Pharmacological protection of CNS white matter during anoxia: actions of phenytoin, carbamazepine and diazepam

The effects of phenytoin (DPH), carbamazepine (CBZ) and diazepam (DZP) on anoxia-induced injury in CNS white matter were studied using the in vitro rat optic nerve preparation. Optic nerves were subjected to 60 min of anoxia and functional recovery was assessed using the area under the compound action potential. Under normoxic conditions, application of DPH, CBZ and DZP reduced compound action potential area over concentration ranges known to block sodium channels. All three compounds, however, protected against anoxic injury at concentrations below those that inhibited the normoxic compound action potential. Thus, the application of 1 microM DPH, CBZ or DZP during anoxia resulted in compound action potential recovery to 60.0, 53.8 and 69.2% of control, respectively, compared to compound action potential recovery of 34.8% in the absence of drugs (P < .05 in all three cases). In the cases of CBZ and DPH, 60% improvement in recovery from anoxia was produced by concentrations well below those employed clinically to treat epilepsy, suggesting a potential role for these drugs in the protection of CNS white matter from anoxic injury.     

Measurement of intracompartmental pressure: a comparison of the slit catheter, side-ported needle, and simple needle

An experimental model of acute compartment syndrome involving the anterolateral compartment of the hindlimb in dogs was used to compare three methods of measurement of intracompartmental pressure: the simple-needle technique, use of the slit catheter, and use of the side-ported needle. No statistical difference was found between the values obtained with the slit catheter and those obtained with the side-ported needle; the mean difference was 1.4 millimeters of mercury throughout the range of compartment pressures that were measured. The side-ported needle appeared to be as accurate as the slit catheter for the measurement of compartment pressures (p = 0.355, 1-beta = 0.9). The values obtained with use of the simple needle were consistently higher than those obtained with the other two methods (p < 0.001): an average of 18.3 millimeters of mercury higher than the values measured with the slit catheter and 19.3 millimeters of mercury higher than those measured with the side-ported needle. Clinically, the side-ported needle or the slit catheter can be used to obtain accurate measurements of compartment pressure. Use of the simple 18-gauge needle is not recommended for this purpose.     

The influence of temperature on the multiple separation of estrogenic steroids using mobile phases modified with beta-cyclodextrin in high-performance liquid chromatography

The effect of temperature on the retention and multiple separation of six estrogenic steroids in reversed-phase liquid chromatography has been studied. Capacity factors (k') of estriol, 17 beta-estradiol, 17 alpha-estradiol, d-equilenin, equilin and estrone were measured using mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5 to 80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in each case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs,min) and relative resolution product (r). The results presented describe precisely the role of temperature in high-performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used. Moreover, the elution order of estrogenic steroids on modified and unmodified mobile phases has been discussed.     

Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.     

Postnatal survival and growth of mouse irradiated in utero with low dose

In order to investigate radiation risks associated with low dose and low dose-rates, pregnant Swiss albino mice were exposed to gamma rays, 0.80 Gy from a cobalt-60 source at two different dose-rates (0.0795 and 0.0012 Gy/min) on 18 day post conception. In females exposed to lower dose-rate (0.0012 Gy/min), litter size was found to be decreased, while those exposed to higher dose-rate (0.0795 Gy/min), it remained unaltered. In both groups, appearance of fur and development of complete fur were delayed, whereas gait was delayed only in higher dose-rate group. Male offspring exhibited a biphasic mode of weight loss, while female offspring after an initial weight loss at 1 week, displayed a continuous recovery, but could not attain the normal weight till 12 weeks of age. It appears that higher dose-rate is more effective in delaying the appearance of physiological markers and weight loss, while in terms of litter size lower dose-rate (0.0012 Gy/min) is more effective.     

Measuring the viscoelastic properties of human platelets with the atomic force microscope

We have measured force curves as a function of the lateral position on top of human platelets with the atomic force microscope. These force curves show the indentation of the cell as the tip loads the sample. By analyzing these force curves we were able to determine the elastic modulus of the platelet with a lateral resolution of approximately 100 nm. The elastic moduli were in a range of 1-50 kPa measured in the frequency range of 1-50 Hz. Loading forces could be controlled with a resolution of 80 pN and indentations of the platelet could be determined with a resolution of 20 nm.