Background: Endogenous steroids are known to be essential for normal brain development. Pre-and postnatal glucocorticoids however, are associated with increased risk for adverse neurological outcome through mechanisms that are not clear, but may lead to alterations of cell structure and function. Objective: The present study tests the hypothesis that prenatal dexamethasone given at critical points in gestation will result in increased neuronal nuclear Ca++-influx, leading to activation of caspase -3 and 9 which triggers the apoptotic pathway and that this mechanism is dependent on gestational age. Design/Methods: Two groups of fetal guinea pigs were studied, at 35 (n=8) and 45 (n=8) days gestation. Saline (Sa), 0.5ml/dose, or dexamethasone (Dx), 0.4mg/kg/dose was injected i.p. into the mother daily x 2 days. Fetal guinea pig brains were harvested at 72 hours post Sa or Dx injection. Nuclei were isolated and ATP-dependent Ca++-influx was determined. Cytosolic caspase-3, and -9 activity was determined. Caspase activity was determined spectrofluorometrically and expressed as nmoles/mg protein/hr. Results: Ca++-influx at 35d was 6.42+/-2.0 (Sa) and 7.42+/-3.4 (Dx), an increase of 16%; at 45d was 5.63+/-2.3 (Sa) and 10.5+/-2.1 (Dx), an increase of 80%. Caspase-3 activity at 35d was 13.3 +/-1.3 (Sa) and 13.9+/-1.9 (Dx); at 45d was 12.8+/-0.8 (Sa) and 9.53+/-1.01 (Dx), a decrease of 25%. Caspase-9 activity at 35d was 3.52+/-0.3 (Sa) and 3.39+/-0.2 (Dx); at 45d was 3.46+/-0.2 (Sa) and 2.49+/-0.2 (Dx), a decrease of 28%. The data show that antenatal Dexamethasone resulted in increased Ca++-influx in neuronal nuclei of fetal guinea pigs without altering caspase-3 or -9 activity. Conclusions: We conclude that Dexamethasone activates neuronal nuclear membrane mechanisms that initiate the apoptotic cascade and increase with gestational age. We speculate that Dexamethasone modifies nuclear membrane mechanisms of Ca++ influx and leads to cellular injury through an apoptotic pathway independent of caspase expression.

We have developed an optical Imaging system, Dual Wavelength Phased Array system (DWLPA) that can be used for testing neonate’s brain function. A phased array system consists of 16 sets of phase cancellation optodes, 0 and 180 degree laser sources with a detector in middle. For this purpose, 9 sets of lasers each with 780 and 830 nms (20 mwatt) and 4 detectors, (PMT-T08 Hamamatsu) are placed on a probe, 4×9 cm. These optodes make 16 combinations of triangle; each has a function of phase cancellation. Thus all the probe areas were filled with 16 triangles and can be seen as sensitive to heterogeneity. The optodes separation is 2.5 cm for each array, and data information is taken by a detector in the middle when each two sources at 1 and 180 degree ignite at once. The actual images are made by subtracting from resting state to active state of cortex when physicians test for neurological examinations, hearing, touching, and visual display. The subtraction can cancel outcome of artifacts caused by inconsistent hair, probe pressure to the head, and slightly dislocated probe angles. The results show that like adults, we saw functions of cortices. The significant is that these blood volume changes by the stimulation can be detected much earlier stage of life than the development of EEG (electrical encephalogram) and Evoked potential. Thus the usefulness of this optical technology can be very beneficial. It implies that neonates already develop circulatory response from the metabolic needs of neurons in the area. We conclude that this technology, namely PA imaging system has a superior S/N ratio by the fact flat it has 180 degree shift for 2.5 cm resolution. These methods can be used clinically to detect infant’s neurological dysfunction before EEG and other test can be used.

Previous studies from our laboratory have shown that prenatal cocaine exposure alters brain cell membrane function by decreasing membrane Na+,K+-ATPase activity and potentiates the glutamate/glycine-dependent activation of the NMDA receptor. Magnesium sulfate(MgSO4) attenuates neuronal membrane dysfunction in hypoxic newborn piglets by preserving N-methyl-D-aspartate (NMDA) receptor number and affinity, and Na+,K+-ATPase activity. Data describing the interaction of cocaine and magnesium on neuronal function are lacking. The present study tests the hypothesis that maternal MgSO4 administration will prevent cocaine-induced NMDA receptor/ion channel modification in the fetus. Studies were performed in 6 pregnant guinea pigs (58 days gestation) assigned to 3 groups: Cocaine-treated (COC, n= 2). Magnesium-treated (MG, n=2) and Magnesium/Cocaine-treated (MG-COC, n=2). Animals in the MG and MG-COC groups received an initial bolus of MgSO4 300 mg/kg i.p. followed by 50 mg/kg q 30 min for 5 hours. Mean maternal [Mg++] was 10.5 mg%. Cocaine HCl 30 mg i.p. was given to the COC and MG-COC groups one hour prior to delivery of the fetuses. 3[H]MK-801 binding studies, an index of NMDA receptor activation, were performed in a concentration range from 0.5 to 50 nM in a reaction medium containing 10mM HEPES (pH 7.0), 100 μM glutamate and glycine, and 75 μg protein. Total number of receptors (Bmax) in the COC, MG and MG-COC groups were 1.08 ± 0.13, 1.34 ± 0.07 and 1.19± 0.09 pmol/mg protein (mean±SD). Kd for the same groups were 2.64 ± 0.22, 5.18 ± 0.87 and 4.65 ± 0.01 nM. The data show that MgSO4 may prevent the cocaine-induced increase in the affinity (decreased Kd) of the NMDA receptor for 3[H]MK-801 in fetal brains. We speculate that cocaine-induced modification of the NMDA receptor ion/channel complex may alter NMDA-mediated developmental processes that are essential for fetal brain function. Furthermore, administration of MgSO4 to cocaine-exposed fetuses may attenuate neurotoxicity by the glutamate-type NMDA receptor in the developing fetal brain. (Funded by NIH-HD-20337, MOD#6- FY94-0135, UCPR 506-93)

Newborns delivered to cocaine-abusing mothers are often exposed to other concurrently consumed illicit drugs, which may alter the hemodynamic and cerebral response to cocaine. This study examined the interaction of ethanol, morphine or barbiturate with cocaine on mean arterial pressure (MAP), cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) in newborn pigs. CBF, CMRO2 and cerebral O2 extraction (CEO2) were measured before and 4 and 10 min after cocaine (1.5 mg/kg i.v.) was administered in piglets that were awake, or pretreated with morphine, ethanol or pentobarbital. In awake piglets, cocaine increased CMRO2 and CEO2 while it had no significant effect on CBF. Conversely, in morphine- and ethanol-pretreated piglets, cocaine decreased CMRO2, decreased CBF and had not effect on CEO2. In awake piglets, cocaine increased MAP, whereas in morphine- or ethanol-pretreated piglets, cocaine decreased MAP. In the pentobarbital group, cocaine had no effect. These data demonstrate that other drugs of abuse alter the hemodynamic and cerebral effects of cocaine in the immature animal and may contribute to the central nervous system abnormalities in 'crack babies'.

The effect of cocaine on brain cell membrane structure and function was studied in the fetal guinea pig. We tested the hypothesis that cocaine, a potent vasoconstrictor, would result in brain cell membrane dysfunction as determined by altered activity of Na+,K+-ATPase and the appearance of products of membrane lipid peroxidation (conjugated dienes (CD) and fluorescent compounds (FC)). A total of 14 pregnant guinea pigs were studied at term (60 days). One hour prior to delivery, the pregnant guinea pigs were divided into 3 groups as follows: cocaine, 30 mg/kg i.p., saline placebo i.p., or 7% FiO2 for 1 h. Following cocaine, brain Na+,K+-ATPase activity decreased (mean±S.D., 25.6 ± 9.2vs.54.6 ± 3.4 μmolPi/mg protein/h, cocaine vs. control, respectively, (P < 0.01) and was similar to the hypoxia group (21.9 ± 2.8 μmolPi/mg protein/h). The products of lipid peroxidation did not change significantly following cocaine hypoxia resulted in a rise in CD from 0 to 0.175 ± 0.015 μmol/g brain, control vs. hypoxia, (P < 0.01), and FC from 1.13 ± 0.15to1.88 ± 0.13 μg quinine sulf brain, control vs. hypoxia, (P < 0.01). These data show that acute fetal cocaine exposure, unlike hypoxia alone, results in a significant decrease in Na+,K+-ATPase activity without a significant increase in the products of lipid peroxidation, suggesting the mechanism by which cocaine affects brain cell membrane integrity is distinct from hypoxia. Inhibition of the enzyme activity may be due to a direct action of cocaine on the enzyme or due to enzyme regulation by cocaine-induced alterations in neurotransmitters.

The present study investigated the effect of cocaine (COC) on cerebral circulation (CBF) and oxidative metabolism (CMRO2) in the newborn piglet and aimed to relate pharmacokinetics of cocaine to cerebrovascular effects. COC decreased CBF and CMRO2 from 75 to 64 and 4.27 to 3.91 ml/min/100 g, respectively, at 4 min with reduced flow to all brain regions (p < 0.05) which returned to baseline by 10 min. COC was rapidly metabolized with a t1/2 of 43 min and peak plasma concentration of 1,172 ng/ml. Norcocaine (NOR) appeared in plasma and CSF within 3 min of cocaine administration and remained elevated for the duration of the study along with COC in the CSF. These data show that the timing of the peak plasma COC level is associated with maximal decreased CBF. Further, the stable elevated level of COC and NOR in the CSF suggests that biotransformation does not occur in the brain. As a result, accumulation of these drugs may occur in the brain with successive COC use and affect the developing CNS in a deleterious manner.

Cocaine abuse by pregnant women is often associated with neurological injury in the newborn. To explore a vascular-related mechanism of injury, we investigated the effect of cocaine on the cerebral circulation in newborn pigs. During normoxic conditions, cocaine administration (1.5 mg/kg i.v.), resulting in peak plasma cocaine levels on the order of 10-6M, decreased cerebral blood flow (CBF) by 14%, as measured by the tracer microsphere method. To elicit the mechanisms by which cocaine decreased CBF, closed cranial windows were placed and the diameter of pial arterioles was measured by intravital microscopy while cocaine (10 -6M) was applied onto the cortical surface. Topically applied cocaine decreased pial arteriolar diameter by 9%. Vasoconstriction induced by topically applied cocaine was blocked by tetrodotoxin (10-7M, Na+ channel blocker), whereas phentolamine (10-5M, nor-adrenergic receptor blocker) had no effect on the arteriolar response to cocaine, which suggested that cocaine effected constriction by an anesthetic and not a sympathomimetic mechanism. To evaluate this hypothesis further, cerebral vessels in the right hemibrain were sympathetically denervated while those in the left hemibrain remained innervated. During normoxia, cocaine (1.5 mg/kg i.v.) decreased CBF equally in both hemibrains, confirming the non-sympathomimetic mechanism. During asphyxia, cocaine administration attenuated cerebral hyperemia in both hemibrains, but in innervated more than in denervated, indicating that anesthetic and sympathomimetic vasoconstriction occurred during asphyxia. We conclude that cocaine constricts the immature cerebrovasculature and decreases CBF by an anesthetic mechanism during normoxic conditions and by both sympathomimetic and anesthetic mechanisms during asphyxia.