Christopher D. Cain

Behavioral sequelae in young rats of acute intermittent antenatal hypoxia

Several studies have examined behavioral sequelae of acute or chronic pre- or postnatal hypoxia. However, few of these tested a large battery of behavioral functions, particularly those following relatively mild, intermittent hypoxia. Also, in few studies were the hypoxic pups cross-fostered or the experimenter blinded as to experimental group. In addition, in almost no studies were concomitant hypoxic-induced brain biochemicals measured. The present study tested the hypotheses that mild, intermittent antenatal hypoxia can lead to long-term alterations in neurobehavioral development, as well as neurochemical changes.

Effect of immobilization and concurrent exposure to a pulse-modulated microwave field on core body temperature, plasma ACTH and corticosteroid, and brain ornithine decarboxylase, Fos and Jun mRNA.

Abstract Stagg, R. B., Hawel, L. H., III, Pastorian, K., Cain, C., Adey, W. R. and Byus, C. V. Effect of Immobilization and Concurrent Exposure to a Pulse-Modulated Microwave Field on Core Body Temperature, Plasma ACTH and Corticosteroid, and Brain Ornithine Decarboxylase, Fos and Jun mRNA. Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (±0.3°C) during the initial 30 min of loose- tube restraint in conditioned animals. When conditioned/tube- trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2–3 min, ACTH increased to 83.2 ± 31.0 pg/dl, compared to 28.1 ± 7.7 pg/dl for cage controls, reaching a maximum at 15–30 min (254.6 ± 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 ± 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3–10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 ± 54.8 compared to 31.2 ± 10.2 pg/dl; corticosterone, 12.2 ± 6.2 μg/dl compared to 37.1 ± 6.4 μg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10–20 fold higher (541.8 compared to 27.2–59.1 pg/dl for ACTH) and 2–5 fold higher (45.7 compared to 8.4– 20.0 μg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF-field exposure when the stress-mediated effects were complete further support our conclusion.

Focus formation of C3H/10T1/2 cells and exposure to a 836.55 MHz modulated radiofrequency field

Disruption of communication between transformed cells and normal cells is involved in tumor promotion. We have tested the hypothesis that exposures to radiofrequency (RF) fields using a form of digital modulation (TDMA) and a chemical tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), are copromoters that enhance focus formation of transformed cells in coculture with parental C3H/10T1/2 murine fibroblasts. RF field exposures did not influence TPAs dose-dependent promotion of focus formation in coculture. Cell cultures were exposed to an 836.55 MHz TDMA-modulated field in TEM transmission line chambers, with incident energies that simulated field intensities at a users head. Specific absorption rates (SARs) of 0.15, 1.5, and 15 muW/g were used during each digital packet, and the packet frequency was 50/s. The TEM chambers were placed in a commercial incubator at 37 degrees C and 95% humidity/5% CO2. The RF field exposures were in a repeating cycle, 20 min on, 20 min off, 24 h/day for 28 days. At 1.5 muW/g, TPA-induced focus formation (at 10, 30, and 50 ng/ml) was not significantly different in RF-exposed cultures compared to parallel sham-exposed cultures in ten independent experiments in terms of the number, density, and area of foci. Similarly, at 0.15 and 15.0 muW/g, in two and four experiments, respectively, RF exposure did not alter TPA-induced focus formation. The findings support a conclusion that repeated exposures to this RF field do not influence tumor promotion in vitro, based on the RF fields inability to enhance TPA-induced focus formation.

Ornithine decarboxylase activity in fetal and newborn rat brain : responses to hypoxic and carbon monoxide hypoxia

In response to acute maternal hypoxia, ornithine decarboxylase (ODC) activity increased significantly in fetal rat brain, peaking at 4 h. This was associated with increased ODC mRNA and elevated polyamine concentrations. To correlate this response with development, we measured ODC activity in the rat from gestational day E 17 to postnatal day P 10. We also examined to what extent hypoxia induces increased ODC activity in adult rat brains and whether the response to chronic hypoxia differed from that to acute hypoxia. To test the hypothesis that this increased activity is due to hypoxic hypoxia per se, we subjected pregnant dams to inspired carbon monoxide concentrations ranging from 150 to 1000 ppm and assayed ODC activity in the fetal brain 4 h later. In the fetus, ODC activity was elevated on E 17 in the cerebrum and cerebellum. It declined gradually to about one-tenth E 17 levels by E 21 and remained low thereafter except for a postnatal elevation in the cerebellum on P 3. In response to 10.5% O2, in the 3-day-old rat, ODC activity peaked between 2 and 3 h of hypoxia, increasing 3-fold in the hippocampus and 2-fold in cerebellum. Similar increases were seen in the hypoxic adult rat brain. In inspired oxygen dose-response studies, exposure of P 3 rat pups to 13.25% O2 for 2.5 h produced a 1.5-fold increase in ODC activity; 10.5% O2 produced a 2-3-fold increase while in response to 9% O2, ODC activity remained at baseline levels. With maternal CO-hypoxia, ODC activity increased in the fetal brain at 4 h, as seen with hypoxic-hypoxia. For example, in hippocampus, ODC activity doubled at 500 ppm and tripled at 600 ppm. We conclude: (1) apparently, the ability to respond thus is not lost as the animal ages and may represent an important cellular response to acute hypoxia; (2) the increase in hypoxic-induced ODC activity is relative to the already elevated activity seen from E 17 to E 20; a vast reserve for the induction of fetal ODC activity probably exists and may indicate the importance of this enzyme during this time frame for differentiation and growth promotion; and (3) the CO-hypoxia studies suggest that some aspects of the cellular responses to CO- and hypoxic-hypoxia are similar.

Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices

In fetal as well as newborn rats, acute hypoxic exposure results in significantly elevated brain ornithine decarboxylase (ODC) activity, polyamine concentrations, and ODC mRNA. The interpretations of these in vivo hypoxic-induced changes, however, are complicated by maternal confounding effects. To test the hypothesis that acute hypoxia will also increase ODC activity in vitro, we developed a brain slice preparation which eliminates such maternal effects. Sections of whole cerebrum, approximately 300-500 microns thick, were made from 3- to 4-day old Sprague-Dawley rat pups. The slices were equilibrated for 1 h in artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O2/5% CO2, prior to induction of hypoxia. We induced hypoxia by changing the oxygen concentration to 40%, 30%, 21%, 15%, 10%, or 0% O2, all with 5% CO2 and balance N2. In the normoxic control brain slices, low but stable basal ODC activity persisted for up to 5 h post-sacrifice. Slices in ACSF treated with bovine serum albumin (BSA), or both BSA and fetal bovine serum (FBS), however, showed stable ODC activity values 2- to 3-fold higher than slices in ACSF alone, for up to 5 h. In response to acute hypoxia (i.e., 15, 21, and 30% O2), ODC activity was elevated 1.5- to 2-fold above control values between 1 and 2 h after initiation of hypoxia. Qualitative light and electron microscopic examination of the neonatal brain slices following 2 h hypoxic exposure suggested that the great majority of cells did not show severe hypoxic damage or necrosis. It was concluded that: (1) in neonatal rat brain slices in vitro, stable ODC activity values approximating the whole brain ODC activity seen at sacrifice, can be maintained for several hours; (2) the in vivo hypoxic-induced increase in ODC activity can be approximated in vitro; (3) the neonatal rat brain slice preparation may be an alternative to other methods for studying hypoxic-induced ODC enzyme kinetics, or other brain enzymes, without maternal confounding effects; and (4) ODC activity may be an indicator of active metabolism within the newborn brain slice both in normoxia and hypoxia.

Ornithine decarboxylase activity and polyamine concentrations in fetal rat brain: response to chronic hypoxic-hypoxia and/or carbon monoxide-hypoxia

Ornithine decarboxylase activity (ODC; E.C. 4.1.1.17), is significantly elevated in fetal and newborn rat brain in response to acute hypoxia. Because relatively little is known about ODC activities and polyamine metabolism in hypoxia and also because ODC and the polyamines are essential for normal growth and development, we examined the effect of chronic maternal hypoxic-hypoxia (16-10.5% O2), carbon monoxide-hypoxia (100-200 ppm CO) and their combination, on fetal weight, fetal brain ODC activity and polyamine concentrations. Time-dated pregnant Sprague-Dawley rats were chronically exposed to hypoxia from gestational day (E-15), to gestational day 21 (E-21), in individual chambers. Pair-fed controls were given an amount equivalent to that eaten by a hypoxic dam over the previous 24 h. We measured fetal weight, as well as brain ODC activity and polyamine concentrations on both E-19 and E-21. Pair-feeding had no effect on fetal weight, ODC activity or polyamine concentrations. On both E-19 and E-21, however, fetal weights were significantly reduced with higher levels of hypoxic-hypoxia (e.g., 10.5% O2). At 100 or 200 ppm, carbon monoxide alone appeared not to affect fetal weight; however, combined with even mild hypoxia (16% O2), fetal weights were reduced almost 20%, suggesting that together, CO- and hypoxic-hypoxia exert a synergistic effect of fetal weight decrements. (1) There was no consistent pattern of ODC activity changes which correlated to the fetal weight losses or levels of hypoxia. These results suggest that ODC activity may not be a good marker for chronic, as opposed to acute hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)