 〉 0.05）, that at 5 minutes was obviously higher than that in the sham-operated group （0.951±0.034, 0.597±0.008, P 〈 0.05）, it decreased gradually 10, 30 and 60 minutes after cerebral ischemia, but had no obvious difference as compared with that in the sham-operated group （P〉 0.05）. In the hypoxic preconditioning group, the NGB mRNA expression increased rapidly 1 minute after cerebral ischemia, which was obviously higher than that in the cerebral ischemia group （0.641±0.010, 0.618±0.015, P 〈 0.05）, and the expressions at 5, 10 and 30 minutes were still obviously higher than those （n the cerebral ischemia group （0.995±0.020 vs, 0.951±0.034; 0.941±0.010 vs. 0.615±0.018; 0.642±0.010 vs. 0.608±0.010, P〈 0.05-0.01 ）, whereas the expression at 60 minutes were not obviously different from that in the cerebral ischemia group （P〉 0.05）. ② Number of NGB positive cells： The numbers of NGB positive cells at 1, 10 and 30 minutes after cerebral ishcemia in the hypoxic preconditioning group [（50.2±3.3）, （67.2±3.3）, （35.0±4.3） cells] were obviously more than those in the cerebral ischemia group [（33.0±2.1 ）, （60.5±1.9）, （23.3±3.1） cells, P 〈 0.05-0.01], whereas those at 5 and 60 minutes had no obvious differences between the two groups （P〉 0.05）. CONCLUSION ： Hypoxic preconditioning can rapidly accelerate the expression of NGB mRNA following cerebral ischemia, and it plays its neuroprotective role in hypoxic preconditioning.