Neuronal free Ca(2+)and BBB permeability and ultrastructure in head injury with secondary insult.

OBJECTIVE: To study changes in free calcium (Ca(2+)), neuronal and blood-brain barrier (BBB) permeability and ultrastructure in brain after diffuse axonal injury (DAI) with secondary brain insults (SBIs). METHOD: One hundred and twenty Sprague-Dawley (SD) rats were randomised into control, DAI alone and DAI with SBI groups which were sub-divided into 5 groups that were 0.5 h, 2 h, 12 h, 24 h, 48 h post trauma. The animal models of DAI and DAI with SBI have been described before (2). Fluorescence probe Fluo-3/Am was used to measure free Ca(2+)in neurons. Laser scan microscopy was used to detect fluorescence intensity. After the animals were anesthetized, Lanthanum nitrate liquid was used for intracardiac perfusion to assess BBB permeability. Under the transmission electron microscope, changes in cerebral ultrastructure and BBB permeability were observed. RESULTS: The fluorescence intensity was weak in the control. The concentration of free Ca(2+)in neurons was obviously increased at 30 min after brain injury, reached a peak at 12 h-24 h (P< 0.01), and appeared to decrease at 48 h after injury. In the DAI alone group, BBB tight junction opening with particles of Lanthanum nitrate outside the vessels was found at 30 min after injury, and peaked at 24 h. In DAI with SBI, the changes in ultrastructure and BBB permeability were more severe than that in the DAI alone group at the same time interval. The shape of the fluorescence concentration curve was basically the same for both kinds of brain injury. The intensity of fluorescence in DAI with SBI was higher than that in the DAI alone group at the same time interval (P< 0.05). CONCLUSION: In DAI alone and DAI with SBI, Ca(2+)overload and BBB permeability changes interact and both play important roles in the aggravation of brain damage.

The expression and changes of heat shock protein 70, MDA and haemorheology in rat cortex after diffuse axonal injury with secondary insults.

In the present study the role of heat shock protein 70 (HSP70) expression, changes of malonyldialdehyde (MDA) in rat cortex and haemorheology with time after diffuse axonal injury (DAI) only and DAI with secondary insults (SI) were studied. The rat DAI and DAI with SI model were made according to our previous work and animals were divided into a control and another five injury groups with time after injury. Immunohistochemical assay was used to detect the neuronal expression of HSP70 at 0.5h, 3h, 12h, 24h, 72h after DAI or DAI with SI. In the meantime, the high (etah ) and low whole blood viscosity (etaL ), haematocrit (HCT) and RBC aggregation index (AI = etaL/etah ) were also detected and calculated. MDA in the homogenised brain tissue was assayed by thiobarbituric acid (TBA) reaction. The results showed that HSP70 positive neurons were not detected at 30 minutes, but the number of HSP70 positive neurons begin to increase obviously at 3 hours, reach a peak at 24 hours (P< 0.01), and decrease at 72 hours (P= 0.05) after brain injury. The trend of expression of HSP70 was alike for both DAI only or DAI with SI. Meanwhile, MDA, etah, etaL, HCT and AI changes showed the same tendency. Compared with DAI only group, MDA and blood viscosity indexes in DAI with SI were significantly higher at respective time points (P< 0.01). It is concluded that HSP70 expression, MDA and haemorheology indices increased after brain injury and brain injury with SI. Free radicals and haemorheological changes play an important role in the aggravation of brain damage and HSP70 expression upregulation.

Prevention of mammary tumorigenesis in acatalasemic mice by vitamin E supplementation.

Adult male and female acatalasemic (C3H/AnLCsbCsb),hypocatalasemic (C3H/AnLCscCsc) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E-deficient (28 animals) and vitamin E-supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E-supplemented diet was 47%, while that in mice given vitamin E-deficient diet was 82% (P < 0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.

Enhanced liver injury in acatalasemic mice following exposure to carbon tetrachloride.

The hypothetical involvement of hydrogen peroxide (H2O2) in carbon tetrachloride (CCl4)-induced acute liver injury and the potential preventive effect of catalase on hepatotoxicity have been studied in acatalasemic (C3H/AnLCsbC2b) mice and compared with normal (C3H/AnLCsaCsa) mice. A single intraperitoneal injection of CCl4 (20% in olive oil/g body weight) caused increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in both mouse groups, but the extents of increases did not show significant differences between the two mouse groups until 12 h. The variation in increases of serum AST and ALT levels in acatalasemic and normal mice turned to be distinctly different from 12 h. At 18 h (peak point for ALT) and 24 h (peak point for AST), the serum enzyme levels in acatalasemic mice were nearly two-fold higher than those in normal ones, the difference being statistically significant (p < 0.01). The liver malondialdehyde (MDA) level in acatalasemic mice was also higher than that in normals at 18 h (p < 0.05). The extent of the centrilobular necrosis was histologically more severe in acatalasemic mice. The catalase activity in livers of acatalasemic mice was one-third to one-fifth those of normal mice (p < 0.05) before and after treatment. The decreased catalase activity in acatalasemic mice might increase tissue or cellular levels of H2O2 during the later phase of the acute liver injury. From these findings, we conclude that H2O2 breakdown in liver would account for the difference in the later stages of the acute liver damage between the two groups of mice, and catalase is important in inhibiting hepatotoxicity of CCl4 in the later stage.

Inhibitory effect of alpha-tocopherol on methemoglobin formation by nitric oxide in normal and acatalasemic mouse hemolysates.

The inhibitory effect of alpha-tocopherol on methemoglobin formation in normal and acatalasemic mice was studied by exposing their hemolysates to nitric oxide. Methemoglobin formation in normal and acatalasemic mouse hemolysates exposed to nitric oxide were significantly inhibited by the addition of alpha-tocopherol at final concentrations ranging from 1.2 to 5.8 mM. Negative correlations were observed between the logarithm of alpha-tocopherol concentration and the methemoglobin formation. The formation of methemoglobin in acatalasemic mouse hemolysates was greater than that in normal mouse hemolysates with or without added alpha-tocopherol. The methemoglobin formation in acatalasemic mice was also significantly inhibited by addition of more than 500 units/ml of catalase, and the methemoglobin formation in normal and acatalasemic mice was also inhibited with sodium diethyldithiocarbamate at a final concentration of 1 M.